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W2317242682.txt
https://zenodo.org/record/2439781/files/article.pdf
de
Über den Einfluß von Substitution in den Komponenten binärer Lösungsgleichgewichte
Monatshefte für Chemie
1,925
public-domain
6,513
863 0bet den Einflu[5 yon Substitution in den Komponenten bin/ rer LSsungsgleiehgewiehte (III. Mitteilung) von R. K r e m a n n . Nach expelimentellen Versuchen der Herren E. Beneseh, W. Deeolle, P. Dolch, K. Kaas, F. Pileh und F. Szherenziss. Aus dem ehemischen Institut der Universit~it Graz. (Mit 10 Textfiguren.) (Vorgelegt in der Sitzung am 9. Juli 1908.) In einer frfiheren Mitteilung hatte ich g e m e i n s a m mit O. R o d i n i s 1 unter a n d e r e m gezeigt, da6: die F~ihigkeit yon Naphthalin, mit den isomeren Dinitrobenzolen und Dinitrotoluolen V e r b i n d u n g e n zu bilden, auger yon der Elektroaffinit~it der betreffenden Nitroverbindung, in erster Linie yon der Stellung der beiden N i t r o g r u p p e n abh/ingt. Es hatte sich damals als Spezialfall einer bei m e h r e r e n a n a l o g e n FS.Ilen zu Recht bes t e h e n d e n Regel ergeben, daft, w/ihrend ~ - und p-Dinitrobenzol mit Naphthalin V e r b i n d u n g e n in ~iquimolekularem Verh~iltnis liefern, o-Dinitrobenzol diese F~ihigkeit nicht besitzt und yon den damals u n t e r s u c h t e n vier Dinitrotoluolen nur diejenigen, bei denen die beiden N i t r o g r u p p e n w e d e r z u e i n a n d e r noch b e i d e z u g l e i c h zur CHa-Gruppe in Orthostellung sich befinden, d. i. bei 1: 2:4- und l:3:5-Dinitrotoluol. Wir erkl~irten dieses unterschiedliche Verhalten durch sterische Valenzbehinderung, die bei diesen Orthostellungen auftritt. Es w a r nun yon einem g e w i s s e n Interesse, die Additionsf~ihigkeit der isomeren Dinitrobenzole und Dinitrotoluole gegen1 Monatshefte fiir Chemie, 27, 125 (1906). Chemie-Heft Nr. 9. 59 864 R. Kremann, fiber anderen aromatischen Kohlenwasserstoffen, etwa Benzol und Phenanthren zu u n t e r s u c h e n und zu erfahren, ob die 0ben erwg.hnte Regel auch bier zu Recht besteht. Zumal write es yon besonderem Interesse gewesen, f~ir das S y s t e m m-Dinitrobenzol-Naphthalin, bei welchem die/iquimolekulare Verbindung dieser Stoffe im Schmelzflul3 so weitgehend dissoziiert, daft der ihr z u k o m m e n d e TeiI n a h e z u eine Horizontale darstellt - - es ist dies ein von mir zum erstenmal beobachteter Fall1 __ ein Analogon zu finden. Bei dem System m-Dinitrobenzol-Phenanthren, bei dem ziemlich analoge Temperaturverh/iltnisse obwalten, wie beim System m-Dinitrobenzol-Naphthalin, wttre yon vornherein ein analoges Verhalten nicht ausgeschlossen. Ich babe deshalb im vorigj~.hrigen und diesj~ihrigen physikalisch-chemischen Praktikum einige Herren veranlai3t; die diesbezfiglichen Schmelzdiagramme aufzunehmen, C~ber welche Versuche im experimentellenTeile u n t e r A n g a b e des betreffenden Beobachters berichtet w i r d . Als allgemeines Ergebnis ist zu verzeichnen, dab aus dem Verlauf der Schmelzkurven keinerlei Andeutungen daftir zu finden sind, dab irgendeines der drei isomeren Dinitrobenzole mit Benzol sowohI, als irgendeines der drei zur Unters u c h u n g verwendeten isomeren DinitrotoIuole (1, 2,4, 1, 2,6 und 1,3,4) als auch mit Phenanthren zu einer Verbindung zusarnmentreten. Von den drei isomeren Dinitrobenzoien gibt mit Phenanthren nicht nut das o-DinitrobenzoI, sondern auch das #n-Dinitrobenzol keine Verbindung. Nur das p-Dinitrobenzol tritt mit Phenanthren, wie aus dem Verlauf des Schmelzdiagrammes und geeigneten Zeit-Abktihlungskurven zu ersehen ist, zu einer Verbindung zusammen, die sich aus 3 MolekCtien Phenanthren und i Molekfil p-Dinitrobenzol zusammensetzt. Ihr Existenzbereich ist nut klein, es Iiegt z w i s c h e n 69 und 79 MolekfiIprozenten Phenanthren. Wir k o m m e n aIso zum Schlusse, dal3 yon den n u n m e h r u n t e r s u e h t e n aromatisehen Kohlenwasserstoffen mit einem, zwei und drei Benzolkernen der zweikernige, das Naphthalin, am f/ihigsten ist zur Bitdung yon Verbindungen. W~.hrend Naphthalin mit A u s n a h m e oben 1 Monatsheftef~irChemic, 25, 1283 (1905). Einflug yon Substitution etc. 865 erwiihnter F/ille sterischerValenzbehinderung mit Dinitrobenzol und Dinitrotoluol Verbindungen liefert, geht Benzol wie Phenanthren diese Fiihigkeit ab. Erst wenn wir die Elel(troaffinitiit der erw~ihnten DinitrokSrper durch Einftihrung einer weiteren Nitrogruppe erh6hen, also Trinitrobenzol, beziehungsweise Trinitrotoluol verwenden, tritt die Additionsf~ihigkeit gegentiber Phenanthren wieder auf. In beiden Fttllen ergibt sich aus dem Verlauf der Schmelzkurven der beiden Systeme PhenanthrenTrinitrobenzol und Phenanthren-Trinitrotoluol, daf3 Phenanthren sowohl mit Trinitrobenzol als auch mit Trinitrotoluol zu Verbindungen im ~quimolekularen Verh/iltnis zusammentritt. Experimenteller Teil. Die Methode der Aufnahme der Schmelzdiagramme war die bekannte, yon mir in der ersten Mitteilung ausftihrlich dargelegte. 1. LSsungsgleiehgewiehte zwischen Benzol und den drei isomeren Dinitrobenzolen. DieVersuchsergebnisse geben beistehendeTabellen wieder. T a b e l l e 1 (Beobachter: W. Decolle). LSsungsgleichgewicht zwischen Benzol und ~r Z u s a t z v o n m - D i n i t r o b e n z o l zu Benzol. Zusatz von m-Dinitrobenzol Gewichtsprozente Benzol Molekiilprozente Benzol Schmelzpunkt a) Menge Benzol: 17"043ff 0"000 2"330 3 "044 4"514 5"936 6'663 7"545 8"647 9"689 11 "224 13,858 lOO'O 88"0 84"8 79" 1 74"2 71 "9 69" 1 66"3 63"7 60; 3 55"1 100'0 94'5 92'7 89'1 86"0 84" 6 82"8 80"9 79"0 76'5 72'5 5"0 2"2 1"1 0"5 4'2 12'0 17'2 20"0 22"5 25"4 30"5 50* 866 R. Kremann, Zusatz yon I Gewichtsprozente m-Dinitrobenzol ] Benzol Molektill?rozente I Benzol I Schmelzpunkt b) Menge Benzol: 1 4 " 7 1 9 g 12'590 15'198 18'118 20'003 22"724 25"609 29"266 31"597 34"264 71 '5 67'4 63"5 61"2 58'1 55"3 52'0 50" 1 48" 1 53'9 49' 2 44' 8 42 "4 39"3 36"5 33"5 31 '8 30' 1 33"0 37'5 42'0 45"0 47'5 51 "0 53"5 55"0 57"0 c) Menge Benzol: 5" 2 7 1 g 15"197 19'446 23'454 27"728 32"478 36"108 39"285 44"815 Tabelle LSsungsgleiehgewicht 25"8 21 '3 18'3 16"0 13"9 12"7 11"8 10"5 43"2 36"8 32"8 29' t 26"2 24"3 22"2 20'5 2 (Beobachter: E, B e n e s c h ) . zwischen Benzol 59"0 64" 5 68 ' 0 74"5 75'0 76'0 77"0 79'0 und o-Dinitrobenzol. Zusatz yon o-Dinitrobenzol zu B e n z o l . Zusatz von Dinitrobenzol Gewiehtsprozente Benzol Molektilprozente Benzol Schmelzpunkt I a) Menge Benzol: 4 ' 7 3 3 g r 0"000 0"454 0"811 1"174 1"508 100"0 91 "8 85"5 80"1 75"8 100'0 96' 1 93" 1 89'7 86' 8 5'0 31 '0 89"0 47 "0 53"0 867 Einflul3 yon Substitution etc. Zusatz von Dinitrobenzol Gewichtsprozente Benzol Molekiilprozente Benzol Sehmelzpunkt 1 "951 2"711 3"295 4"373 5"591 6"813 8"263 9"971 11 '370 13" 347 70"8 83"9 78"9 75"6 69'9 64"5 59'8 55'2 50"8 47'3 43'5 58'0 65'0 69'0 75'0 80'0 84"0 87"0 90'0 93"0 95"5 63"6 59'0 51 "9 45' 8 41 '0 36'4 32"2 29"4 26'2 I b) Menge Benzol: 6"644 3" O" 142 0"275 97"9 94" i 98"7 97'2 4"3 23"0 T a b e I l e 3 (Beobachter: F. Pitch). LSsungsgleichgewicht zwischen p-Dinitrobenzol und Benzol Z u s a t z y o n y - D i n i t r o b e n z o l zu Benzol. Zusatz von Dinitrobenzoi Gewichtsprozente Benzol Molekiilprozente Benzol Schmelzpunkt a) Menge Benzol: 9"318gr 0"000 0"111 O" 349 O' 498 O" 730 0'906 100'0 98"8 96 "4 94"9 92"7 91'1 lO0'O 5"2 99'4 98"3 4"8 28'01 97"6 96"5 95"7 40" 6 54"0 61 '51 1 LSsungen angegebener Konzentration schieden Krystalle aus vom Sehmelzpunkt 172 ~ und erwiesen sich als reines p-Dinitrobenzol. 868 R. K r e m a n n , I I --... / 50 I ! ~ 3O 2~ r I ! i 84 , i I ! I ~ I i ,~0 ~0 70 L 9 t 4 t o I l 1 .-+ 89 9~ JQG Mole~lprozente Benzol Fig. i. LSsungsgleichgewicht zwischen Benzol und den drei isomeren Dinitrobenzolen. Einflul3 yon Substitution etc, Zusatz yon Dinitrobenzoi I Gewichtsprozente Molektilprozente Benzol BenzoI 869 Sehmeizpunkt i b) Menge Benzol: 10' 168g t'275 1"677 88"7 85'7 94'5 92'8 70"5 78'91 1 Bei 80"9~ tritt Sieden des Benzols ein. Es ist also hier die sogenannte ,Kochgrenze< erreicht. Bei Zusammenbringen yon Benzo! und p-DinitrobenzoI im Verhiiltnis yon 92" 0 : 8'0 Molekiilprozenten tritt eben noch LSsung des p-Dinitrobenzols ein. Bei 92'0 MoIekiilprozenten Benzol betr~igt der Sehmelzpunkt also 80"9~ Dieser Punkt ist im Diagramm mit * bezeichnet. Aus den die Versuchsergebnisse veranschaulichenden Diag r a m m e n Fig. 1 ergibt sich, dat~ keines der drei Dinitrobenzole mit Benzol zu einer Verbindung zusammentritt. Die LSslichkeitslinien derselben in Benzol stellen stetige Kurven dar; die sich auch bei tieferen Temperaturen, etwa bei 100, a u s s c h e i d e n d e n Krystalle enthielten kein Benzol und erwiesen Temperatur sich durch B e s t i m m u n g des S c h m e l z p u n k t e s als reine Dinitrobenzole. Bei einer Mischung von 70 Molekfil] prozenten Benzol und 30 Molektilprozenten m-Dinitrobenzol nahm ich eine Zeit-Abkiihakl l u n g s k u r v e auf. Ihr stetiger Verlauf zeigt, dal3 die geringen Unregelm~tl3igkeiten, die das S e h m e t z d i a g r a m m m - DinitrobenzoI- Benzol Fig. 2. etwa bei 80 Molekiilprozenten Benzol aufweist, nicht d u t c h das Auftreten einer U m w a n d l u n g s e r s c h e i n u n g z u r 0 c k z u f f i h r e n ist, sondern seinen Grund in Beobachtungsfehlern hat. Die B e o b a c h t u n g des T h e r m o m e t e r s w/ihrend des Erstarrens einer Mischung obiger Z u s a m m e n s e t z u n g ergab: Zeit in Minuten Temperatur 0 0"5 l'O 44 40 36 Zeit in Minuten I 95 2"0 2'5 Temperatur 34 33 31 870 R. Kremann, Zeit in Minuten 3"0 4"0 5"0 Temperatur 30 27 24 Zeit in Minuten 6"0 7"0 Temperatu/ 22 20 Die in Fig. 2 g e g e b e n e Zeit-Abkfihlungskur~e ergibt den E r s t a r r u n g s p u n k t , d. i. das A u s s c h e i d e n der ersten Krystalle bei 34 ~ w a s sich dem Verlauf der S c h m e l z k u r v e sehr gut einpal3t, bei tieferer T e m p e r a t u r keine A n d e u t u n g ffir irgendeine U m w a n d l u n g s e r s c h e i n u n g . 2. L 6 s u n g s g l e i z h g e w i e h t e z w i s z h e n Benzol u n d den i s o m e r e n D i n i t r o t o l u o l e n : 1, 2, 4; 1, 2, 6; 1, 3, 4. Die folgenden T a b e l l e n g e b e n die V e r s u c h s e r g e b n i s s e wieder, welche die S c h m e l z k u r v e n der Fig. 3 v e r a n s c h a u l i c h e n . Tabelle 4 (Beobachter: E. B e n e s c h ) . L S s u n g s g l e i c h g e w i c h t z w i s c h e n B e n z o l und 1, 2, 4-Dinitrotoluol. Zusatz yon Dinitrotoluol zu Benzol. Zusatz von Dinitrotoluol Gewichtsprozente Molekiilprozente Benzol Benzol Schmelzpunkt a) Menge Benzol: 15"6883" 0"000 lO0'O lOO'O 5'0 95"4 2"5 1'511 90'4 2"358 86"9 93"9 1'5 2"846 84"6 92'6 1"0 3"218 83"0 92'0 0'5 4'289 78"5 89'0 --0'5 5"239 75"0 87"1 +3'5 6"099 72"0 85'6 -I-5"5 7'196 68"6 83'6 +11"0 871 Einflul3 yon Substitution etc. i Zusatz von Dinitrotoluol Gewichtsprozente i Molekfilprozente Benzol I Benzol Schmelzpunkt I J 81"i -+-15'0 61'0 78'6 +18"0 ~ 58"0 76'4 --I-22'0 74'7 +24"0 72"8 +25'0 8'485 64"9 10"022 11"346 12'422 13'773 I ! ~i ! I ! I 55'8 53"3 I I I b) Menge Benzol : 17' 291 ff 19'457 47'0 67"5 30'0 20'343 45"9 66"5 30"5 21'302 44'7 65'4 31 "5 23"017 42'9 63'7 33"0 24'806 41'1 62'0 35'0 26"021 39'9 60'8 35'0 27'666 38"5 59"4 37"0 29"908 36"5 57"3 38'5 32"378 34"7 55"4 40'0 34'591 33'3 53"8 41 '5 c) Menge Benzol: 2'460g 5"677 30"2 50'2 46'0 6"643 27"0 46"6 48"0 7'817 24'0 42'3 50"5 9"679 20'3 37"3 53'0 12"954 16"0 31 "0 57"0 17 "416 12'4 24'6 59"0 23"379 9'5 19"2 62 "0 28' 804 7'9 15"6 63 ' 0 872 R. K r e m a n n , Tabelle 5 (Beobachter LSsungsgleichgewicht F. S c h e r e n z i s s ) . zwischen Benzol und 1, 2, 6 - D i n i t r o - toluol. a) Z u s a t z v o n B e n z o l z u D i n i t r o t o l u o l . Menge Dinitrotoluol : i0" 508 ft. Zusatz v o n Dinitrotoluol o'ooo o.t51 1"265 1'726 2'155 2'657 3.681 4"297 5"596 6"828 9"071 10'408 Gewichtsprozente Benzol Molekfilprozente Benzol Schmelzpunkt 0"0 1"4 10:7 14"I 17"0 20'5 26"1 29"2 34"7 39" 4 46'3 49'8 0"0 2"8 21 '2 27'8 32'5 37"5 45"0 49 "0 55"3 60"3 66'8 69'8 65 "0 63"2 53'3 49'4 46"5 43'5 38'0 36 '4 33"0 30'0 25'0 22"6 b) Z u s a t z y o n D i n i t r o t o l u o l z u B e n z o l . M enge Benzol: 8" 5112". Zusatz von Diiiitrotoluoi Gewichtsprozente Benzol Molekiilprozente Benzol Schmelzpankt 0"0O0 0"789 1'837 100'0 91.5 82'3 75"3 70" 4 63"3 58"2 53"8 50" 4 100"0 96"2 91 "3 87'3 84"9 80'2 76"6 73"0 70'3 5"3 2"8 0'1 3'8 9"0 13 "0 17 "0 19"5 21 '8 2"787 3'817 4"924 6"121 7"323 8"375 873 Einflutl von Substitution etc. Tabelle L6sungsgleichgewicht 6 ( B e o b a c h t e r F. P i l c h ) . zwischen Dinitrotoluol (1,3,4) und Benzol. a) Zusatz yon Dinitrotoluol zu Benzol. Zusatz yon Dinitrotoluol Gewichtsprozente Benzol Molekiilprozente Benzol Sehmelzpunkt Menge Benzol : 9" 862 g 0"000 0"542 0"890 1"397 1'996 3"109 4"579 5"573 7'904 8"654 10'328 10O'O 94'8 91'8 87'7 83'3 76"2 68"5 63'3 55"8 54"6 49'1 100"0 97"7 96"3 94"3 92"1 88"0 83' 8 80"1 74"6 73'7 69'4 5'1 4'0 3'4 2"7 2"0 0'2 --2"0 --3'2 --2'0 --0'6 --6"6 Menge Benzol : 3" 323 07 2'520 57"0 75"5 --5"01 1 Dureh Aufnahme einer Zeit-Abkiihlungskurve wurde der eutektische Punkt bei - - 5 " 0 festgelegt, was im Diagramm Fig. 3 durch die eutekfische Horizontale e es angedeutet ist. 5) Z u s a t z y o n B e n z o l zu D i n i t r o t o l u o l . Menge Dinitrototuol : 9" 499 07. Zusatz yon Benzol Gewichtsprozente Benzol Molekfilprozente Benzol Schmelzpunkt O' 000 1"308 I "663 2'383 3"261 3" 993 4"813 5"639 7"739 0"0 12"1 14-9 19"6 25"7 29"6 33'6 37"2 44"9 0"0 24' 3 29"5 36"3 44"6 49" 5 54'2 58" I 65"5 59"0 46"0 42"5 38'2 32"5 28"6 25"0 20"5 11"8 874 R. Kremann, [ ? ao I [ o 1o t - =o 3Iolekiilprozente so ~o ~o eo w eo 90 I00 Benzol Fig. 3. L~Ssungsgleichgewichtezwischen Dinitrotoluoleqund Benzol. Man sieht, dal3 auch bier keinerlei Andeutung sich ffir die Existenz von Verbindungen vorfindet. Die L6slichkeitskurven der Dinitrotoluole ill Benzol verlaufen stetig. Die Lage der eutektischen Punkte ist ohne, weiters aus den Diagrammen ersichtlich. Bei der Kurve Benzol-I, 2,4-Dinitrotoluol finden sich bei zirka 75 Molekfilprozent Benzol einige Unregelm/iI3igkeiten. DaB dies jedoch nur auf Beobachtungsfehler zurCtckzuffihren ist und "jede Umwandlungserscheinung fehlt, beweist das aufgenommene Zeit-AbkfihIungsFig. 4. diagramm. Eine Mischung yon 60 MolekfilproZeit in Minute14 zenten Benzol und 40 Molekfilprozenten Dinitrotoluol gab bei der langsamen Abkflhlung unter stetem r\l 875 Einfluf~ von Substitution etc. Rtihren der Reihe nach folgende T e m p e r a t u r e n bei Ablesung des T h e r m o m e t e r s w~ihrend je einer Minute: 46, 40, 37, 31, 26, 22, 19, 17"5 ~ . Die graphisehe Darstellung der Zeit-Abktihlungskurve in Fig. 4 zeigt, dab nur ein kurzes Anhalten des T h e r m o m e t e r s bei dem Auftreten der ersten Krystalle, d. i. bei 37 ~ zu beobachten war. Unterhalb dieser T e m p e r a t u r ist bis 17"5 ~ eine U m w a n d l u n g s e r s c h e i n u n g nicht zu b e o b a c h t e n gewesen. Bemerken mSchte ich nut, dal3 die LSslichkeitskurven von 1, 2, 4-Dinitrotoluol und 1,2, 6-Dinitrotoluol bei 9 ~ sich schneiden. W i t haben es also unterhalb 9 ~ mit einer A b w e i c h u n g der Regel yon T h o m s o n und C a r n a l l e y 1 zu tun. 3. LSsungsgleiehgewiehte zwisehen Phenanthren und den drei isomeren Dinitrobenzolen. Die Versuchsergebnisse sind in den folgenden drei Tabellen w i e d e r g e g e b e n und in Fig. 5 graphisch dargestellt. Tabelle 7 (Beobachter: W. D e c o l l e ) . LSsungsgleichgewicht zwischen o-Dinitrobenzol und Phenanthren. a) Z u s a t z y o n P h e n a n t h r e n z u D i n i t r o b e n z o l . M e n g e Dinitrobenzol : 5' 829 g'. Zusatz von Ph enanthren 0"000 1 ' 273 1 "862 3'052 4 ' 606 6"759 8'187 Gewichtsprozente Phenanthren Molektilprozente Phenanthren Schmelzpunkt 0"0 17"9 24' 2 34"4 O'O 17"1 23"1 33"1 42'7 51 "2 56'9 116'0 107' 1 104'0 98'0 91 ' 5 83"5 79 "0 44"2 53'7 58"4 1 Journ. Chem. Soc., 53, 782 (1888), u n d Monatshefte ftir Chemie, 28, 7 (1907). 876 R. K r e m a n n , b) Z u s a t z y o n D i n i t r o b e n z o l z u P h e n a n t h r e n . Menge Phenanthren: 5 '294g. Zusatz von Dinitrobenzol Oewichtsprozente Phenanthren Molekfilprozente Phenanthren Schmelzpunkt O'000 1O0"0 100"0 103 '0 0"591 89'9 89'3 96"5 1'191 81'6 80"7 90'2 2'520 67"8 66"5 79"0 3"805 58"2 56"7 79"5 T a b e l l e 8 (Beobachter: K. Kaas). L6sungsgleichgewicht zwischen ~4~-Dinitrobenzol und Phenanthren. a) Zusatz yon m-Dinitrobenzol zu P h e n a n t h r e n . Menge Phenanthren : 8' 787 o Zusatz v o n Dinitrobenzol Gewichtsprozente Phenanthren Moleki_ilprozente Phenanthren Schmelzpunkt 0'00O 100"0 10O'0 103"5 0'420 95 "4 95'2 100"0 0'939 90'2 89"7 96"0 1"791 82'9 82'1 89'0 2"545 77"4 76"3 84'5 3'622 70'6 69'3 77"0 4"774 64'5 63"2 70"0 5'957 59'4 57"9 64"5 7'199 54'7 53'2 58'0 8"194 51 "5 50"O 51"0 9"05O 49 '0 47'5 48"5 10"440 45 "4 44'0 53"0 877 Einflut3 yon Substitution etc. b) Z u s a t z y o n P h e n a n t h r e n z u m - D i n i t r o b e n z o l . Menge m-Dinitrobenzol : 5' 086 g. Zusatz von Phenanthren Gewichtsprozente Phenanthren Molekiilprozente Phenanthren Schmelzpunkt 0"000 0"253 0"596 0'975 1'299 1"773 2"243 2'580 3'056 3"576 4'234 4"8419 5"425 5"777 6"154 6"445 6"716 7'089 7'695 0"0 4'7 10"5 16"1 20'3 25"9 30"6 33'7 37'5 41"3 45"4 48"8 51"6 53"2 54"8 55'9 56'9 58"3 60'2 0'0 4'5 10'0 15'3 19'4 24"7 29'4 32"4 36"2 39"9 44"0 47"3 50"2 51"8 53'3 54"5 55"5 56'8 58"8 89'5 87'0 83 "O 80'0 76'5 72'0 69"0 65"5 62'5 58"0 53'0 48"5 53'0 55"5 57"0 60"0 62'0 65 '0 T a b e l l e 9 (Beobachter: F. Pilch). LSsungsgleichgewicht zwischen p-Dinitrobenzol und Phenanthren. a) Z u s a t z v o n p - D i n i t r o b e n z o l z u P h e n a n t h r e n . [ Zusatz yon Dinitrobenzol Gewichtsprozente i Molekiilprozente Phenanthren Phenanthren Schmelzpunkt / c~) Menge Phenanthren: 11" 0242' 0"000 0"649 1"277 1"914 3'026 4'138 1O0'0 94"4 89"7 85"2 78'5 72"7 100'0 94"1 89'1 84"4 77"5 71"5 103'0 98'5 94'0 89'5 81 "0 80"3 878 R. K r e m a n n , Zusatz yon Dinitrobenzol Gewichtsprozente Phenanthren Molekiilprozente Schmelzpunkt Phenanthren I I ~) Menge Phenanthren : 4" 208 3" 1"731 ] 70'9 69' 6 2'498 62'8 61 '4 2'986 58'5 57' 1 3"402 55'3 53'9 3"789 52"6 51 '2 4"284 49"6 48' 1 81'0 i , 104'0 i 113'0 I 119"0 I i i I I 124"0 129"5 7) Menge Phenanthren: 5 ' 7 4 7 f f 1'758 76'6 75'5 81 '0 1'890 75'3 76"2 80'5 5'263 71 '8 70'6 79'0 2'462 70'0 68'8 84'0 b) Z u s a t z von Phenanthren zu Dinitrobenzol. Menge y-Dinitrobenzol: 11" 184g'. Phenanthren Gewichtsprozente Phenanthren Molekiilprozente Phenanthren Schmelzpunkt 0"000 0"0 0"0 172'0 0"513 4'4 4"1 170'3 ZHsatz v o n 1'042 8'5 8"0 168"0 1'749 13'5 12'8 i65'0 2"829 20" l 19'2 160'0 3"901 25"9 24"8 155'5 5"221 31 '8 30'6 150'0 6"348 36"2 34'9 145"5 7'373 39'7 38"4 141'5 8'438 43"0 41"6 137'6 c~ ~:~ co , ? = 2 co 0 ~q ~'~ ~3 o c~ Oq C31 -+ S c h m d z l e m p e r a t u r e ~ in Celsiusgraden Co o 880 R. Kremann, stellen je zwei stetig verlaufende Kurven dar, die LOsliehkeitslinien yon Phenanthren einerseits, der beiden Dinitrotoluole andrerseits. Der eutektische Punkt beider Systeme ergibt sich ohneweiters aus den Diagrammen. Bei dem System p-Dinitrobenzol-Phenanthren finder Sich ein Sttick alb zwischen 69 und 77 Molektilprozent Phenanthren, das weder der Lt3slichkeitskurve von Phenanthren b c noeh der yon p - D i n i t r o b e n z o I angeh~Srt. Es dtirfte sich hier um das Auftreten einer Verbindung handeln, die naeh der Lage im Schmelzdiagramm die Zusammensetzung yon 3 Molektilen Phenanthren und 1 Molektil Dinitrobenzol haben dtirfte. Ich habe deshalb g: % 7' "O~ -.~ Zeil i~ Mim#e~ Fig. 6. zur Festlegung der beiden eutektischen Punkte a und b sowie des Schmelzpunktes der Verbindung f Zeit-Abktihlungskurven aufgenommen. Denn wenn keine Verbindung zwischen Phenanthren und p-Dinitrobenzol existierte, so wtirde sich nur ein eutektischer Punkt, und zwar im Schnittpunkt der beiden L6slichkeitslinien e, d. i.:bei 71 ~ realisieren lassen mtissen. Eine Mischung, die 80 MolekCflprozent Phenanthren enthielt, also auf der L~Sslichkeitskurve von Phenanthren liegend, reines Phenanthren ausscheidet, wurde langsam unter stetem R/_ihren erstarren gelassen und die Temperatur der Schmelze w~ihrend jeder Minute abgelesen. Es wurden der Reihe nach folgende Temperaturwerte erhalten: 108, 102, 97 , 93~ 90, 86, 85, 84, 88, 82, 81, 80, 79, 79, 79, 79, 79, 79, 79, 78"5, 78, 77, 76. (Bei diesen Ietzten Bestimmungen war bereits alles lest.) Einflufl von Substitution etc. 881 Man sieht aus der Zeit-Abktih!ungskurve der Fig. 6, daI3 die erste Krystallausscheidung bei 85 ~ eintritt, wie sich auch aus dem Verlauf der LSslichkeitskurve P h e n a n t h r e n bc ableiten i/~fgt, der eutektische P u n k t b bei 79 ~ liegt und nicht tiefer bei 71 ~ Eine Mischung von 74 Molekfilprozent gab w/ihrend der E r s t a r r u n g folgende T e m p e r a t u r w e r t e : 106, 101, 97, 92"5, 88"5, 86, 84, 82, 81, 81, 81, 80'5, 80, 80, 79"5, 79"5. 79"5, 79"5, 79, 78"5, 78, 77"5, 77, 76"5. Wir befinden uns bei der Mischung dieser Z u s a m m e n setzung auf der LSslichkeitskurve a b der Verbindung. W i r sehen aus dem Verlauf der Zeit-Abkfihlungskurve Fig. 7, I daft bei 81 ~ ein Anhalten d e s T h e r m o m e t e r s stattfindet, nach einiger tritt Sinken ein, bis bei 79"5 ~ wieder ein neuerliches Anhalten der T e m p e r a t u r zu beobachten ist. Dann tritt unter gleichzeitigem vollkommenen Festw e r d e n der Masse steres -+ Zei~ in Minulen Sinken ein. Fig. 7. Es scheidet sich eben bei 81" 0 ~ die Verbindung aus; damit n/ihert sich die Z u s a m m e n setzung der Mutterlauge immer mehr dem eutektischen Punkte ~, was mit dem Sinken der T e m p e r a t u r von 81 ~ ab Hand in H a n d geht. Ist schliel31ich die Z u s a m m e n s e t z u n g der eutekfischen Mischung a erreicht, so erstarrt diese h o m o g e n bei 79"5 ~ welche T e m p e r a t u r die des eutektischen Punktes a darstellt. Lassen wir eine Mischung yon 67 Molekfilprozent Phenanthren erstarren, so erhalten wit bei der T e m p e r a t u r b e o b achtung jeder Minute wS.hrend des Erstarrens der Reihe n a c h 108, 105, 101"5, 99, 97'5, 95, 92, 90, 87"5, 87, 86, 85'0, 83"9, 82-8, 82, 81, 80, 79"5, 79"5, 79"5, 79, 78"5, 78, 77"5, 77, 76'5, 76. Die Mischung obiger Z u s a m m e n s e t z u n g liegt bereit~ a u f der LSslichkeitskurve p-DinitrobenzoI. Bei 87 ~ beginnt, wie aus der Zeit-Abkfihlungskurve Fig. 8 zu ersehen ist, die 60* 882 R. Kremann, Ausscheidung yon reinem Dinitrobenzol. Nun ~indert sich unter steter langsamer Temperaturabnahme infolge der Ausscheidung yon Dinitrotoluol die Zusammensetzung der Mutterlauge inaolange, als die Zusammensetzung derselben der eutektischen Mischung a der Fig. 5 entsprieht; dann wird alles feat, was dem erneuten Anhalten der Zeit-Abktihlungskurve (Fig. 8) bei 79" 5 ~ entspricht. Wir kommen also zum Resultat, dal3 Phenanthren mit p-Dinitrobenzol zu einer Verbindung, bestehend aus 3 Molekfilen Phenanthren und 1 Molekfil Dinitrobenzol vom Schmelzpunkt 81 bis 81"5 ~ zusammentritt. Der eutektische Punkt \ r i Zeit in Minuten Fig. 8. zwischen der Verbindung und p-Dinitrobenzol liegt bei einer Zusammensetzung yon 70 Molekfilprozenten Phenanthren und 30 Molekfilprozenten p-Dinitrobenzol und bei 79"5 ~ (Punkt a tier Fig. 5). Der eutektische Punkt zwischen der Verbindung und Phenanthren liegt bei einer Zusammensetzung yon 77 Molektilprozenten Phenanthren und 23 Molekfilprozenten p-Dinitrobenzol und bei 79 ~ (Punkt b d e r Fig. 5). Die beiden anderen Dinitrobenzole geben, wie eingangs erw/ihnt, mit Phenanthren k e in e Verbindungen. 4. L6sungsgleichgewiehte zwischen P h e n a n t h r e n und den isomeren Dinitrotoluolen 1, 2, 4; 1, 2, 6; 1, 3, 4. Die VersuchSergebnisse geben die folgenden Tabellen wieder. 883 Einflul~ yon Substitution etc. T a b e l l e 10 (Beobachter: K. Kaas und P. Doleh). LSsungsgleichgewicht zwischen Dinitrotoluol (1, 2, 6) und Phenanthren. a) Zusatz y o n D i n i t r o t o l u o l z u P h e n a n t h r e n . Menge Phenanthren: 8" 252ff. Zusatz von Dinitrotoluol Gewichtsprozente Phenanthren Molekiilprozente Phenanthren 0'000 160"0 100"0 103'5 0"426 95-1 95"2 100"5 0"818 91'0 91"2 97'2 92'0 Sehmelzpunkt 1'526 84'4 84"7 2'276 78'4 78"7 87"5 3"223 71"9 72"3 81"5 4"317 65"7 66'2 75"5 5"171 61'5 62"0 72'5 6'159 57'3 57'8 68"0 7"255 53"2 53'8 63'0 8"625 48"9 49"4 57'5 9"930 45'4 11"365 42'1 .... 46"0 53"0 42'6 " 48"0 b) Z u s a t z y o n P h e n a n t h r e n z u D i n i t r o t o l u o l . Menge Dinitrotoluol: 7"540g. ZHsatz von Phenanthren Gewichtsprozente Phenanthren Molekiilprozente Phenanthren Sehmelzpunkt O'OOO 0"0 0'0 65'0 0"156 2"0 2"I 64'0 0"669 8'2 8'3 60'5 1"324 14'9 15"2 57'0 884 R. K r e m a n n , c) Zusatz v o n P h e n a n t h r e n zu Dinitrotoluol. Menge Dinitrotoluol: 5" 290g. Phenanthren Gewichtsprozente Phenanthren Molekfilprozente Phenanthren 1 "32 1 '61 2"01 2 "40 2'97 3"58 4" 10 4"47 20"0 23"3 27"5 31 "2 35"9 40'6 43"6 45'9 20"3 23"7 28 "0 31 '7 36"5 Zusatz v o n Tabelle L6sungsgleichgewieht 54"0 53 "0 48'0 45'0 40'0 44" 0 49"0 52'0 i 41 "2 44" 2 46 '5 11 ( B e o b a c h t e r : zwischen Schmelzpunkt K. K a a s ) . Dinitrotoluol ( 1 , 2, 4 ) und Phenanthren. a) Zusatz yon P h e n a n t h r e n zu Dinitrotoluol. Menge Dinitrotoluol : 8" 900 g. Phenanthren Gewichtsprozente Phenanthren Molekfilprozente Phenanthren 0'000 0"291 0"714 1"091 1"489 1'927 2'301 0'0 3"2 7"4 10"9 14'3 17"8 20'6 2"818 3"169 3"460 3'855 4'512 5"040 24'0 26'3 27"9 30"2 33'7 36"2 5"565 6"306 7"284 8"341 38"5 41"5 45"0 48'4 0'0 3'2 7'6 11"1 14"6 18"1 20'9 24'4 26"7 18 '4 30"7 34"2 36'2 39'0 42'0 45"6 Zusatz yon 48'9 Schmelzpunkt 69'0 68'0 65'5 64"0 61"5 60"0 58 "0 56'0 54'5 53'0 51 '0 49 "0 46' 7 9 44'0 41 '.0 30"0 44" 5 885 Einflul3 von Substitution etc. b) Zusatz y o n Dinitrotoluol zu Phenanthren. Menge Phenanthren: 8" 095 g. Zusatz yon Phenanthren Gewichtsprozente Phenanthren Molekfilprozente Phenanthren Schmelzpunkt 0"000 0'680 1'152 1'602 2'204 2"986 3"436 4'159 4'902 5'559 6"159 7'359 8"541 9"424 10'332 100'0 92"2 87"5 84"6 78'6 73'4 70"2 61"1 62"3 59'3 56'8 52"4 48"6 46'2 43"9 100"0 92'4 87"8 84'9 79'0 73'8 70"7 66'6 62"8 59"8 57"3 52"9 49'2 46"8 44"3 103'5 98'0 95'0 91"7 87"0 81'5 78"0 73"5 68"0 63"0 59'5 53'0 46'0 42'0 37"5 Tabelle 12 (Beobachter: F. Scherenziss). LSsungsgleichgewicht zwischen Dinitrotoluol (1, 3, 4) und Phenanthren. a) Zusatz von Dinitrotoluol zu P h e n a n t h r e n . Menge Phcnanthren: 8" 245 2". Zusatz VOI~ Dinitrotoluol Gewichtsprozente Phenanthren Molekfilprozente Phenanthren Schmelzpunkt 0.000 0'543 1"037 1"595 2"311 3"300 4"230 5'580 7"170 8'563 100'0 93'8 88"8 83'8 78"2 71"4 66"1 58"9 53"8 49"0 iO0"0 94"0 89' 1 84"1 78'6 71 '8 66"6 59"4 5~'3 49" 6 103' 5 99"5 96"5 92"2 87"5 82"0 77"0 69'0 61 '0 55"0 886 R. Kremann, b) Z u s a t z y o n P h e n a n t h r e n zu Dinitrotoluol. Menge Dinitrotoluol : 8" 262 g. Zusatz yon Phenanthren Gewichtsprozente Phenanthren Molek~ilprozente Phenanthren 0"000 1"159 i'384 1'654 2"206 3'074 8"827 4'219 5"854 7"035 8'328 0"0 12'3 14'4 16"7 21"1 27'2 31"6 32'3 41"4 44"7 50'5 0'0 12'5 I4"7 17'0 21"5 27'6 32'1 32"8 41"9 45"3 51"1 Schmelzpunkt 59"0 51 "5 49" 0 47"0 44'0 40"0 36'0 34' 0 42"8 50"9 57"6 ~00 i - I & ~so__. ~ . .-+ Molekiilproze.te Phena~thren 0 t.0" 20 30 ~0 60 70 ~0 9O Fig. 9. LSsungsgleichgewicht zwischen Dinitrotoluolen und Phenanthren. .100 Einflul3 von Substitution etc. {{87 Die graphische Darstellung in Fig. 9 stellt die Versuchsdaten anschaulich dar. Ich kann reich bei der Diskussion der V e r s u c h s e r g e b n i s s e g a n z k u r z fassen. Aus dem Verlauf der S c h m e l z k u r v e n gelat eindeutig hervor, dab keines der drei untersuchten Dinitr0toluole mit Phenanthren zu einer Verbindung zusammentri!t. Die Lage und Z u s a m m e n s e t z u n g de:" eutektischen Punkte ergil?t sich ohneweiters aus den S c h m e l z k u r v e n der Fig. 9. 5. L i J s u n g s g l e i c h g e w i c h t e zwiSchen P h e n a n t h r e n u n d T r i n i t r o benzol, b e z i e h u n g s w e i s e T r i n i t r o t o l u o l . Die Versuchsergebnisse Tabellen 13 und 14 wieder. Tabelle geben die beiden folgenden 13 (Beobaci:ter: P. D o l c h ) . L S s u n g s g l e i c h g e w i c h t z w i s c h e n P h e n a n t h r e n , und Trinitrotoluol~ a) Zusatz y o n Trinitrotoluol zu Phenanthren. Menge Phenanthren: 7' 68 g. Zusatz y o n Trinitrotoluol 0'00 0'42 0'77 1;13 1"87 2'49 4'06 4"62 5"10 5"78 6"67 7"82 8' 29 9"18 Gewichtsprozente Molekiilpr0zente Phenanthren Phenanthren 100"0 94"8 90"9 87"2 80"4 75 "5 65 "4 62'4 60'1 57"1 53 "4 49'6 48' 1 45'6 100'0 95"9 92;8 89' 8 84" 1 79"6 70"7 67'8 65.8 62'9 59 '4 55'6 54"1 51 "7 Schmelzpunkt 103 "0 i01 "5 98"5 96"0 92"0 87 "0 77"0 77"5 79 "0 82"5 85"0 86"5 87'0 87'5 888 R. K r e m a n n , b) Zusatz yon P h e n a n t h r e n zu Trinitrotoluol. Menge Trinitrotoluol : 6 '97 g. Zusatz von Phenanthren Gewichtsprozente ] M01ekiilprozente Phenanthren Phenanthren 0"00 0"00 0"63 1" 14 1 '48 2' 24 2"70 3 "48 4"49 4'80 Schmelzpunkt 0'0 10"4 17"2 21'3 29"i 33"1 39"0 44"9 49"5 8.a 14"0 17"4 24" 3 27'9 3324 89 "0 43 ' 4 78'0 74"0 70"0 72"0 78"5 82'0 85"0 87"0 87"5 T a b e l l e 14 (Beobach;er: P. Dolch). LSsungsgleichgewicht zwischen Trinitrobenzol und Phenanthren. a) Zusatz yon Trlnitrobenzol zu P h e n a n t h r e n . Menge Phenanthren : 5" 11 g'. Zusatz von Trinitrobenzol GewichtsProzente Phenanthren Molekiilprozente Phenanthren 0"00 0"37 0"57 0"85 1.04 1 "26 1'77 2"28 2' 84 3:36 4-41 5"42 100'0 93"2 89'9 85"8 83"1 lO0'O 94'2 91 "5 87 "8 85"6 82"6 78"7 72"8 68"2 64"5 58"I 802 75"5 69'1 64'3 60'3 53'7 48"5 52.9 Schmelzpunkt 103 100 97 94 91 91 1()2 111 t17 120 124 125 889 Einflul3 von Substitution etc. b) Z u s a t z y o n P h e n a n t h r e n zu Trinitrobenzol. Menge Trinitrobenzoi: 5" 67 g. Zusatz von Phenanthren Gewichtsprozente Phenanthren Molektilprozente Phenanthren Schmelzpunkt 0'0 10'0 15'9 24'6 38"0 0'0 1t "7 18'5 27" 1 42' 1 121 111 106 115 124 0"00 0"63 I "07 1 "85 3'47 130 f \ J 910 < / i r I [ I ~o so I I 7o 10 -+ 20 30 ~0 50 60 70 ~o 00 ~aO Molekiilprozer162 P h e n a n l h r e n Fig. 10. L6sungsgleichgewichte zwischen Trinitrobenzol und -toluol und Phenanthren. In F i g . 10 s i n d die e x p e r i m e n t e l l e n D a t e n g r a p h i s c h vera n s c h a u l i c h t . M a n s i e h t , daft P h e n a n t h r e n s o w o h l m i t T r i n i t r o b e n z o l als a u c h m i t T r i n i t r o t o l u o l z u V e r b i n d u n g e n im /iquim o l e k u l a r e n VerhS, ltnis z u s a m m e n t r e t e n , die S c h m e l z p u n k t e v o n 125, b e z i e h u n g s w e i s e 8 7 " 5 ~ a u f w e i s e n . W i r h a b e n in b e i d e n F/illen d a h e r z w e i e u t e k t i s c h e P u n k t e . Der eutektische Punkt zwischen TrinitrobenzoI und der V e r b i n d u n g l i e g t bei 17 M o l e k f i l p r o z e n t e n P h e n a n t h r e n u n d 890 R. Kremann, Einflul~yon Substitution etc. bei 104 ~, der zwischen der Verbindung uncl P h e n a n t h r e n liegt bei einer Z u s a m m e n s e t z u n g von 84"5 Molekfilprozenten Phenanthren und 85"5 ~ . . . . . . . . Zwischen 17 und 84"5 Molektilprozenten Phenanthren liegt das Existenzbereich der Verbindung Phenanthren-Trinitrobenz01. " ....... Das Existenzbereich d e r V e r b i n d u n g Trinitrotoluol-Phenanthren liegt zwischen Z u s a m m e n s e t z u n g der Mischung von 18 bis 7 0 Molekiilprozenten Phenanthren. Die~ sind auch die Z u s a m m e n s e t z u n g e n der eutektischen Punkte von Trinitrotoluoi und der Verbindung :einerseits, der V e r b i n d u n g und Phenanthren andrerseits. Die entsprechenden T e m p e r a t u r w e r t e der beiden eutektischen Punkte liegen bei 69 ~ beziehungsweise 70 ~
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Production of thyrotropin receptor antibodies in acute phase of infectious mononucleosis due to Epstein–Barr virus primary infection: a case report of a child
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Nagata et al. SpringerPlus (2015) 4:456 DOI 10.1186/s40064-015-1236-8 Open Access CASE STUDY Production of thyrotropin receptor antibodies in acute phase of infectious mononucleosis due to Epstein–Barr virus primary infection: a case report of a child Keiko Nagata1*, Keisuke Okuno2, Marika Ochi1, Keisuke Kumata1, Hitoshi Sano2, Naohiro Yoneda2, Jun‑ichi Ueyama2, Michiko Matsushita1, Satoshi Kuwamoto1, Masako Kato1, Ichiro Murakami1, Susumu Kanzaki2 and Kazuhiko Hayashi1 Abstract Various autoantibodies have been reported to be detected during the progression of infectious mononucleosis. We observed a case of infectious mononucleosis due to Epstein–Barr virus primary infection for 2 months, and noticed the transiently increased titer of thyrotropin receptor autoantibodies detected at the acute phase on the 3rd day after admission. At that time, real-time quantitative PCR also revealed the mRNA expressions of an immediate early lytic gene, BZLF1, and a latent gene, EBNA2. The expression of BZLF1 mRNA means that Epstein–Barr virus infects lyti‑ cally, and EBNA2 protein has an important role in antibody production as well as the establishment of Epstein–Barr virus latency. These results suggest that Epstein–Barr virus lytic infection is relevant to thyrotropin receptor autoanti‑ body production. Thyrotropin receptor autoantibodies stimulate thyroid follicular cells to produce excessive thyroid hormones and cause Graves’ disease. Recently, we reported the thyrotropin receptor autoantibody production from thyrotropin receptor autoantibody-predisposed Epstein–Barr virus-infected B cells by the induction of Epstein–Barr virus lytic infection in vitro. This case showed in vivo findings consistent with our previous reports, and is important to consider the pathophysiology of Graves’ disease and one of the mechanisms of autoimmunity. Keywords: Epstein–Barr virus (EBV), Infectious mononucleosis, Lytic infection, Reactivation, Autoantibody, Thyrotropin receptor antibody (TRAb), Graves’ disease Background Various autoantibodies have been reported to be detected in the serum, during the clinical course of infectious mononucleosis (IM), which can be one of the factors explaining the relevance of Epstein–Barr virus (EBV) to autoimmune diseases (Sutton et al. 1974; Longnecker et al. 2013). EBV is a common virus of Herpesviridae, and over 90 % of adults have its serum antibody. However, in developed *Correspondence: nanamon@theia.ocn.ne.jp 1 Division of Molecular Pathology, Department of Pathology, Faculty of Medicine, Tottori University, 86 Nishi‑cho, Yonago, Tottori 683‑8503, Japan Full list of author information is available at the end of the article countries, 50 % of children and young adults are seronegative and primary infection even in infants and young children is usually symptomatic or has nonspecific symptoms (Longnecker et al. 2013). Since a report by Henle et al. (1974), the serum levels of EBV antibodies from onset to the convalescent or chronic phase have been well investigated (Longnecker et al. 2013; Henle et al. 1974; Cohen 2006; Luzuriaga and Sullivan 2010). Recently, real-time PCR for EBV viral load has become available, as well as serum titers of EBV antibodies for diagnosis. However, there are few reports monitoring the expression of EBV latent gene mRNA and lytic gene mRNA. We have been studying EBV reactivation (lytic infection) and autoimmune hyperthyroidism, © 2015 Nagata et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Nagata et al. SpringerPlus (2015) 4:456 and reported that EBV lytic infection induction in vitro causes the production of thyrotropin (TSH) receptor antibodies (TRAbs) from host TRAb-predisposed EBVinfected (TRAb(+)EBV(+)) B cells (Nagata et al. 2014, 2015). We diagnosed this case of IM, as an EBV primary infection and followed it up for 2 months. Blood sampling was performed three times, once in the acute phase and the others in the convalescent phase. Investigation of these samples revealed that mRNA of an immediate early lytic gene, BZLF1, and of a latent gene, EBNA2, was expressed in the acute phase, and that heterogeneous autoantibodies against TSH receptor, TRAbs, were also detected in the same acute lytic phase. This case is important for considering the mechanisms of autoantibody production induced by EBV lytic infection. Case description A boy who was 3 years and 10 months old was admitted to our hospital on July 23. He had fever, appetite loss, and bilateral cervical lymph node enlargement with pain from July 21. We noticed that the tonsils were swollen with white exudates, the infra-auricular lymph nodes (right 5 × 2 cm, left 4 × 2 cm) were swollen and painful, and several neck lymph nodes (1 cm) were palpable. Neither axillar nor inguinal lymph nodes were swollen. The abdominal region was soft and flat, and hepato-splenomegaly was not apparent. Laboratory data are shown in Table 1 and Fig. 1. C-reactive protein (CRP) increased to 3.63 mg/dl on the day of admission, but granulocytes were not increased. Lymphocytes increased to 50 %, with 9 % atypical lymphocytes. The patient was anemic and had slight liver dysfunction. Negative cytomegalovirus (CMV)-IgM and high titer of CMV-IgG indicated past infection of CMV, and positive EBV VCA-IgM was consistent with IM due to EBV primary infection. Except for antibiotics against Moraxella Catarrhalis 4+ and Haemophilus Influenzae 3+ detected from throat swab culture, rest alone was effective for recovery and the patient left hospital on the 5th day. He was observed for 2 months as an outpatient. From the blood samples, one taken in the acute phase and two in convalescent phase, we measured serum titers of EBV-VCA, -EBNA, -EA-D, and total immunoglobulin by ELISA and EBV copy numbers by real-time PCR (Kimura et al. 1999). In the examination on the 36th day, the condition of the patient was good, and the serum data indicated that he was already in the convalescent phase, with a decrease of VCA-IgM and an increase of VCA-IgG. EBV copy number was still high, but it usually remains high for 6 weeks Page 2 of 5 Table 1 Laboratory data July 23 July 25 Admission 3rd day August 27 September 24 36th day 64th day Hgb (g/dl) 10.9 3.86 × 106 3.71 × 106 4.24 × 106 4.18 × 106 Platelet (/μl) 220 × 103 231 × 103 259 × 103 12,400 4600 239 × 103 Segmented (%) 32 15 41 38 Band (%) 0 2 0 0 Lymphocytes (%) 50 64 54 57 Monocytes 9 8 3 2 Atypical lym‑ phocytes (%) 9 11 1 0 Reticulocyte (%) 3.8 3.1 1.3 1.2 Total bilirubin (mg/dl) 0.4 0.5 0.4 AST (IU/l) 35 36 32 31 ALT (IU/l) 23 24 20 19 LDH (IU/l) 537 567 260 268 CRP (mg/dl) 3.63 2.2 0.02 0.02 RBC (/μl) WBC (/μl) 9800 10.7 11.6 11.3 5600 after onset, so we considered that this would be reasonable given the development of immunity in this child (Longnecker et al. 2013). Through the follow-up, total immunoglobulin changed almost in parallel with VCA antibodies. We examined serum autoantibodies of the 3 stored samples (at day 3, 36, and 64): anti-nuclear antibody (ANA) and anti-smooth muscle antibody (SMA), using fluorescent antibody (FA), and TRAbs by radio-receptor assay, at the same time for each antibody (Table 2). TRAb radio-receptor assay (DYNOtest TRAb Human; Yamasa Corporation, Choshi, Japan) were performed according to manufacturer’s instruction (clinical cut-off is 1 IU/l). We constructed primers and probes for EBV latent genes and lytic genes (Table 3); then, we performed realtime quantitative PCR to detect the mRNA expression of EBV latent genes and lytic genes (Table 2) (Kubota et al. 2008; Ryan et al. 2004). The results indicated that mRNA of an immediate early lytic gene, BZLF1, and a latent gene, EBNA2, was detected in the acute phase, and that TRAbs were also detected in the same acute phase. As a complication, this case had coagulopathy due to deficit of von Willebrand factor, but this disease is not accompanied by immune disorder and rarely has an effect on viral infection. Discussion and evaluation In EBV primary infection, most B cells become latently infected lymphoblastoid cells, and some B cells become Nagata et al. SpringerPlus (2015) 4:456 Page 3 of 5 Table 2 EBV copy numbers, serum antibodies, and expression of EBV mRNA BZLF1 mRNA (70.09 copies/µgDNA) and TRAbs (0.24 IU/L) were detected 15 Atypical lymphocyte (%) 10 6 EBV copy number (/10 WBC) 5 EBV-EA-IgG (index*) 0 EBV-EBNA-IgG (index*) 4 CRP (mg/dl) 2 0 EBV copy number (x 103/106WBC) 4 August 27 September 24 3rd day 36th day 64th day 3 4.2 × 10 2.6 × 103 0.2 (−) 1.9 (+) 4.1 × 10 0.4 (−) 0.1 (−) 3 0.3 (−) 0.3 (−) EBV-VCA-IgM (index*) 1.5 (+) 0.6 (±) 1.4 (+) EBV-VCA-IgG (index*) 1.1 (+) 6.6 (+) 8.9 (+) Total-IgG (mg/ml) 11.15 13.37 9.49 Total-IgM (μg/ml) 836.2 815.05 825.1 Autoantibodies 2 0 10 IgG 8 EBV VCA-IgG EBV VCA-IgM (index*) July 25 ANA (x) (−) (−) (−) SMA (x) (−) (−) (−) TRAbs (IU/l) 0.24 (−) (−) EBV mRNAs (copies/μgDNA) 6 LMP1 (−) (−) (−) 4 LMP2 (−) (−) (−) 2 IgM 0 July July 23 25 (admission) (3rd day) acute phase August 27 (36th day) September 24 (64th day) convalescent phase Fig. 1 Time-course change in this case. According to the decrease of inflammation represented by CRP, the number of atypical lympho‑ cytes decreased. The copy number of EBV began to decline in the convalescent phase. In the acute phase, EBV VCA-IgM was higher than EBV VCA-IgG, but in the convalescent phase, EBV VCA-IgG was higher. BZLF1 mRNA (70.09 copies/μgDNA) and TRAbs (0.24 IU/l) were detected in the acute phase. *Index stands for sample absorbance/ absorbance of cut-off serum. EBV Epstein–Barr virus, VCA viral capsid antigen, BZLF1 one of the EBV-immediate-early lytic genes, TRAb thyrotropin receptor antibody lytic infected cells (Longnecker et al. 2013; Cohen 2000). The lymphocytes of this case expressed mRNA of BZLF1, the immediate early gene of EBV lytic infection, and mRNA of EBNA2, the latent gene, in the acute phase (Table 2; Fig. 1). The expression of BZLF1 mRNA indicates that EBV lytic infection occurred in the acute phase of IM. EBNA2 is important for B cell transformation when EBV establishes its latent infection and is the transactivator of various genes, including LMP1 that activates NF-κB (Longnecker et al. 2013); thus, it is related to antibody production and the expression of activationinduced cytidine deaminase (Tran et al. 2010). During IM, mRNA for latent genes as well as lytic genes could be expressed, but in this case, we could not detect mRNA other than BZLF1 and EBNA2. This might be related to the probably low potential of the immune system of this case of 3-year-old child to react to the EBNA1 (−) (−) (−) EBNA2 4.29 (−) (−) BZLF1 70.09 (−) (−) EA-D (−) (−) (−) Each value of mRNA is normalized to the expression of β-actin We used EBV mRNA from B95-8 strain as a reference EBV Epstein–Barr virus, EA early antigen, EBNA, Epstein–Barr nuclear antigen, VCA viral capsid antigen, LMP latent membrane protein, TRAbs thyrotropin receptor antibodies, SMA smooth muscle antibody, ANA antinuclear antibody * Index stands for sample absorbance/absorbance of cut-off serum invasion of viruses (Longnecker et al. 2013; Piątosa et al. 2010; Parham 2009). Children at this age are also known to have weak antibody production (Longnecker et al. 2013; Parham 2009). In this case, we could not detect EA-D serum antibody and EA-D mRNA. The levels of EBV VCA-IgM and serum total IgM were high in the acute phase and those of EBV VCA-IgG and serum total IgG rose in the convalescent phase (Table 2). These results imply that IgM production may be caused by EBV acute infection (Nakamura et al. 1988; Casali et al. 1990). Sutton et al. (1974) showed that SMA was detected at the onset of IM and declined in the convalescent phase, and that ANA and rheumatoid factor (RF) are rarely present and do not preferentially appear in the acute phase (Sutton et al. 1974; Longnecker et al. 2013). These increases of antibody production are considered to be the result of polyclonal B-cell activation in IM (Longnecker et al. 2013). In our case, ANA and SMA were negative throughout the observation period, but we could detect TRAbs at a low titer (Table 2). FA tests for ANA and SMA are common, but not sensitive compared with TRAb Nagata et al. SpringerPlus (2015) 4:456 Page 4 of 5 Table 3 Primers and probes for real-time quantitative PCR of EBV Assay Sequence References Sense 5′-CCC TTT GTA TAC TCC TAC TGA TGA TCA C Kubota et al. (2008) Anti‑ sense 5′-ACC CGA AGA TGA ACA GCA CAA T Kubota et al. (2008) Probe 5′-CTC ATC GCT CTC TGG AAT TTG CAC GG Kubota et al. (2008) LMP1 LMP2 Sense 5′-AGC TGT AAC TGT GGT TTC CAT GAC Anti‑ sense 5′-GCC CCC TGG CGA AGA G Probe 5′-CTG CTG CTA CTG GCT TTC GTC CTC TGG EBNA1 Sense 5′-TAC AGG ACC TGG AAA TGG CC Ryan et al. (2004) Anti‑ sense 5′-TCT TTG AGG TCC ACT GCC G Ryan et al. (2004) Probe 5′-AGG GAG ACA CAT CTG GAC CAG AAG GC EBNA2 Sense 5′-TCT TGC GTT ACA TGG GGG AC Anti‑ sense 5′-CCT GGT AGG GAT TCG AGG GA Probe 5′-AAT TGT TGA CAC GGA TAG TCT TGG BZLF1 Sense 5′-AAA TTT AAG AGA TCC TCG TGT AAA Ryan et al. (2004) ACA TC Anti‑ sense 5′-CGC CTC CTG TTG AAG CAG AT Probe 5′-ATA ATG GAG TCA ACA TCC AGG CTT GGG C Ryan et al. (2004) EA-D Sense 5′-CGT GCC AAT CTT GAG GTT TT Anti‑ sense 5′-CAC CCG GGG ACT TTT ATC TT Probe 5′-TTT ATT TAA CCA CGC CTC CG β-Actin Sense 5′-CCT GGC ACC CAG CAC AAT G Anti‑ sense 5′-GCC GAT CCA CAC GGA GTA CT Probe 5′-ATC AAG ATC ATT GCT CCT CCT GAG CGC radio-receptor assay. Using a more sensitive ELISA system for ANA and SMA, a certain amount of antibodies might be detected, despite a low titer. Several viral infections are recognized to induce autoantibody production, but the antibodies are often low level, and do not develop any clinical symptoms (Mandel et al. 2011; Poole et al. 2011; Camarero et al. 2008). Our case is too young to produce sufficient antibodies, and did not show any specific autoimmune symptoms. However, there are some cases reported to have developed Graves’ disease related to IM (Akahori et al. 2010), and this child needs long-term follow-up. We observed that EBV lytic infection and autoantibody production occurred in the same period, which suggests that EBV lytic infection stimulates autoantibody production. EBV is a latent virus and persists mainly in B lymphocytes. B cells differentiate into antibody-producing cells; thus, persistent EBV may stimulate the antibody production of host cells (Nagata et al. 2011, 2014, 2015). We have been investigating TRAb-production in Graves’ disease induced by EBV reactivation (lytic infection) (Nagata et al. 2011, 2014, 2015). Our hypothesis is that lytic change of EBV in TRAb-predisposed and EBVinfected (TRAb(+)EBV(+)) cells would stimulate host B cells to promote TRAb production with different level of efficiency between patients and controls, and may cause the development or exacerbation of Graves’ disease. We showed that TRAb(+)EBV(+) cells really exist in the peripheral blood of Graves’ disease patients and healthy controls (Nagata et al. 2014), and these cells released TRAbs in culture fluid when we induced lytic change in persistent EBV (Nagata et al. 2015). In EBV primary infection, some B cells become lytic (Longnecker et al. 2013; Cohen 2000). According to our hypothesis, these lytic infected B cells with TRAb predisposition, differentiate to plasma cells and produce TRAbs. The result of research of this case in which EBV lytic gene expression and autoantibody production occurred simultaneously in the acute phase is consistent with our previous data in vitro and provides a suggestive example in vivo. Conclusions We have reported a 3-year-old patient with IM due to EBV primary infection. This case simultaneously showed EBV lytic gene expression and autoantibody (TRAb) production in the acute phase of the disease. This case is important to consider the pathophysiology of Graves’ disease and the mechanisms of autoantibody production induced by EBV lytic infection. Abbreviations EBV: Epstein–Barr virus; IM: infectious mononucleosis; TSH: thyrotropin; TRAb: thyrotropin receptor antibody; CMV: cytomegalovirus; EA: early antigen; EBNA: Epstein–Barr nuclear antigen; VCA: viral capsid antigen; LMP: latent membrane protein; ANA: antinuclear antibody; SMA: smooth muscle antibody; FA: fluo‑ rescent antibody; RF: rheumatoid factor; MS: multiple sclerosis; SLE: systemic lupus erythematosus. Nagata et al. SpringerPlus (2015) 4:456 Authors’ contributions KN designed the study and performed real-time PCR analysis, and drafted the manuscript. KO examined and observed the patient in his office. MO constructed PCR primers for EBV mRNA expression. KK carried out enzymelinked immunosorbent assay. HS examined and took care of the patient in his admission. NY and JU performed physical examination of the patient. MM and SK carried out the laboratory examinations. MK and IM participated in the design of the study and in interpretation of the result. SK participated in clini‑ cal examination and therapy. KH conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Author details 1 Division of Molecular Pathology, Department of Pathology, Faculty of Medi‑ cine, Tottori University, 86 Nishi‑cho, Yonago, Tottori 683‑8503, Japan. 2 Divi‑ sion of Pediatrics and Perinatology, Department of Multidisciplinary Internal Medicine, Tottori University, 36‑1 Nishi‑cho, Yonago, Tottori 683‑8504, Japan. Acknowledgments We thank Dr. Yukio Satoh (Tottori University) of his kind guidance for PCR. We also thank Medical English Service (Kyoto, Japan) for proofreading this manu‑ script. One of the authors, KO has the research funding from The Morinaga Foundation for Health & Nutrition. Compliance with ethical guidelines Competing interests K. Okuno has the research funding from The Morinaga Foundation for Health & Nutrition. Other authors have no competing interest associated with this manuscript. Ethics, consent and permissions This case research was approved by the Medical Ethics Committee for Human Subject Research (No. 2283) at the Faculty of Medicine, Tottori University, Yonago, Japan. The patient showed assent and the parents provided written informed consent for participation in this study. Consent to publish The patient showed assent and the parents provided written informed con‑ sent for publication of this case report. Received: 20 March 2015 Accepted: 11 August 2015 References Akahori H, Takeshita Y, Saito R, Kaneko S, Tamura T (2010) Graves’ disease asso‑ ciated with infectious mononucleosis due to primary Epstein–Barr virus infection: report of 3 cases. Intern Med 49:2599–2603 Camarero C, Ramos N, Moreno A, Asensio A, Mateos ML et al (2008) Hepatitis C virus infection acquired in childhood. Eur J Pediatr 167:219–224 Casali P, Nakamura M, Ginsberg-Fellner F, Notkins AL (1990) Frequency of B cells committed to the production of antibodies to insulin in newly diagnosed patients with insulin-dependent diabetes mellitus and generation of high affinity human monoclonal IgG to insulin. J Immunol 144:3741–3747 Page 5 of 5 Cohen JI (2000) Epstein–Barr virus infection. N Engl J Med 343(7):481–492 Cohen JI (2006) Epstein–Barr virus: clinical hematology. Elsevier, Philadelphia Henle W, Henle G, Horwitz C (1974) Epstein–Barr virus specific diagnostic tests in infectious mononucleosis. Hum Pathol 5(5):551–565 Kimura H, Morita M, Yabuta Y, Kazushima K, Kato K et al (1999) Quantitative analysis of Epstein–Barr virus load by using a real-time PCR assay. J Clin Microbiol 37(1):132–136 Kubota N, Wada K, Ito Y, Shimoyama Y, Nakamura S et al (2008) One-step mul‑ tiplex real-time PCR assay to analyse the latency pattern of Epstein–Barr virus infection. J Virol Methods 147:26–36 Longnecker RM, Kieff E, Cohen J (2013) Epstein–Barr virus. In: Knipe DM, How‑ ley PM (eds) Fields virology, vol 2, 6th edn. Lippincott Williams & Wilkins, Philadelphia, pp 1898–1959 Luzuriaga K, Sullivan JL (2010) Infectious mononucleosis. N Engl J Med 362:1993–2000 Mandel SJ, Larsen PR, Davies TF (2011) Thyrotoxicosis. In: Melmed S, Polonsky KS, Larsen PR, Kronenberg HM (eds) Williams textbook of endocrinology, 12th edn. Saunders, Philadelphia, p 372 Nagata K, Fukata S, Kanai K, Satoh Y, Segawa T et al (2011) The influence of Epstein–Barr virus reactivation in patients with Graves’ disease. Viral Immunol 24(2):143–149 Nagata K, Higaki K, Nakayama Y, Miyauchi Y, Kiritani Y et al (2014) Presence of Epstein–Barr virus-infected B lymphocytes with thyrotropin receptor antibodies on their surface in Graves’ disease patients and in healthy individuals. Autoimmunity 47(3):193–200 Nagata K, Nakayama Y, Higaki K, Ochi M, Kanai K et al (2015) Reactivation of persistent Epstein-Barr virus (EBV) causes secretion of thyrotropin recep‑ tor antibodies (TRAbs) in EBV-infected B lymphocytes with TRAbs on their surface. Autoimmunity 48(5):328–335. doi:10.3109/08916934.2015.1022 163 Nakamura M, Burastero SE, Ueki Y, Larrick JW, Notkins AL et al (1988) Probing the normal and autoimmune B cell repertoire with Epstein–Barr virus. J Immunol 141:4165–4172 Parham P (2009) The immune system, Chap 9, 3rd edn. Garland Science, New York, pp 248–287 Piątosa B, Wolska-Kuśnierz B, Pac M, Siewiera K, Gałkowska E et al (2010) B cell subsets in healthy children: reference values for evaluation of B cell matu‑ ration process in peripheral blood. Cytom B Clin Cytom 78B(6):372–381 Poole BD, Kivovich V, Gilbert L, Naides S (2011) Parvovirus B19 nonstructural protein-induced damage of cellular DNA and resultant apoptosis. Int J Med Sci 8(2):88–96 Ryan JL, Fan H, Glaser SL, Schichman SA, Raab-Traub N et al (2004) Epstein– Barr virus quantitation by real-time PCR targeting multiple gene seg‑ ments. J Mol Diagn 6(4):378–385 Sutton RNP, Emond RTD, Thomas DB, Doniach D (1974) The occurrence of autoantibodies in infectious mononucleosis. Clin Exp Immunol 17:427–436 Tran TH, Nakata M, Suzuki K, Begum NA, Shinkura R et al (2010) B cell-specific and stimulation-responsive enhancers derepress Aicda by overcoming the effects of silencers. Nat Immunol 11(2):148–155
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The COVID-19 Gene and Drug Set Library
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The COVID-19 Gene and Drug Set Library Maxim V. Kuleshov1, Daniel J.B. Clarke1, Eryk Kropiwnicki1, Kathleen M. Jagodnik1, Alon Bartal1, John E. Evangelista1, Abigail Zhou1, Laura B. Ferguson2, Alexander Lachmann1, Avi Ma’ayan1,* Maxim V. Kuleshov1, Daniel J.B. Clarke1, Eryk Kropiwnicki1, Kathleen M. Jagodnik1, Alon Bartal1, John E. Evangelista1, Abigail Zhou1, Laura B. Ferguson2, Alexander Lachmann1, Avi Ma’ayan1,* 1Department of Pharmacological Sciences; Mount Sinai Center for Bioinformatics; Big Data to Knowledge, Library of Integrated Network-based Cellular Signatures, Data Coordination and Integration Center (BD2K-LINCS DCIC); Knowledge Management Center for Illuminating the Druggable Genome (KMC-IDG); Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1603, New York, NY 10029, USA 2Department of Neurology; Dell Medical School; University of Texas at Austin; 1601 Trinity Street, Bldg B, Austin, TX 78712, USA. To whom correspondence should be addressed: avi.maayan@mssm.edu Abstract The coronavirus (CoV) severe acute respiratory syndrome (SARS)-CoV-2 (COVID-19) pandemic has received rapid response by the research community to offer suggestions for repurposing of approved drugs as well as to improve our understanding of the COVID-19 viral life cycle molecular mechanisms. In a short period, tens of thousands of research preprints and other publications have emerged including those that report lists of experimentally validated drugs and compounds as potential COVID-19 therapies. In addition, gene sets from interacting COVID-19 virus-host proteins and differentially expressed genes when comparing infected to uninfected cells are being published at a fast rate. To organize this rapidly accumulating knowledge, we developed the COVID-19 Gene and Drug Set Library (https://amp.pharm.mssm.edu/covid19/), a collection of gene and drug sets related to COVID-19 research from multiple sources. The COVID-19 Gene and Drug Set Library is delivered as a web-based interface that enables users to view, download, analyze, visualize, and contribute gene and drug sets related to COVID-19 research. To evaluate the content of the library, we performed several analyses including comparing the results from 6 in-vitro drug screens for COVID-19 repurposing candidates. Surprisingly, we observe little overlap across these initial screens. The most common and unique hit across these screen is mefloquine, a malaria drug that should receive more attention as a potential therapeutic for COVID-19. Overall, the library of gene and drug sets can be used to identify community consensus, make researchers and clinicians aware of the development of new potential therapies, as well as allow the research community to work together towards a cure for COVID-19. Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that was first detected in Wuhan, Hubei Province, China in November 2019. Infection with SARS-CoV-2 causes the coronavirus disease (COVID-19). Globally, there are more than 2.9 million confirmed COVID-19 cases and 203,000 reported deaths (as of April 25, 2020). The World Health Organization declared COVID-19 a pandemic on March 11, 2020. Many biomedical researchers have been shifting their efforts to battle the coronavirus COVID-19 pandemic. One area of activity is computationally prioritizing and experimentally testing approved drugs for repurposing as candidate therapies for attenuating COVID-19 infection. Drug repurposing studies present a promising avenue for quickly offering a treatment for COVID-19 because these drugs have known safety profiles. So far, drug repurposing studies can be categorized into three groups: in-vitro screens (1-6), computational predictions based on structural biology methods (7-9), and computational predictions based on network models and transcriptomics (10-12). Few studies have validated top computational predictions in cell-based models for COVID-19 cell based models (7,10,11). The lists of drugs mentioned in these studies can be analyzed for consensus, and suggested drugs can be grouped by their type. At the same time, many researchers attempt to understand the molecular mechanisms of the COVID-19 virus life cycle. Much attention has been given to a study that profiled, with mass-spectrometry proteomics, host proteins that interact with each of the COVID-19 proteins (11). Another important dataset produced RNA-seq gene expression signatures from various relevant human cell lines, ferret model lungs, and human lung biopsies before and after COVID-19 infection (13). These are just two examples of the many studies that produce gene sets that can be organized and compared. In the past, we developed a crowdsourcing project where we asked the community to identify gene expression signatures from drug, gene, and disease perturbations (14). The collection of over 6,000 signatures that we collected with the help of >70 contributors from around the world enabled us to produce a useful database called CREEDS. Similarly, for this project, we developed a crowdsourcing project to integrate gene and drug sets related to COVID-19 research collected with the assistance of the research community. Collecting COVID-19-Related Gene Sets with Geneshot Geneshot (17) is a platform that we developed to convert PubMed searches into gene sets. Using Geneshot, gene sets associated with the search terms SARS, SARS-CoV, MERS-CoV, ACE2, and TMPRSS2 were created using both the AutoRIF and GeneRIF (18) methods. Additionally, top COVID-19 drug repurposing candidates reported in recent literature (Table 1), including chloroquine and hydroxychloroquine, were included. Predictions of additional genes potentially associated with these terms were also added to the COVID-19 gene set library. These predictions were based on the literature-associated genes using each of five strategies: Co-occurrence via AutoRIF, GeneRIF, Enrichr (19), Tagger (20), and co-expression using data from ARCHS4 (21). Collecting COVID-19 Drug Sets from Drug Repurposing Publications Since the emergence of the COVID-19 epidemic, tens of thousands of new publications related to COVID-19 research have emerged in a very short period (2 months). We continually survey these publications to identify research that describes drug repurposing efforts, and manually extract drug sets from these studies to populate the drug set library. We also submit to the platform published drug sets from historical sources such as those from studies that listed drugs showing antiviral activity for other related viruses. So far, we have collected 20 drug repurposing publications (Table 1). An updated version of this table is maintained here: .google.com/spreadsheets/d/1x6aKaZGadfLqNrQoFQwLlRhCXfUGYiRbzwYipIon h i To assist us with developing and maintaining the collection, we have received help from the research community by allowing researchers to upload gene and drug sets to the database. These submissions are manually evaluated before making them publicly available. Collecting SARS Signatures from GEO with GEO2Enrichr and GEN3VA A set of 35 gene expression signatures resulting from infections by different coronaviruses for different cell types and tissues, with expression data originating from the gene expression omnibus (GEO) database, was processed using the GEO2Enrichr tool (15) and stored on the GEN3VA platform (16). The 70 entries were submitted to the COVID-19 crowdsourcing platform, with an upregulated and a downregulated gene set associated with each signature. The GEN3VA report for these signatures is available here: https://amp.pharm.mssm.edu/gen3va/report/646/SARS. Developing the COVID-19 Gene and Drug Set Library Website The COVID-19 gene and drug set library website has five sortable and searchable tables that list the drug and gene sets (Fig. 1). Sorting can be based on the date of submission, alphabetical ordering, or list size. The two tables are searchable via metadata terms such as title, authors, and descriptions, as well as via data search for specific gene or drug names. Users can download each gene set or drug set as well as the entire library. In addition, each gene set is provided with the option to perform gene set enrichment analysis with Enrichr (19), while genes are linked to Harmonizome (24) for further interrogation. The individual drugs that map to known compounds are linkable to their corresponding DrugBank landing pages (22). The website enables users to submit drug and gene sets related to COVID-19 research by completing a simple form. The form includes a dataset title, a URL source, and a description that explains how the set is relevant to COVID-19 research. The submitter is also provided with mechanisms to add additional metadata terms that can describe the cell type, tissue, organism, and other critical information about the submitted set. Users can specify the category of metadata provided, allowing for a broad set of additional metadata about each set. Users can also opt to submit their contact information; this information is kept private, but users can opt-in to make it public. Once a user submits a contribution to the site, their dataset is directed to a review queue in which we can examine the validity and relevance of the contribution. The reviewing process enables an administrator to approve or reject the submitted set. If approved, the set is added to the library. To make it easy for contributors to submit multiple sets, users can access the site via API. The code behind the site is open source and available at: https://github.com/MaayanLab/covid19_crowd_library Expression Analysis of In-Vitro Screens Hits Drug sets extracted from 3 in-vitro screens (1-3) were first identified. The drugs were matched to drugs profiled by the L1000 assay available from GSE92742. Average signatures for each drug were computed by taking the z-score mean for each gene. Clusters were identified based on the average signatures using hierarchical clustering. Differential z-scores of genes relative to the two clusters were identified using the t-test statistic. The top up and down differentially expressed genes in each cluster were submitted to Enrichr for gene set enrichment analysis. To quantify the z-scores of genes co-expressed with ACE2, we calculated the correlation over 2,000 randomly sampled drug signatures from the L1000 database. We then calculated the mean z-scores of the top 50 correlated genes to ACE2 and compared those values against a distribution calculated from sampling 50 random genes, repeatedly 10,000 times. The p-values were calculated against the sampled distribution and corrected for multiple hypothesis testing by applying the Bonferroni correction method. The code behind this analysis is open source and available at: https://github.com/maayanlab/covid19l1000 Collecting COVID-19 Drug Sets from Twitter Twitter is an important source for timely discussions related to therapeutics for COVID-19, including drug repurposing efforts and clinical trials. Using the Twitter API, we query Twitter daily with a list of more than 14,000 drug terms and their synonyms to collect tweets that mentioned these drugs in context of COVID-19. The drug search list was curated from DrugBank (22), L1000FWD (23), and the list of drugs submitted to the COVID19 drug and gene set library website. We then filter the identified tweets for those that are co-mentioned with COVID-19, and SARS linguistic variations. For each drug, we counted the occurrences of tweets and recorded a tally of mentions for each day. Data collection continues with daily reports, tweet IDs of the tweets originating the discussions, and the longitudinal drug trends. These data are shared publicly on GitHub:https://github.com/MaayanLab/COVID19DrugsTrendTracker/tree/master/daily_reports Each day the set of discussed drugs on Twitter are automatically deposited into the COVID-19 drug set library via an API. This approach enables real time trend detection of the the most discussed drugs as potential therapeutics for COVID-19 while enriching the content of the COVID- 19 drug and gene set library. Developing the COVID-19 Gene and Drug Set Library Website Results Analysis and Visualization of Consensus Drug and Gene Sets Analysis and Visualization of Consensus Drug and Gene Sets So far, we have collected 87 drug sets composed of 1265 unique drugs, and 361 gene sets consisting of 13,347 unique genes. The drug sets are subdivided into four categories: experimental (20), computational (31), Twitter (31), and other (5). The top 20 most frequent drugs and genes across all sets are displayed in Fig. 2A-E. The list of experimental drugs with most supportive evidence are hydroxychloroquine, mefloquine, chloroquine, and remdesivir (Fig. 2A). While hydroxychloroquine, chloroquine, and remdesivir are the most discussed drugs on Twitter, mefloquine received so far less attention (Fig. 3). While mefloquine is consistently ranked within the top 100 drugs each day, the anti-malaria drug mefloquine (25) is much less discussed and as such may be a good candidate for further experimental investigation (Fig. 3). The top 20 most frequently submitted genes are all members of the innate immune response (Fig. 2E). These genes include the typical interferon and cytokine response genes observed to be involved in the response of human cells to most pathogens. These genes are also listed as the top differentially expressed upregulated genes from the GEN3VA report of 35 signatures (Fig. 4). However, the list here also includes the immediate early gene (IEG) module composed of the transcription factors EGR1 and FOS and the phosphatase DUSP1. Based on enrichment analysis with Enrichr (19), the top 10 downregulated genes from the GEN3VA report include five genes (SH3BGRL, LGALS1, COX7A2, CRIP1 and LYZ) that are highly expressed in dendritic cells (p- value < 0.003, Fisher exact test), suggesting that there may be a depletion of dendritic cells due to SARS infections. Our ability to collect tweets about drugs using the Twitter API enables us to track trends about new drugs that are increasingly discussed on this social media platform. During the period between April, 2nd to April 24th, 2020, we noticed the rise and fall of discussions about the drug galidesivir (Fig. 5). Galidesivir (BCX4430) is an adenosine analog that was previously suggested as a potential antiviral drug for several related viral diseases (26-28). While most of the drug sets in the library are from studies that utilized computational methods to predict drugs, few are from large-scale approved drug screens (1-6). Results Using the Venn diagram tool developed for ad-hoc analysis of the sets in the library, we compared the results from six in-vitro COVID-19 drug screen studies (Fig. 6). Although, there is very little overlap across these drug screens, where only one or two drugs are shared across these experimental studies, some interesting less discussed hits emerge. Namely, these are amuvatinib, proscillaridin, mefloquine, hexachlorophene, clofazimine, and almitrine. Amuvatinib is a multi-targeted tyrosine kinase inhibitor (29) and proscillaridin is an organic compound that is used an old cardiotonic steroid but more recently suggested as a cancer agent (30). Hexachlorophene is a disinfectant that is used in dermatological products (31). Clofazimine is a drug used to treat leprosy and its mechanisms of action suggest that it interferes with DNA synthesis (32). Almitrine is a drug that stimulates respiratory respiration by activating receptors of carotid bodies (33). It is used in the treatment of chronic obstructive pulmonary disease (34), and as such it is most relevant to the COVID-19 symptoms. ACE2 Up- or Down-Regulation Effects of Drug Hits? To further explore the molecular effects of positive hits from the in-vitro drug screens, and to demonstrate the utility of the collected library, we developed a case study that asks whether drugs that have been shown to inhibit COVID-19 infection of human cells in-vitro, up- or down-regulate the ACE2 gene. ACE2 is the suspected cell surface receptor for COVID-19 (35), and cells that do not express this gene have been shown to be less prone to COVID-19 infection. Since it is still undetermined whether it is desired to up- or down-regulate ACE2 expression, we queried each drug hit from two published in-vitro drugs screens against the LINCS L1000 data (36). We identified 23 drug hits from the screens that have been profiled by L1000 comprising a total of 1251 differential gene expression profiles. 17 of these drugs display, on average, an increase in ACE2 expression, while 6 drugs display a decrease in expression of ACE2. Overall, the gene expression signatures for these 23 drugs can be categorized into two distinct clusters (Fig. 7). The observed difference in gene expression signatures suggest different modes of action for these drugs. The drugs that are most similar to chloroquine exhibit consistent up-regulation of genes highly correlated to ACE2. Chloroquine gene expression signatures on average up- regulate the top 50 most correlated genes to ACE2 (p=2.55e-03). In addition, cepharanthine, fluspirilene, bazedoxifene, and amuvatinib also up-regulate the same set of ACE2 most correlated genes (p=3.17e-17, p=7.01e-13, p=2.74e-03, and p=4.94e-03, respectively). These 50 genes are available in Table 2. The strong dysregulation of ACE2 associated genes could point to a similar mode of action regarding antiviral activity. Enrichment analysis of the consensus genes up- regulated by the drugs that co-cluster with chloroquine dislpay up regulation of immune response genes and down-regulation of cell cycle genes. This suggests that the drugs in the chloroquine cluster are pro-inflammatory while the other cluster of drugs contains anti-inflammatory drugs. Up genes in the chloroquine cluster: https://amp.pharm.mssm.edu/Enrichr/enrich?dataset=d9201bbba4dff47598bcf66f3db3e93e Down genes in the chloroquine cluster: https://amp.pharm.mssm.edu/Enrichr/enrich?dataset=698f9a31fbc85e13da57bce68a543083 Up genes in the chloroquine cluster: Discussion A list of computationally predicted drugs is not as useful towards identifying a therapy for COVID- 19 when compared with a list of experimentally validated drugs. A list of upregulated genes after COVID-19 infection of cells may provide more useful information about the virus life cycle when compared with a list of genes returned from a Geneshot search using the term SARS. Hence, the users of the data collected into the library should be aware of such limitations. With these limitations in mind, we hope that researchers will be able to develop or refine hypotheses from the snapshot overview our platform provides, and then delve deeper into the studies most relevant to their questions. For example, the GEN3VA report revealed a down-regulation of genes known to be enriched in dendritic cells (DCs) after infection with SARS. SARS-CoV-1 is known to infect DCs and impair DC function (40). It should be clear that the consensus analysis results should be viewed with caution. The most common drugs are not necessarily the most efficacious or promising treatments. At the same time, the most common genes may not be the most relevant to understand COVID-19 research. It should be noted that not all drug sets and gene sets have equal weight in quality and relevancy. A list of computationally predicted drugs is not as useful towards identifying a therapy for COVID- 19 when compared with a list of experimentally validated drugs. A list of upregulated genes after COVID-19 infection of cells may provide more useful information about the virus life cycle when compared with a list of genes returned from a Geneshot search using the term SARS. Hence, the users of the data collected into the library should be aware of such limitations. With these limitations in mind, we hope that researchers will be able to develop or refine hypotheses from the snapshot overview our platform provides, and then delve deeper into the studies most relevant to their questions. For example, the GEN3VA report revealed a down-regulation of genes known to be enriched in dendritic cells (DCs) after infection with SARS. SARS-CoV-1 is known to infect DCs and impair DC function (40). In a period of rapid development of methods and data related to COVID-19 research, it is critical to provide means to organize the accumulated information in a way that it can be summarized and reused. Discussion The COVID-19 gene set and drug set library provides such utility. The library of drug and gene sets can be used to identify community consensus and make researchers and clinicians aware of the development of new potential therapies as they become available, as well as allow the research community to work together towards a cure for COVID-19. Social media has played a critical role in enabling scientists to communicate results rapidly and exchange ideas. By mining the trends about discussions on Twitter about drugs in context of COVID-19, we are able to keep track with global trends before these are reported in scientific journals. Discussion Here we describe a platform created to collect drug and gene sets related to COVID-19 research using various methods of data accrual. All of the top 10 genes associated with COVID-19 are interferon-related (ISG15, IRF7, OAS1, IFITM3, MX1, IFIH1, STAT1, IFIT3, and EIF2AK2). This is consistent with our knowledge that type I (IFN-α, IFN-β) and type III interferon (IFN-λ) systems are the primary defense against viral infections. It has been hypothesized that hyper-inflammation in COVID-19 could drive disease severity and would be amenable to treatment with drugs that reduce inflammation (37). However, this remains controversial as the high level of antiviral response could be reflective of increased viral burden rather than an inappropriate host response (38). The most striking result from the meta-analysis applied to the content of the library is the little overlap across drug screen studies. It is expected that experimental validation of drugs to inhibit COVID-19 in-vitro will be more consistent. The inconsistency across these studies could be due to a need to produce results quickly due to the urgency for discovering potential treatments. Regardless, there is some overlap, and there is expectation that more screens will be published and top leads will advance to animal models and human trials for further testing. To prioritize compounds that may treat COVID-19, some researchers have used the strategy of finding drugs that modulate genes related to ACE2 gene expression (39). We found a highly significant up-regulation of the genes most correlated with ACE2 by chloroquine and related compounds in the L1000 database. This finding suggests that identifying compounds that up- regulate genes correlated with ACE2 expression could be a useful approach for identifying more compounds that inhibit COVID-19 in mammalian cells. However, it should be noted that other considerations must be taken into account, including the known side effects for the compounds and whether dosing would allow for high enough drug concentration to achieve therapeutic effects. It should be clear that the consensus analysis results should be viewed with caution. The most common drugs are not necessarily the most efficacious or promising treatments. At the same time, the most common genes may not be the most relevant to understand COVID-19 research. It should be noted that not all drug sets and gene sets have equal weight in quality and relevancy. Acknowledgements We would like to thank Russ Altman, Anne Carpenter, Pedro Bellester, and Tudor Oprea for contributing information about missing publications. This project is partially funded by U54HL127624 and U24CA224260. Figures and Figure Legends Figures and Figure Legends Fig. 1 Screenshot from the landing page of the COVID-19 Drug and Gene Set Library Fig. 1 Screenshot from the landing page of the COVID-19 Drug and Gene Set Library Fig. 1 Screenshot from the landing page of the COVID-19 Drug and Gene Set Library Fig. 2A Counts of most common drugs from the collection of experimental studied that reported lists of drugs that inhibit COVID-19. Fig. 2A Counts of most common drugs from the collection of experimental studied that reported lists of drugs that inhibit COVID-19. Fig. 2B Counts of most common drugs from the collection of computational studied that reported lists of drugs that may inhibit COVID-19. Fig. 2B Counts of most common drugs from the collection of computational studied that reported lists of drugs that may inhibit COVID-19. Fig. 2C Counts of most common drugs discussed each day on Twitter during the period from April 4, 2020 to May 6, 2020. Fig. 2C Counts of most common drugs discussed each day on Twitter during the period from April 4, 2020 to May 6, 2020. Fig. 2C Counts of most common drugs discussed each day on Twitter during the period from April 4, 2020 to May 6, 2020. Fig. 2C Counts of most common drugs discussed each day on Twitter during the period from April 4, 2020 to May 6, 2020. Fig. 2D Counts of most common drugs from the collection of all drug sets in the library. Fig. 2D Counts of most common drugs from the collection of all drug sets in the library. Fig. 2D Counts of most common drugs from the collection of all drug sets in the library. Fig. 2D Counts of most common drugs from the collection of all drug sets in the library. Fig. 2E Counts of most common genes from the collection of all gene sets in the library. Fig. 2E Counts of most common genes from the collection of all gene sets in the library. Fig. 2E Counts of most common genes from the collection of all gene sets in the library. Fig. 3 Ranks of drugs based on their mentions on Twitter in context of COVID-19 over time. Fig. Acknowledgements 3 Ranks of drugs based on their mentions on Twitter in context of COVID-19 over time. Fig. 3 Ranks of drugs based on their mentions on Twitter in context of COVID-19 over time. Fig. 4 The SARS GEN3VA report gene view. The heatmap displays the most consistent up and down-regulated genes from 35 signature created from microarray studies where mammalian cells and tissues were challenged with SARS. The GEN3VA report is available from here: http://amp pharm mssm edu/gen3va/report/646/SARS Fig. 4 The SARS GEN3VA report gene view. The heatmap displays the most consistent up and down-regulated genes from 35 signature created from microarray studies where mammalian cells and tissues were challenged with SARS. The GEN3VA report is available from here: http://amp.pharm.mssm.edu/gen3va/report/646/SARS Fig. 4 The SARS GEN3VA report gene view. The heatmap displays the most consistent up and down-regulated genes from 35 signature created from microarray studies where mammalian cells and tissues were challenged with SARS. The GEN3VA report is available from here: http://amp.pharm.mssm.edu/gen3va/report/646/SARS Fig. 4 The SARS GEN3VA report gene view. The heatmap displays the most consistent up and down-regulated genes from 35 signature created from microarray studies where mammalian cells and tissues were challenged with SARS. The GEN3VA report is available from here: http://amp.pharm.mssm.edu/gen3va/report/646/SARS Fig. 5 Ranks of drugs based on their mentions on Twitter in context of COVID-19 over time. Fig. 5 Ranks of drugs based on their mentions on Twitter in context of COVID-19 over time. Fig. 5 Ranks of drugs based on their mentions on Twitter in context of COVID-19 over time. Fig. 6 Overlap analysis across six in-vitro drug repurposing screens for COVID-19 inhibitors Fig. 6 Overlap analysis across six in-vitro drug repurposing screens for COVID-19 inhibitors Fig. 7 Hierarchical clustering of gene expression signatures for 23 drug hits from 3 published in-vitro screens that also have L1000 profiling gene expression data. The row represent genes where red represents high expression and yellow low expression. Fig. 7 Hierarchical clustering of gene expression signatures for 23 drug hits from 3 published in-vitro screens that also have L1000 profiling gene expression data. The rows represent genes where red represents high expression and yellow low expression. Fig. 7 Hierarchical clustering of gene expression signatures for 23 drug hits from 3 published in-vitro screens that also have L1000 profiling gene expression data. The rows represent genes where red represents high expression and yellow low expression. Table 2 SLC13A1, LRRC19, HAVCR1, HHLA2, CT62, LDHAL6B, SLC22A11, DDX4, CLCN5, SLC25A31, GIPC2, GUCY2C, ABCB1, TMPRSS15, RBMXL2, TM4SF20, KDM4D, SLCO4C1, PRM2, RNF32, CNTN6, ACSL6, UBQLN3, HNF4G, ANKRD7, MXD1, SLC17A3, IL12RB2, ANKRD40CL, SLC6A20, SLC5A12, SLC10A2, ACRV1, ALPI, EPPIN, IL17A, AKAP4, HKDC1, BRDT, TSBP1, SLC22A2, P2RY1, FOXD2, NAT8B, ASCL2, IL12A, CREB5, DMRT1, PHF14 Table 2 SLC13A1, LRRC19, HAVCR1, HHLA2, CT62, LDHAL6B, SLC22A11, DDX4, CLCN5, SLC25A31, GIPC2, GUCY2C, ABCB1, TMPRSS15, RBMXL2, TM4SF20, KDM4D, SLCO4C1, PRM2, RNF32, CNTN6, ACSL6, UBQLN3, HNF4G, ANKRD7, MXD1, SLC17A3, IL12RB2, ANKRD40CL, SLC6A20, SLC5A12, SLC10A2, ACRV1, ALPI, EPPIN, IL17A, AKAP4, HKDC1, BRDT, TSBP1, SLC22A2, P2RY1, FOXD2, NAT8B, ASCL2, IL12A, CREB5, DMRT1, PHF14 Acknowledgements Tables and Table Legends Table 1 First author Journal Drugs Exper. Comp. Method Target Cells Jeon biorxiv 24 Yes No Inhibition assay Vero cells Gordon biorxiv 63 No Yes Mass-spec Docking Multiple HEK293T Farag chemrxiv 71 No Yes Docking Mpro Wang chemrxiv 21 No Yes Docking Mpro Contini chemrxiv 19 No Yes Docking Mpro & C3Lpro Kumar chemrxiv 10 No Yes Docking Mpro Zhou Cell Discovery 16 No Yes Network Analysis Aly chemrxiv 7 No Yes Docking Mpro Jin Nature 7 Yes Yes Docking Mpro Rensi chemrxiv 21 No Yes Docking TMPRSS2 Xing biorxiv 11 Yes Yes L1000 Inhibition assay Vero cells Touret biorxiv 90 Yes No Inhibition assay Vero cells Ko biorxiv 35 Yes No Inhibition assay Vero cells Nguyen biorxiv 84 No Yes Docking Mpro Ge biorxiv 1 Yes Yes L1000 Network Analysis PARP1 PBMCs Alakwaa mSystems 4 No Yes L1000 scRNA-seq Beck Comput Struct Biotech J. 8 No Yes Docking Multiple Chen F1000Res. 15 No Yes Docking C3Lpro Cava MDPI 36 No Yes Network Analysis Ellinger Research Square 64 Yes No Inhibition assay Caco-2 Heiser biorxiv 100 Yes No Image-based assay HRCE cells Riva biorxiv 30 Yes No Inhibition assay Vero cells References 1. 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In-Pile Qualification of a Fast-Neutron-Detection-System
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In-pile qualification of the fast-neutron-detection-system L. Barbot, D. Fourmentel, J.-F. Villard, C. Destouches, B. Geslot, L. Vermeeren, M. Schyns In-pile qualification of the fast-neutron-detection-system L. Barbot, D. Fourmentel, J.-F. Villard, C. Destouches, B. Geslot, L. Vermeeren, M. Schyns To cite this version: L. Barbot, D. Fourmentel, J.-F. Villard, C. Destouches, B. Geslot, et al.. In-pile qualification of the fast-neutron-detection-system. EPJ Web of Conferences, 2017, 170, pp.04025. ￿10.1051/epj- conf/201817004025￿. ￿cea-02437051￿ Distributed under a Creative Commons Attribution 4.0 International License I. INTRODUCTION A significant part of this effort have been conducted in the framework of the Joint Instrumentation Laboratory between the CEA and the Belgian Nuclear Research Centre (SCK•CEN). F AST neutron flux (E ≥ 1 MeV) is a key neutron parameter in nuclear reactors. Its measurement is particularly relevant to assess material damage under irradiation. In very low power research reactors, such as ZPRs (Zero Power Reactors), the fast neutron flux is usually measured on-line with specific fission chambers, containing for example 238U coating. In more powerful reactors, such as Material Testing Reactors (MTRs) or nuclear power plants (NPPs), techniques used in ZPRs are not appropriate. This is mainly due to the rapid evolution of the fissile coating under high neutron flux that reduces the sensitivity of the sensor to the ‘fast’ spectrum domain (cf. II.B). This neutron parameter is therefore not accessible on-line and is generally evaluated afterward by activation dosimetry or by Monte Carlo calculations. However, in the current context of increasing requirements for in-pile measurements, particularly for experiments in MTRs, the on-line measurement of fast neutron flux has become a major objective. F In order to improve measurement techniques for neutron flux assessment, a unique system for online measurement of fast neutron flux has been developed and recently qualified in-pile. The Fast-Neutron-Detection-System (FNDS) has been designed to monitor accurately high-energy neutrons flux (E > 1 MeV) in typical Material Testing Reactor conditions, where overall neutron flux level can be as high as 1015 n.cm-2.s-1 and is generally dominated by thermal neutrons. Moreover, the neutron flux is coupled with a high gamma flux of typically a few 1015 .cm-2.s-1, which can be highly disturbing for the online measurement of neutron fluxes. The patented FNDS system is based on two detectors allowing the simultaneous detection of both thermal and fast neutron flux. Thermal neutrons can be measured using a Self-Powered- Neutron-Detector (SPND) or a 235U miniature fission chamber, while fast neutron detection requires a miniature fission chamber with a special fissile material presenting an energy threshold near 1 MeV, which can be 242Pu for MTR conditions. Fission chambers are operated in Campbelling mode for an efficient gamma rejection. HAL Id: cea-02437051 https://cea.hal.science/cea-02437051v1 Submitted on 13 Jan 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License ANIMMA-69105 ANIMMA-69105 I. INTRODUCTION FNDS also includes a specific software that processes measurements to compensate online the fissile material depletion and to adjust the sensitivity of the detectors, in order to produce a precise evaluation of both thermal and fast neutron flux even after long term irradiation. In order to ensure the quality and the relevance of irradiation programs in the future Jules Horowitz Reactor (JHR), the French Alternative Energies and Atomic Energy Commission (CEA) has significantly increased its research and development effort in the field of in-pile instrumentation during the last decade. In this context and in the framework of the Joint Instrumentation Laboratory between the CEA and the Belgian Nuclear Research Centre (SCK•CEN), a measurement system dedicated to on-line measurement of fast neutron flux has been developed. FNDS has been validated through a two-step experimental program. A first set of tests was performed at BR2 reactor operated by SCK•CEN in Belgium. Two FNDS prototypes were operated in-pile during nearly 1000 hours. These tests exhibited the consistency of the measurement of thermal to fast neutron flux ratio with Monte Carlo calculations, as well as the right compensation of fissile material depletion. Then a second test was recently completed at ISIS reactor operated by CEA in France. For this irradiation, FNDS signal was compared to reference thermal and fast neutron flux measurements using activation dosimeters analyzed under COFRAC® Quality Certification. During this latter test, FNDS proved its ability to measure online the fast neutron flux with an overall accuracy better than 5%. Next paragraphs describe the innovative features of the Fast-Neutron-Detection-System (FNDS) and discuss the results of its final in-pile qualification. a better and real-time evaluation of the fast neutron flux received by material and fuel samples. Abstract— In order to ensure the quality and the relevance of irradiation programs in the future Jules Horowitz Reactor (JHR), the French Alternative Energies and Atomic Energy Commission (CEA) has significantly increased its research and development effort in the field of in-pile instrumentation during the last decade. Major progresses have thus been achieved in the capability to perform accurate in-pile measurements using reliable and updated techniques. Index Terms—Nuclear Measurements, Fission Chamber, Reactor Instrumentation In-Pile Qualification of the Fast-Neutron- Detection-System mentel1, J-F. Villard1, C. Destouches1, L. Barbot1, B. Geslot2, L. Vermeeren3, M. Schyns3 a better and real-time evaluation of the fast neutron flux received by material and fuel samples. a better and real-time evaluation of the fast neutron flux received by material and fuel samples. ANIMMA-69105 2 Fig. 2. Overview of FNDS components FNDS thus consists in 2 detectors for ‘fast’ and ‘thermal’ spectrum domains, an acquisition system operating in Campbelling mode and a calculation module for on-line analysis of neutron fluxes (Figure 2). These innovations were patented for the detectors [4] and for the entire measurement system [5]. Fig. 2. Overview of FNDS components because of the low neutron sensitivity of this type of fission chamber, the high gamma flux of MTRs leads to a high gamma contribution to the signal. Furthermore, neutron captures on the nuclei of the fissile coating create isotopes that have a high cross-section in the thermal spectrum domain. Thus the ‘fast’ sensitivity of the detector decreases rapidly. Isotopic impurities of the coating must also be taken into account. Figure 1 illustrates this difficulty for the use of 242Pu, which has a suitable response in the fast spectrum domain [1]. Fig. 2. Overview of FNDS components FNDS thus consists in 2 detectors for ‘fast’ and ‘thermal’ spectrum domains, an acquisition system operating in Campbelling mode and a calculation module for on-line analysis of neutron fluxes (Figure 2). These innovations were patented for the detectors [4] and for the entire measurement system [5]. Fig. 1. Fission cross sections of a typical 242Pu fissile coating showing that impurities and isotopes created by capture must be taken into account for fast neutron flux measurements B. Detectors An important bibliography study was carried out to establish the state of the art of neutron and photonic measurements in MTRs [6, 7], and led us to select fission chamber sensors. It was shown that the most suitable fissile isotope for the estimation of the fast neutron flux is 242Pu under the typical irradiation conditions of MTRs, such as OSIRIS (CEA Saclay – France), BR2 (Mol – Belgium) and JHR reactors [1, 8]. Figure 3 illustrates the raison why 242Pu coating is the best choice compared to other fissile isotopes; it shows the stability of its fast neutron sensitivity at high fluence. Fig. 1. Fission cross sections of a typical 242Pu fissile coating showing that impurities and isotopes created by capture must be taken into account for fast neutron flux measurements We can notice that impurities and isotopes created by neutron captures induce an increasingly high sensitivity to thermal neutrons. Fig. 3. Sensitivity to fast neutrons for initially pure deposits under typical BR2 neutron spectrum [3] II. FNDS SPECIFICATIONS This paper describes the innovative features of FNDS and discusses the results of its final in-pile qualification. FNDS is now operational and is assumed to be the first and unique acquisition system able to provide an online measurement of the fast neutron flux in MTR conditions. This system will of course be used to perform spectral neutron characterization of JHR channels, but it may also be implemented in future irradiation experiments, for On-line measurements of fast neutron flux are based on the use of a fission chamber with a specific fissile coating, presenting an energy threshold near the fast domain (E > 1 MeV). If the use of such fission chamber is not a problem at very low neutron flux, it faces major difficulties under intense mixed neutron and gamma fluxes. First of all, C. Acquisition system Φé𝑝𝑖= 𝛼⋅Φ𝑟𝑎𝑝 The acquisition system has 2 channels for the sensor couple. Each channel is equipped with a high-voltage power supply for polarization with adjustable voltage (typically 250 V to reach the saturation plateau of a 3 mm fission chamber). The measured low-level current signals pass through a preamplifier (manufactured by CEA Saclay), amplification stage and then are filtered and digitalized for processing with the measurement interpretation software. This hypothesis was verified by calculations [10]. It allows to reduce our system to 2 equations with 2 unknown variables and to establish the following equation of the fast neutron flux: Φ𝑟𝑎𝑝= 𝑅1 𝑆𝑡ℎ1 −𝑅2 𝑆𝑡ℎ2 𝛼⋅𝑆é𝑝𝑖1 + 𝑆𝑟𝑎𝑝1 𝑆𝑡ℎ1 −𝛼⋅𝑆é𝑝𝑖2 + 𝑆𝑟𝑎𝑝2 𝑆𝑡ℎ2 There are typically 3 modes to operate fission chambers: pulse, fluctuation (or Campbelling) and current modes. In MTRs conditions, the advantage of the Campbelling mode is the radical limitation of the gamma contribution on the signal [2] compared to current mode. Indeed, MTR neutron fluxes are too high to operate such fission chambers in pulse mode. However, in a light water reactor, 235U coating is predominantly (~99% initially) sensitive to thermal neutrons [6, 7] and makes it possible to measure thermal neutron flux without calculation assumptions. In this case, fast neutron flux can then be written in the following reduced form: In Campbelling mode, the measurement of the variance of low fission chamber signal with a specific bandwidth has required the development of a dedicated acquisition system. As such a system was not available from Industry; prototypes were specially designed at CEA. Studies are on-going for the industrialization of such acquisition system in a short term [9]. Φ𝑟𝑎𝑝= 𝑅1 −𝑆𝑡ℎ1 ⋅Φ𝑡ℎ 𝑆𝑟𝑎𝑝1 + 𝛼⋅𝑆é𝑝𝑖1 ANIMMA-69105 by the CEA, to compute fission cross sections of each isotope present in the fissile coating and to calculate the isotopic evolution of these coatings with fluence and time. The method to combine measurements and estimate fast neutron flux is the following [7, 8]: signal requires a precise knowledge of its isotopic composition and as well as the effective deposited mass of fissile materials. It is necessary to quantify with the best accuracy their contribution to the signal. For the in-pile qualification of FNDS (§.III), analyzes of fissile coatings give the following results: If R1 is the fission rate of the ‘fast’ fission chamber and R2 the fission rate of the ‘thermal’ fission chamber, the following two equations can be written: m(235U): 10.4 μg isotope: 234U 235U 236U 238U % mass: 1.66 97.65 0.15 0.54 m(242Pu): 98.3 μg isotope: 238Pu 239Pu 240Pu 241Pu 242Pu % mass: 0.003 0.009 0.019 0.006 99.963 𝑅1 = 𝑆𝑡ℎ1 ⋅Φ𝑡ℎ+ 𝑆é𝑝𝑖1 ⋅Φé𝑝𝑖+ 𝑆𝑟𝑎𝑝1 ⋅Φ𝑟𝑎𝑝 𝑅2 = 𝑆𝑡ℎ2 ⋅Φ𝑡ℎ+ 𝑆é𝑝𝑖2 ⋅Φé𝑝𝑖+ 𝑆𝑟𝑎𝑝2 ⋅Φ𝑟𝑎𝑝 with Si thermal, epithermal and fast neutron sensitivity [1/n.cm-2] and j thermal, epithermal and fast neutron fluxes [n.cm-2.s-1]. As a reminder, the odd isotopes of plutonium are mostly fissile in the ‘thermal’ spectrum domain and have very high fission cross sections compare to 242Pu in the ‘fast’ spectrum domain (cf. Figure 1). To solve this system of 2 equations with 3 unknown variables we assume that the ratio between epithermal and fast fluxes is constant during irradiation: A. FNDS general description In order to compensate difficulties mentioned above, FNDS includes an acquisition system allowing measurements with fission chambers in Campbelling mode. This mode allows a very efficient rejection of the gamma contribution [2] while offering a very wide dynamic range. FNDS also integrates signal processing software that firstly calculate in real time the evolution of the fissile coating of the fission chamber using an evolution code DARWIN [3], and that secondly deduce the corresponding evolution of the neutron sensitivity of the fast fission chamber. This software extracts in real-time the value of the fast neutron flux from the signal. In order to allow a precise evaluation of thermal neutron fluence received by the ‘fast’ fission chamber, FNDS is equipped with a second detector that is sensitive to thermal neutron flux and positioned near the ‘fast’ fission chamber. This second detector may be a fission chamber with 235U coating or a Self-Powered Neutron Detector (SPND). Fig. 3. Sensitivity to fast neutrons for initially pure deposits under typical BR2 neutron spectrum [3] A typical ‘fast’ fission chamber selected for MTRs contains 100 μg of 242Pu while the typical associated ‘thermal’ detector contains 10 μg of 235U. Both sensors have a 3 mm outer diameter body and integrated mineral insulated cable. These sensors have been developed and manufactured at the Fission Chamber Manufacturing Workshop located in CEA Cadarache. As explained previously, the analysis of fission chamber 3 C. Monte Carlo and experimental results - 7 cobalt (AlCo1% - diameter 10 mm, thickness 100 µm) including 1 in a cadmium shield (for the epithermal ratio on cobalt). Activity measurements of the EFICAF dosimeters were carried out at the MADERE platform of CEA Cadarache under COFRAC® Quality Accreditation [16]. Table 1 below summarizes the neutron flux results calculated by TRIPOLI4 code and measured by activation dosimetry at the locations of the two fission chambers. Finally, Monte Carlo calculations were performed with TRIPOLI4 code [12] for FNDS processing software and for activation dosimeter analysis. TABLE I THERMAL AND FAST NEUTRON FLUXES TRIPOLI4 CALCULATIONS AND DOSIMETRY MEASUREMENTS C/E : TRIPOLI4/Dosimeter type -1 [%] TRIPOLI® (n.cm-2.s-1) AlCo 1% (n.cm-2.s-1) C/E Thermal neutron flux 1,04E+12 1,00E+12 3,3% TRIPOLI® (n.cm-2.s-1) Fe (n.cm-2.s-1) C/E Ni (n.cm-2.s-1) C/E Fast neutron flux 4,74E+11 4,58E+11 3,4% 4,58E+11 3,4% A. EFICAF specifications It was decided to measure the absolute thermal and fast neutron fluxes in a single irradiation channel of ISIS core. Fission chambers were located on both sides of the maximum neutron flux height in order to receive the same neutron flux with a simple experimental device and to avoid a moving device. Reactor power was specified at 250 kWth, on one hand for easy implementation of the experiment and on the other hand to minimize the fluence and the corresponding evolution of fissile materials. According to the relation between fission rate ‘R’ and variance measurements 𝜎𝑉 2 there is a second calibration coefficient 𝐾𝑒 to be calculated. 𝐾𝑒 links the current signal delivered by the fission chambers to the corresponding variance [V2] for each measurement channel. The calibration coefficients of the electronics for the 2 measurement channels of FNDS were the following: For this experiment, reference neutron flux measurements were performed using activation dosimeters. They provided a precise evaluation of the neutron flux profile in the experimental channel. These dosimeters were the following: K𝑒(𝑐ℎ𝑎𝑛𝑛𝑒𝑙1) = 5.45. 1015Ω2. Hz K𝑒(𝑐ℎ𝑎𝑛𝑛𝑒𝑙2) = 4.91. 1015Ω2. Hz 7 pure iron (diameter 10 mm, thickness 100 µm) - 7 pure nickel (diameter 10 mm, thickness 125 µm), ANIMMA-69105 the second opportunity to use this calibration method. The calibration coefficient obtained for both fission chambers is: Flux in Isis by Fission Chamber) experiment in ISIS reactor (CEA Saclay, France). ISIS reactor allows stable and reproducible irradiation conditions. Its neutron flux level is also sufficient to operate fission chambers in Campbelling mode. Moreover, its flexibility allowed to carry out all the measurements that were necessary for the qualification (on- line measurements and activation dosimetry measurements without external disturbance). Flux in Isis by Fission Chamber) experiment in ISIS reactor (CEA Saclay, France). ISIS reactor allows stable and reproducible irradiation conditions. Its neutron flux level is also sufficient to operate fission chambers in Campbelling mode. Moreover, its flexibility allowed to carry out all the measurements that were necessary for the qualification (on- line measurements and activation dosimetry measurements without external disturbance). Kd = 3.75. 10−27A2. Hz−2 We can notice that the calibration coefficient is the same for both fission chambers. Indeed, the average charge of a fission product between the electrodes is mostly similar for both coatings. This calibration coefficient value also depends on the signal bandwidth. It is therefore important to characterize the bandwidth of the analogue-to-digital processing for an appropriate use of the calibration coefficient. Details about the calibration are given in [13, 15]. D. Processing software FNDS has been validated through a two-step experimental program. A first set of tests was performed at BR2 reactor through the FICTIONS-8 experiment. Two FNDS prototypes were operated in-pile during nearly 1000 hours. This experiment exhibited the consistency of the measurement of thermal to fast neutron flux ratio with Monte Carlo calculations, as well as the right compensation of fissile material depletion [11]. The FNDS processing software calculates the sensitivity of each fission chamber to the thermal, epithermal and fast neutrons from specific input data and then calculates the thermal and fast neutron fluxes versus time. Input data required for the processing are the following: - typical neutron spectrum at the location of the detectors, - nuclear data library, - nuclear data library, - mass and isotopic composition of the fissile coatings, A second set of tests was then necessary to qualify the absolute fast neutron flux measurement process. The main requirement was to perform measurements in well-controlled conditions (neutron spectrum, flux gradient, etc.) in order to minimize external sources of uncertainty and bias. In this context, it was decided to carry out the EFICAF (Estimation of - calibration coefficient of the fission chambers (§.III.B), - calibration coefficient of the electronic (§.III.B), - variance measurements. - variance measurements. Processing software includes DARWIN code [6], developed Processing software includes DARWIN code [6], developed 4 IV. CONCLUSION y [8] P. Filliatre, L. Oriol, C. Jammes et al., “Joint estimation of the fast and thermal components of a high neutron flux with a two on-line detector system”, Nuclear Instruments And Methods in Physics Research Section A, 603 (3), p. 415- 420, 2009. EFICAF experiment demonstrated the ability of FNDS to measure on-line thermal and fast neutron fluxes. Deviations between fast neutron flux measured by FNDS and activation dosimetry results are below 5%. These results confirm the excellent accuracy of the analysis method using a couple of 235U and 242Pu fissile coatings and special calibration in Campbelling mode. [9] M. Thevenin et al., “Digital Real-Time Multiple Channel Multiple Mode Neutron Flux Estimation on FPGA-based Device”, International Symposium on Reactor Dosimetry (ISRD 2015), 18-23 May 2015, Aix-en-Provence, France. [10] B. Geslot, A. Legrand, C. Jammes et al., “Qualification du système d’acquisition FNDS lors de l’expérience FICTIONS-8”, CEA Cadarache DER/LDCI/NT/09-032, 2010. FNDS is now operational and is assumed to be the first and unique acquisition system able to provide an on-line measurement of the fast neutron flux in MTR conditions. This system will of course be used to perform spectral neutron characterization of JHR channels, but it may also be implemented in future irradiation experiments, for a better and real-time evaluation of the fast neutron flux received by material and fuel samples. [11] B. Geslot, et al., “New measurement system for on line in core high- energy neutron flux monitoring in materials testing reactor conditions” Review of Scientific Instruments, Vol. 82, 033504 (2011). ( ) [12] E. Brun et al., “TRIPOLI-4®, CEA, EDF and AREVA reference Monte Carlo code”, Annals of Nuclear Energy, Vol. 82, pp. 151–160, 2015. [13] B. Geslot, T. C. Unruh, P. Filliatre et al., “Method to calibrate fission chambers in Campbelling mode”, IEEE Trans. Nucl. Sci., vol. 59, no. 4, pp. 1377–1381, 2012. pp [14] D. Fourmentel, P. Filliatre, L. Barbot et al., “Comparison of Thermal Neutron Flux Measured by 235U Fission Chamber and Rhodium Self- Powered Neutron Detector in MTR”, IEEE Transactions on Nuclear Science, Vol. 61, No. 4, 2014. Fig. 4. Illustration of updated FNDS based on the Multichannel Acquisition System MONACO [9]. [15] D. Fourmentel, B. Geslot, “Méthode et protocole d'étalonnage des chambres à fission en mode fluctuation”, CEA Cadarache DER/LDCI/ NT/14-009, 2014. [16] COFRAC accreditation certificate n° 1-0736 available at https://www.cofrac.fr/annexes/sect1/1-0736.pdf [17] L. REFERENCES TABLE III FAST NEUTRON FLUX EVALUATIONS AND DEVIATIONS BETWEEN FNDS AND TRIPOLI4 CALCULATIONS AND BETWEEN FNDS AND FE, NI MEASUREMENTS CF Pu242 (n.cm-2.s-1) (TRIPOLI/CF Pu242)-1 (Fe-Ni/CF Pu242)-1 1st measurements 4.39E+11 7.9% 4.4% 2nd measurements 4.40E+11 7.7% 4.1% [1] P. Filliatre et al., “Reasons why Plutonium 242 is the best fission chamber deposit to monitor the fast component of a high neutron flux”, Nuclear Instruments and Methods in Physics Research A 593 (2008) 510– 518. [2] L. Vermeeren et al., “Experimental Verification of the Fission Chamber Gamma Signal Suppression by the Campbelling Mode”, IEEE Transactions on Nuclear Science, vol. 58, n°2, April 2011. The deviation between FNDS fast neutron flux measurements and dosimetry results is mainly covered by the uncertainties of the activation dosimetry measurements (typically 4% for fast neutron flux evaluation). The results of this experiment confirm the quality of the on-line fast neutron flux measurements at a few 1011 n.cm-2.s-1 (E > 1 MeV) with this type of miniature fission chambers and therefore confirm FNDS capability to measure in real-time the absolute thermal and fast neutron fluxes with an excellent accuracy. [3] A.Tsilanizara, C.M.Diop et al., J.Nucl.Sci.Technol.37 (2000)845 [4] L. Oriol, P. Filliatre, L. Vermeeren, “Détecteur pour la mesure en ligne des neutrons rapides dans un réacteur” Patent FR 2925750 A1, 2009. [5] L. Oriol, B. Lescop, L. Vermeeren, “ Dispositif de mesure en ligne d’un flux de neutrons rapides et épithermiques” Patent WO 2010043554 A1, 2010. [6] D. Fourmentel, “Etat de l’art des mesures neutroniques et photoniques en réacteur d’irradiation pour la cartographie du cœur du réacteur Jules Horowitz”, CEA Cadarache DER/LDCI/RT/10-010 ind.B DR. [7] D. Fourmentel, “Mesures neutroniques et photoniques combinées pour la caractérisation précise des canaux expérimentaux du futur réacteur d’irradiation Jules Horowitz (RJH)”, PhD thesis, Aix-Marseille University, 2013. B. Fission chambers calibration in Campbelling mode A method to calibrate fission chambers in Campbelling mode has been developed [13]. This calibration was carried out for FNDS sensors in MINERVE zero power reactor at CEA Cadarache. In this calibration it is assumed that the measured signal is equal to the product of the coating fission rate, a calibration coefficient of the electronic and a calibration coefficient of the detector. The variance of the signal can then be expressed according to the following relation: The results of TRIPOLI4 calculations and dosimetry measurements are in good agreement to assess neutron fluxes values given by FNDS. 𝜎𝑉 2 = 𝐾𝑒∙𝐾𝑑∙𝑅 Thermal and fast neutron fluxes measured by FNDS are compared with values given the TRIPOLI4 code and activation dosimetry respectively in Table 2 and Table 3. with: 𝜎𝑉 2: variance measurement [V2], 𝐾𝑒: coefficient of the electronic channel [Ω2.Hz], 𝐾𝑑: calibration coefficient of the detector [A2.Hz-2] and 𝑅: total fission rate of the coating [Hz]. TABLE II THERMAL NEUTRON FLUX EVALUATIONS AND DEVIATIONS BETWEEN FNDS AND TRIPOLI4 CALCULATIONS AND BETWEEN FNDS AND ALCO1% MEASUREMENTS CF U235 (n.cm-2.s-1) (TRIPOLI/CF U235)-1 (AlCo 1%/CF U235)-1 1st measurements 9.34E+11 11.0% 7.5% 2nd measurements 9.15E+11 13.3% 9.7% This method was used for the first time in 2012 for the CARMEN-1 experiment led in OSIRIS reactor at CEA Saclay. It exhibited excellent results for 235U fission chamber used in Campbelling mode by comparison with rhodium SPND and activation dosimeters [14]. EFICAF experiment was therefore ANIMMA-69105 5 These results confirm the good agreement for absolute thermal neutron flux using Campbelling mode operation and the corresponding calibration process [14]. shortly be performed to evaluate FNDS ability to measure also the epithermal neutron flux. shortly be performed to evaluate FNDS ability to measure also the epithermal neutron flux. shortly be performed to evaluate FNDS ability to measure also the epithermal neutron flux. IV. CONCLUSION Barbot et al., “On Line Neutron Flux Mapping in Fuel Coolant Channels of a Research Reactor”, IEEE Transactions on Nuclear Science, vol. 62, n°2, April 2015. Fig. 4. Illustration of updated FNDS based on the Multichannel Acquisition System MONACO [9]. Further studies are on-going to update the FNDS acquisition system using a multi-channel and multi-mode acquisition system called MONACO (Multichannel Online Neutron Acquisition in Campbell mOde) [9,17]. Studies are also carried out to assess the uncertainty associated with neutron flux analysis calculations, mainly for the calibration coefficients of the detectors. Finally, additional tests will
https://openalex.org/W4220874515
https://zenodo.org/records/6227158/files/147-150.pdf
Quechua
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NEMIS TILIGA O'ZLASHGAN SO'ZLARNING IMLOSI
Zenodo (CERN European Organization for Nuclear Research)
2,022
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PEDAGOGS international research journal PEDAGOGS international research journal NEMIS TILIGA O’ZLASHGAN SO’ZLARNING IMLOSI Berdimurodov Adham Musurmonovich Qarshi davlat universiteti Nemis tili va adabiyoti kafedrasi o'qituvchisi Annontatsiya: G’arbiy Yevropa tillaridan biri hisoblangan nemis tili ham yillar davomida qo’shni davlatlarning tillari, jumladan, qadimgi lotin tili, shuningdek italyan, fransuz, ispan, rus, ingliz tillaridan olingan so’z va iboralar asosida o’z lug’at boyligini boyitgan. Nemis tilining ilk shakllanish davrida lotin tilining lug’at boyligi yetakchilik qilgan bo’lsa, 9-13 asrlarda fransuz va ispan tillaridan, Yevropadagi uyg’onish davrida italyan tilidan, Ikkinchi jahon urushi natijasida rus tilidan, fan-texnika jadal sur’atlar bilan rivojlanayotgan bugungi kunda esa ingliz tilidan o’zlashgan so’z va iboralar nemis tilining lug’at tarkibidan muqum o’rin egallaganligi e’tiborga molikdir. Kalit so’z: Job, jobben, Computer, Lyzeum, College, Chef, Charakter Kalit so’z: Job, jobben, Computer, Lyzeum, College, Chef, Charakter Ammo bir tildan ikkinchi bir tilga o’zlashgan so’zlarning grammatik belgilari, tovush tizimi hamda imloviy ko’rinishi ayrim qiyinchiliklarni ham keltirib chiqarishi mumkin. Masalan, nemis tiliga boshqa tillar asosida o’zlashgan ot so’z turkumiga mansub quyidagi so’zlardan fe’llar hosil qilish mumkin: jobben - nemis so’zlashuv tilida “ishlamoq” ma’nosida; joggen - “gimnastika bilan shug’ullanmoq”, “yugurmoq” ma’nolarida. joggen - “gimnastika bilan shug’ullanmoq”, “yugurmoq” ma’nolarida. Vaholanki, ingliz tilidagi “Job” so’zi hamda kelib chiqishi Sharqiy Osiyo hududiga tegishli bo’lgan “Joga” atamasi faqat ot so’z turkumidagina qo’llanila oladi. Yoki, nemis tiliga o’zlashgan “Lyzeum, College, Chef, Charakter” kabi so’zlardagi ba’zi bir harflarning tovush xususiyatiga alohida ahamiyat berish talab etiladi. Zero, nemischa so’zlarda “eu” diftongining talaffuzi “oy” deb , “g” va “ch” undoshining o’qilishi esa qoidaga ko’ra |g|, |ɞ|, |x| tarzida talaffuz etiladi. Shu sabab, Volume-4, Issue-1, February–2022 www.pedagoglar.uz 147 PEDAGOGS international research journal PEDAGOGS international research journal nemis tilini o’rganayotgan talabalarga bunday so’zlarni qaysi tillardan nemis tiliga o’zlashganligini va qanday talaffuz qilinishini alohida uqtirib o’tish talab etiladi. Shuningdek, nemis tilida chop etilayotgan darslik va adabiyotlarda o’zlashgan ba’zi bir so’zlarning ikki xil shaklda yozilishini ham talabalarda tushunmovchilik keltirib chiqarishi mumkin deb hisoblaymiz. Jumladan, “kompyuter, kreativ” kabi so’zlar hozirgi nemis tilida imloviy ko’rinishi bo’yicha ikki xil shaklda ifoda etilmoqda: “Komputer - Computer”, “kreativ - creativ”. Olinma so’zlarning qo’llanilishi borasidagi fikrimizning isboti sifatida biz o’rta maktabning yuqori sinf o’quvchilari hamda akademik litsey va kollej o’quvchilari uchun mo’ljallangan darsliklarda ifoda etilgan olinma so’zlar va ularning imlosi borasida to’xtalib o’tamiz. Sh.S.Imyaminova, S.Ya.Abdullayeva, L.T.Xoliyorovlar tomonidan yaratilgan “Deutsch” darsliklarida 100 ga yaqin o’zlashma so’zlar qo’llanilgan. Ammo bu so’zlarning nemis tilida ifoda etishda mualliflar tomonidan ba’zi bir imloviy xatoliklarga ham yo’l qo’yilganligini ta’kidlab o’tishimiz zarur. Jumladan, o’rta maktablarning 10-sinflari uchun mo’ljallangan darslikda quyidagicha kamchiliklar uchraydi: Darslikning 40-betida “DJ” qisqartmasining transkripsiyasi berilmagan. 62- betda ifodalangan “joggen” so’zining nemischa ma’nosi berilmagan. 73-betda “E- learning” atamasi bilan bir qatorda bu so’zdan hosil qilingan nemischa so’zlar – “E- Lernen”, “E-Lerner” so’zlari izohsiz ifoda etilgan. 83-betda ingliz tilidan nemis tiliga kirib kelgan “party” so’zi izohsiz tarzda ikkita gapda ishlatilgan. 93-betda ifoda etilgan “Feed-Back” so’zining nemischa muqobilini topish topshirig’i ham, bizningcha, o’quvchilarga qiyinchilik keltiradi. Darslikning 110-betida inglizcha “Non-Profit-Organisation” (notijorat tashkiloti) ifodasi nemis tiliga “Die nichtstaatliche und nichtkommerzielle Organisationen” (nodavlat va notijorat tashkilotlari) shaklida ham mantiqan, ham grammatik jihatdan noto’g’ri talqin etilgan. 131-betda esa ”Info-point” ifodasi qo’shtirnoq ichiga olinmagan. 138-betda Volume-4, Issue-1, February–2022 www.pedagoglar.uz Volume-4, Issue-1, February–2022 148 PEDAGOGS international research journal PEDAGOGS international research journal olgan “Mein Plan/Mein Ziel” matnida qo’llanilgan “ins Visier” birikmasi nafaqat o’quvchilarga, balki o’qituvchilarga ham tushunarli emas. “Deutsch 2. Zum Ziel” darsligida o’zlashma so’z va birikmalarning imlosi bo’yicha nisbatan kam xato kuzatiladi. Jumladan, darslikning 17-betidagi “I-Phone, Evernote” kabi olinma so’zlar qo’shtirnoq ichiga olinmagan. Shuningdek, darslikning 3-Lektioniga sarlavha sifatida chiqarilgan “Recherche” so’zining va bu so’zdan hosil qilingan “recherchieren” fe’lining (24-bet) transkripsiyasisiz berilishi ham talabalarga bu so’zlarni to’g’ri talaffuz qilishda qiyinchilik tug’diradi, deb o’ylaymiz. O’z navbatida darslikda berilgan matnlarning birida “Profi” (65-bet) qisqartma so’zi o’rnida “Professional” olinmasi yoki “Master” so’zi qo’llanilganda o’quvchilarga bu so’zning ma’nosi yanada tushunarli bo’lardi. Xulosa sifatida shunga alohida ahamiyat berishimiz kerakki, o’zlashgan so’z va iboralardan ma’lum bir tilda to’g’ri foydalanish uchun, eng avvalo, bu ifodalarning lug’aviy ma’nosini bilgan holda ulardan samarali foydalanish talab etiladi. Shuningdek, olinma so’zlarning talaffuzi va imlosida xato bo’lmasligini ta’minlash uchun, bizningcha, ular tegishli transkripsiyalari bilan, shakli o’zgarmagan ko’rinishda ifodalanishi kerak bo’ladi. PEDAGOGS international research journal PEDAGOGS international research journal “CV” qisqartmasining, shuningdek, 170-betda “Science-Fiction” qo’shma so’zining izohi va transkripsiyasi berilmagan. “CV” qisqartmasining, shuningdek, 170-betda “Science-Fiction” qo’shma so’zining izohi va transkripsiyasi berilmagan. Yuqoridagi mualliflarning 11-sinf o’quvchilariga mo’ljallab yaratgan darsligida quyidagi kamchiliklarni ko’rish mumkin: Darslikning 29-betida ingliz tilidan olingan “recycling” so’zi asosida nemischa qo’shma so’z yasalgan: Recyclingssysteme, biroq bu o’rinda “recycling” so’zining o’qilishi va anglatgan ma’nosi qayd etilmagan. 50-betda “joggen” (gimnastika bilan shug’ullanmoq) o’zlashma so’zi nemischa “Laufen” (yugurmoq) so’ziga ma’nodosh sifatida berilgan. 91-betda Roksana ismli qizning xatida ifoda etilgan “Azubis” so’zining ma’nosi o’quvchilarga tushunarli emas. Darslikda “sartarosh” ma’nosini anglatuvchi o’zlashma so’z ikki xil shaklda 98-betda “Frisur”, 100-betda esa “Friseur” shaklida berilgan. Akademik litsey va kasb-hunar kollejlarining o’quvchilariga mo’ljallab yozilgan va mualliflari S.Ya.Abdullayeva, L.T.Holiyarov, S.A.Ziyayeva, Sh.A.Hojiyevalar bo’lgan darsliklarda ham nemis tiliga o’zlashgan so’zlarning talaffuzi va imlosi bo’yicha bir qator kamchiliklar ko’zga tashlanadi. Quyida “Deutsch 1/2 Zum Ziel” darsliklarida o’zlashma so’zlar bo’yicha yo’l qo’yilgan kamchiliklarni e’tirof etamiz: “Deutsch 1. Zum Ziel” darsligida “Chef” (17-bet), “Job” (18-bet), “Feed-Back” (32-bet), “E-Learning” (43-bet), “Chance” (45-bet) kabi so’zlarning transkripsiyasi ko’rsatilmagan. Bu narsa esa bunday o’zlashma so’zlarning to’g’ri talaffuz qilinishida qiyinchiliklarni keltirib chiqradi. 78-betdagi “Heiss oder Nicht?” nomli matnda ifodalangan “die Studenten – ID-Images”, Website von Facemash” kabi birikmalar o’quvchilarga biroz tushunarsizdir. Shuningdek, 134-betda “qisqacha xulosa” ma’nosini anglatuvchi “Resume” so’zi “Resümee” shaklida ishlatilgan, nemischa “bequem” (qulay) so’zi o’rnida esa “komfortabel” o’zlashma so’zi qo’llanilgan. Darslikdagi 12-Lektionga tegishli 1-darsda “Evalution” olinma so’zi imlo xatosi bilan sarlavhaga chiqarilgan. Aynan shu so’z darslikka ilova qilingan “Glossar” da “Evaluation” ko’rinishida berilgan. Bundan tashqari 151-betdan o’rin Volume-4, Issue-1, February–2022 www.pedagoglar.uz 149 PEDAGOGS international research journal olgan “Mein Plan/Mein Ziel” matnida qo’llanilgan “ins Visier” birikmasi nafaqat o’quvchilarga, balki o’qituvchilarga ham tushunarli emas. Аdabiyotlar: 1. Мюллер В.К. Англо-русский словарь. – Москва:Русский язык, 1985 – 864 c. 2. Abdullayeva S.Ya.,.Holiyarov L.T,.Ziyayeva S.A, Hojiyeva Sh.A. Deutsch 1. Zum Ziel. – Toshkent: Davr, 2014. – 168 b. 3 Abd ll S Y H li L T Zi S A H ji Sh A D t h 2 1. Мюллер В.К. Англо-русский словарь. – Москва:Русский язык, 1985 – 864 c. 2. Abdullayeva S.Ya.,.Holiyarov L.T,.Ziyayeva S.A, Hojiyeva Sh.A. Deutsch 1. Zum Ziel. – Toshkent: Davr, 2014. – 168 b. 3. Abdullayeva S.Ya.,.Holiyarov L.T,.Ziyayeva S.A, Hojiyeva Sh.A. Deutsch 2. Zum Ziel. – Toshkent: Davr, 2015. – 104 b. 1. Мюллер В.К. Англо-русский словарь. – Москва:Русский язык, 1985 – 864 c. 2. Abdullayeva S.Ya.,.Holiyarov L.T,.Ziyayeva S.A, Hojiyeva Sh.A. Deutsch 1. Zum Ziel. – Toshkent: Davr, 2014. – 168 b. 3. Abdullayeva S.Ya.,.Holiyarov L.T,.Ziyayeva S.A, Hojiyeva Sh.A. Deutsch 2. Zum Ziel. – Toshkent: Davr, 2015. – 104 b. 3. Abdullayeva S.Ya.,.Holiyarov L.T,.Ziyayeva S.A, Hojiyeva Sh.A. Deutsch 2. Zum Ziel. – Toshkent: Davr, 2015. – 104 b. Volume-4, Issue-1, February–2022 www.pedagoglar.uz 150
https://openalex.org/W2537410218
https://europepmc.org/articles/pmc5087551?pdf=render
English
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A Nanoporous Alumina Membrane Based Electrochemical Biosensor for Histamine Determination with Biofunctionalized Magnetic Nanoparticles Concentration and Signal Amplification
Sensors
2,016
cc-by
9,407
sensors sensors Weiwei Ye 1,2,*, Yifan Xu 2, Lihao Zheng 2, Yu Zhang 2, Mo Yang 3 and Peilong Sun 2 Weiwei Ye 1,2,*, Yifan Xu 2, Lihao Zheng 2, Yu Zhang 2, Mo Yang 3 and Peilong Sun 2 1 Institute of Ocean Research, Zhejiang University of Technology, Hangzhou 310014, China 2 Department of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310014, China; Lvanwork@163.com (Y.X.); zhenlihaoll.2008@163.com (L.Z.); zhangyuzjut@163.com (Y.Z.); sun_pl@zjut.edu.cn (P.S.) 3 1 Institute of Ocean Research, Zhejiang University of Technology, Hangzhou 310014, China 2 Department of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310014, China; Lvanwork@163.com (Y.X.); zhenlihaoll.2008@163.com (L.Z.); zhangyuzjut@163.com (Y.Z.); sun_pl@zjut.edu.cn (P.S.) 3 Interdisciplinary Division of Biomedical Engineering, The Hong Kong Polytechnic University, HungH K l H K Chi @ l d hk 3 Interdisciplinary Division of Biomedical Engineering, The Hong Kong Polytechnic University, Hun Kowloon, Hong Kong, China; mo.yang@polyu.edu.hk p y g g g g y y g Kowloon, Hong Kong, China; mo.yang@polyu.edu.hk * Correspondence: yeweiwei0@163.com or yeweiwei@zjut.edu.cn; Tel.: +86-571-8833-2832 Academic Editors: Anton Ficai and Alexandru Mihai Grumezescu ceived: 15 September 2016; Accepted: 17 October 2016; Published: 22 October 2016 Received: 15 September 2016; Accepted: 17 October 2016; Published: 22 October 2016 Abstract: Histamine is an indicator of food quality and indispensable in the efficient functioning of various physiological systems. Rapid and sensitive determination of histamine is urgently needed in food analysis and clinical diagnostics. Traditional histamine detection methods require qualified personnel, need complex operation processes, and are time-consuming. In this study, a biofunctionalized nanoporous alumina membrane based electrochemical biosensor with magnetic nanoparticles (MNPs) concentration and signal amplification was developed for histamine determination. Nanoporous alumina membranes were modified by anti-histamine antibody and integrated into polydimethylsiloxane (PDMS) chambers. The specific antibody modified MNPs were used to concentrate histamine from samples and transferred to the antibody modified nanoporous membrane. The MNPs conjugated to histamine were captured in the nanopores via specific reaction between histamine and anti-histamine antibody, resulting in a blocking effect that was amplified by MNPs in the nanopores. The blockage signals could be measured by electrochemical impedance spectroscopy across the nanoporous alumina membrane. The sensing platform had great sensitivity and the limit of detection (LOD) reached as low as 3 nM. This biosensor could be successfully applied for histamine determination in saury that was stored in frozen conditions for different hours, presenting a potentially novel, sensitive, and specific sensing system for food quality assessment and safety support. Keywords: nanoporous alumina membrane; histamine; magnetic nanoparticles; electrochemical biosensor Article A Nanoporous Alumina Membrane Based Electrochemical Biosensor for Histamine Determination with Biofunctionalized Magnetic Nanoparticles Concentration and Signal Amplification Weiwei Ye 1,2,*, Yifan Xu 2, Lihao Zheng 2, Yu Zhang 2, Mo Yang 3 and Peilong Sun 2 www.mdpi.com/journal/sensors 1. Introduction Histamine, one of the biogenic amines, is synthesized by basophils, mast cells, enterochromaffine cells, platelets, and histaminergic neurons in vivo. It is stored in vesicles and released on stimulation. Histamine is also formed by enzymatic decarbonylation of histidine by bacterial action with improper processing procedures or inappropriate storage conditions of food, and hence could be present in substantial amounts in fermented food stuffs and seafood, especially saury, mackerel, sardine, tuna, herring, and anchovy [1,2]. Therefore, it is often used as one of the indicators for food quality control Sensors 2016, 16, 1767; doi:10.3390/s16101767 www.mdpi.com/journal/sensors 2 of 12 Sensors 2016, 16, 1767 in the processes of production, storage, transportation, and transaction [3]. Histamine can enter the human body by dietary sources and can bind to receptors on cellular membranes in the gastrointestinal, cardiovascular, and haemotological immunological system, thereby initiating allergic reactions such as hypotension, flushing, diarrhea, vomiting, and headaches [4,5]. The symptoms may vary between individuals who are exposed to the same level of histamine in contaminated food [6]. High levels of histamine can even cause cancer [7]. Thus, rapid and sensitive determination of histamine is urgently needed in clinical and food analysis. y The current histamine detection methods include high-performance liquid chromatography (HPLC), chromatographic analysis, and capillary zone electrophoresis [8–10]. These methods for histamine analysis often require qualified personnel, extensive sample cleanup, and derivatization procedures. Immuno-enzymatic techniques such as enzyme-linked immunosorbent assay (ELISA) are usually used to perform screening for histamine poisoning, allowing a more rapid analysis method with less operation procedures being required and offering a quick screening method for industrial food quality evaluation, although the method is qualitative or semi-quantitative [11]. In recent years, miniaturized and integrated biosensors have been developed for direct, fast, specific, and sensitive histamine detection in seafood spoilage [12–15]. Various biosensors have been explored for histamine detection, such as fluorescence biosensors, molecularly imprinted polymer (MIP) histamine sensors, and electrochemical histamine sensors [16–22]. Among these methods, MIP and electrochemical histamine sensors could provide rapid histamine determination methods with low costs. MIP histamine sensors are pH dependent, limiting their wide application in clinical diagnostics and food analysis. Electrochemical biosensors are inexpensive techniques for histamine binding event detection by monitoring electrochemical spectra. Metal electrodes are used in most of the currently developed electrochemical sensing platforms. The electrode polarization effect limits the performance and application of these sensors [23]. 2.2. Biofunctionalization of Nanoporous Alumina Membrane (N S), t ic o oacetic acid (99%), sodiu yd o ide (98%) from Sigma Aldrich. Dimercaptosuccinic acid (DMSA) mo XFNANO (Nanjing China) Toluene (99 8%) anhydrous The biofunctionalization process of nanoporous alumina membranes was based on our previous study [38]. The scheme for surface modification of nanoporous alumina membrane by GPMS and anti-histamine antibody conjugation was shown in Figure 1. Nanoporous alumina membranes were kept in boiling hydrogen peroxide for 30 min to clean the membranes and hydroxyl groups formed on the nanopore surfaces. The membranes were immersed in deionized (DI) water on shakers for 15 min. The dried membranes were functionalized with the mixture of 2% GPMS and toluene at the temperature of 60 ◦C overnight, and alternatingly rinsed by toluene and anhydrous ethanol. After rinsing three times, the membranes were cured at 60 ◦C for 2 h to form a self-assembled monolayer on the nanopore surface. To immobilize anti-histamine antibody on silanized nanoporous membranes, antibody in phosphate buffer solution (PBS) buffer (pH 7.4) was injected into the nanoporous alumina membranes for coupling under 4 ◦C for about 4 h. The antibody was immobilized in the nanopores by chemical reaction between amino groups and epoxy groups. In addition, bovine serum albumin (BSA) was used as the blocking agent to reduce non-specific binding. The nanoporous alumina membranes were rinsed and then immersed in 1% BSA (w/w) in PBS (pH 8.0) for about 4 h. The biofunctionalized nanoporous membranes were then rinsed with PBS (pH 7.4) for later use. (30%) were purchased from Sigma Aldrich. All of these chemicals were used as received without further purification. 2.2. Biofunctionalization of Nanoporous Alumina Membrane The biofunctionalization process of nanoporous alumina membranes was based on our previous study [38]. The scheme for surface modification of nanoporous alumina membrane by GPMS and anti-histamine antibody conjugation was shown in Figure 1. Nanoporous alumina membranes were kept in boiling hydrogen peroxide for 30 min to clean the membranes and hydroxyl groups formed on the nanopore surfaces. The membranes were immersed in deionized (DI) water on shakers for 15 min. The dried membranes were functionalized with the mixture of 2% GPMS and toluene at the temperature of 60 °C overnight, and alternatingly rinsed by toluene and anhydrous ethanol. After rinsing three times, the membranes were cured at 60 °C for 2 h to form a self-assembled monolayer on the nanopore surface. To immobilize anti-histamine antibody on silanized nanoporous membranes, antibody in phosphate buffer solution (PBS) buffer (pH 7.4) was injected into the nanoporous alumina membranes for coupling under 4 °C for about 4 h. 1. Introduction Compared to flat substrates, nanostructured substrates with through nanopores have large surface affinity areas and allow ions to pass through. With the ability of integrating electrochemical impedance spectroscopy, nanostructured substrates have been applied for biosensing across a wide range. Nanoporous alumina membranes have become a hot research area because of their established fabrication processes, nano-ordered structures, dramatically large surface areas, easy surface modifications, high surface reaction rate, as well as their intensified output signals [24]. Wide applications of nanoporous alumina membranes are in areas of DNA detection, lipid membrane-based biosensors, foodborne pathogen biosensing, and virus and cancer cell detection [25–31]. However, the application of nanoporous alumina membranes for histamine determination has not yet been explored. Due to the easy surface modification and non-conductive frame with through nanopores, nanoporous alumina membranes are suitable candidates for constructing electrochemical sensing platforms for histamine investigation. g The sensing mechanism for histamine study is based on the blocking effect of molecules in the nanopores. However, it is quite challenging for small histamine molecules to block nanopores with large efficiency. Nanoparticles are needed as tags to increase the blocking effect and amplify signals. In our previous study, a gold nanoparticle label based sensing platform was developed for sensing DNA with great blockage efficiency and detection sensitivity [32]. Magnetic nanoparticles (MNPs) can be easily modified for biomolecule capture and concentration under magnetism field [33]. Paramagnetic microparticles and nanoparticles with nanomaghemite cores were used to off-line couple with ion exchange liquid chromatography for isolation of biogenic amines and detection of histamine in silages, respectively [34,35]. MNPs with the diameter of about 5–20 nm have the ability of entering 100 nm nanopores [36,37]. These properties make MNPs good candidates for concentrating histamine from samples to nanoporous membranes and increasing the blocking effect in the nanopores as well. In this paper, we reported the use of nanoporous alumina membranes based electrochemical biosensor with biofunctional MNP concentration for histamine determination in seafood. Nanoporous alumina membranes were functionalized with anti-histamine antibody. Biofunctional MNPs 3 of 12 Sensors 2016, 16, 1767 concentrated histamine under the external magnetic field to conjugate in the nanopores. The application of MNPs brought enhanced detection sensitivity to the electrochemical biosensor, whose output signals were measured by the change of impedance spectra. 2.2. Biofunctionalization of Nanoporous Alumina Membrane (N S), t ic o oacetic acid (99%), sodiu yd o ide (98%) from Sigma Aldrich. Dimercaptosuccinic acid (DMSA) mo XFNANO (Nanjing China) Toluene (99 8%) anhydrous The antibody was immobilized in the nanopores by chemical reaction between amino groups and epoxy groups. In addition, bovine serum albumin (BSA) was used as the blocking agent to reduce non-specific binding. The nanoporous alumina membranes were rinsed and then immersed in 1% BSA (w/w) in PBS (pH 8.0) for about 4 h. The biofunctionalized nanoporous membranes were then rinsed with PBS (pH 7.4) for later use. Figure 1. Scheme for surface modification of nanoporous alumina membrane by (3-glycidoxypropyl) trimethoxysilane (GPMS) and conjugation with anti-histamine antibody. 2.3. Magnetic Nanoparticles Functionalization Figure 1. Scheme for surface modification of nanoporous alumina membrane by (3-glycidoxypropyl) trimethoxysilane (GPMS) and conjugation with anti-histamine antibody. Figure 1. Scheme for surface modification of nanoporous alumina membrane by (3-glycidoxypropyl) trimethoxysilane (GPMS) and conjugation with anti-histamine antibody. 2 3 Magnetic Nanoparticles Functionalization Figure 1. Scheme for surface modification of nanoporous alumina membrane by (3-glycidoxypropyl) trimethoxysilane (GPMS) and conjugation with anti-histamine antibody. 1. Introduction This nanoporous alumina membrane based sensing platform with MNPs for concentrating target molecules and increasing blocking effect shows the ability for rapid and sensitive histamine determination in field. 2.1. MaterialsSen Nanoporous alumina membranes were purchased from Whatman, Inc., Maidstone, UK. The membranes were round with 13 mm diameter and were 60 µm thick. The nanopores were about 100 nm in diameter. Histamine and anti-histamine antibody produced in rabbits were purchased from Sigma Aldrich (St. Louis, MO, USA). (3-glycidoxypropyl) trimethoxysilane (GPMS, 98%), N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), trichloroacetic acid (99%), sodium hydroxide (98%), and n-butanol (99.8%) were also bought from Sigma Aldrich. Dimercaptosuccinic acid (DMSA) modified Fe3O4 MNPs were obtained from XFNANO (Nanjing, China). Toluene (99.8%), anhydrous ethanol (99%), and hydrogen peroxide (30%) were purchased from Sigma Aldrich. All of these chemicals were used as received without further purification. application of MNPs brought enhanced detection sensitivity to the electrochemical biosensor, whose output signals were measured by the change of impedance spectra. This nanoporous alumina membrane based sensing platform with MNPs for concentrating target molecules and increasing blocking effect shows the ability for rapid and sensitive histamine determination in field. 2. Materials and Methods 2.1. Materials Nanoporous alumina membranes were purchased from Whatman, Inc., Maidstone, UK. The membranes were round with 13 mm diameter and were 60 μm thick. The nanopores were about 100 nm in diameter. Histamine and anti-histamine antibody produced in rabbits were purchased from Sigma Aldrich (St. Louis, MO, USA). (3-glycidoxypropyl) trimethoxysilane (GPMS, 98%), N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide Dimercaptosuccinic acid (DMSA) mo were activated by EDC/NHS for anti-histam 2.3. Magnetic Nanoparticles Functionalization y y μ g Dimercaptosuccinic acid (DMSA) modified MNPs with an average diameter of about 10 nm were activated by EDC/NHS for anti-histamine antibody immobilization. MNPs (100 µL, 4 mg/mL) were 4 of 12 Sensors 2016, 16, 1767 contained in a tube and added with PBS (100 µL, 0.01 M, pH 6) for thorough mixing. A magnetic separator was placed under the tube which pulled MNPs to the bottom under the magnetic field. The supernatant was pipetted carefully out of the tube and nanoparticles were resuspended in PBS for rinsing. This was repeated for three times and the nanoparticles were then suspended in a 100 µL PBS (0.01 M, pH 6) solution. The rinsed DMSA modified MNPs (600 µL, 0.4 mg/mL) were activated by EDC/NHS [39]. EDC (0.26 M, 3 µL) and NHS (0.26 M, 1.5 µL) were added to MNPs (600 µL, 0.4 mg/mL) and mixed by vortex in a centrifuge tube (1.5 mL). After half an hour, the centrifuge tube was put on a magnetic separator to pull the activated nanoparticles to the bottom of the tube. The supernatant was pipetted out of the tube and the unreacted EDC/NHS was removed by magnetic field. Nanoparticles were rinsed and dispersed in PBS (600 µL, 0.01 M, pH 8.0). Antibody solution (12 µL, 0.73 mg/mL) was immediately added and mixed with the magnetic nanoparticles at room temperature for 4 h. Then, the antibody-nanoparticles conjugation was separated by magnetic field and the supernatant was pipetted out of the tube carefully. The conjugation was rinsed with PBS three times and dispersed in 1% BSA (w/w) in PBS (600 µL, 0.01 M, pH 8.0) for 4 h to block the unspecific binding sites. The biofunctionalized MNPs were rinsed with PBS (600 µL, 0.01 M, pH 7.4) and resuspended in the PBS solution for later use. 2.5. Histamine Concentration to Biofunctionalized Nanoporous Alumina Membrane Anti-histamine antibody modified MNPs (100 µL, 0.4 mg/mL) were mixed with the above histamine extracts (100 µL) for 30 min for conjugation. The MNPs-histamine conjugation was pulled to the tube bottom by magnetic field with supernatant removed and added to the antibody biofunctionalized nanoporous alumina membrane. The membrane was placed on a small permanent magnet and left for 30 min. Histamine conjugated with magnetic nanoparticles were concentrated on the membrane sensing surface and captured by the antibody. The membrane was rinsed with PBS three times to wash away the unbounded histamine-MNP conjugation. 2.4. Histamine Extraction from Saury Fish Fish samples of saury were purchased at the local market. They were cleaned with the giblets removed. Extraction was performed using 5 g of homogenized saury according to the previous study [4]. The flesh of the fish was directly weighed in 50 mL capped polyethylene centrifuge tubes and mixed with trichloroacetic acid solution (15 mL, 100 g/L). The mixture was sonicated for 30 min and filtered through a 0.45 mm membrane to obtain a 50 mL filtrate. Sodium hydroxide was added to the filtrate (2 mL) to make the solution alkaline, and n-butanol (3 mL) was also added. The mixture was mixed in a vortex mixer for 1 min. Histamine in n-butanol solution was obtained by separation. The procedure was repeated three times. The three extracts were gathered, adjusting the volume to a final 10 mL with n-butanol. The extracts in n-butanol (2 mL) were added with hydrochloric acid (3 mL, 3%) and mixed thoroughly. Histamine was extracted in hydrochloric acid by separation. The procedure was repeated five times and the extracts were gathered, adjusting the volume to a final 10 mL with hydrochloric acid (3%). Sodium hydroxide was used to adjust the pH of the extracts to pH 7.4. 2.5. Histamine Concentration to Biofunctionalized Nanoporous Alumina Membrane 3.1. Mechanism of Histamine Detection by Electrochemical Biosensor p g ( g ) 3 Results and Discussion The sensing mechanism of the proposed nanoporous alumina membrane based electrochemical biosensor for histamine sensing assisted by MNP concentration is shown in Figure 2. DMSA modified Fe3O4 MNPs were activated by EDC/NHS and modified with anti-histamine antibody. When the biofunctionalized MNPs conjugated histamine from samples, they were concentrated and separated under magnetic field. Nanoporous alumina membranes were modified by GPMS and immobilized with anti-histamine antibody. BSA was used as a blocking agent to reduce non-specific binding of biomolecules. The membrane was anchored in the PDMS chamber. The MNPs-histamine conjugation was concentrated to the nanoporous membrane and captured in nanopores by the reaction between histamine and the anti-histamine antibody (Figure 2a). MNPs-histamine conjugation caused blocking effect in the nanopores that can be measured by impedance analyzer (Figure 2b). Two platinum electrodes were immersed in electrolyte solution in the upper and lower chamber for current measurement using an electrochemical impedance analyzer. For nanoporous membranes immobilized with antibody, ions passed through nanopores and large current was detected. The capture of target histamine conjugated MNPs increased the blocking effect in the nanopores leading to the decrease of the electrolyte current and increase in the impedance signals. By recording the impedance spectra across the membranes, histamine was detected. In this system, the measured current was mainly based on electrolyte ion flow in the solution through the nanopores. The main effect of MNPs was to concentrate histamine from samples and to amplify blocking of ion flow in the nanopores. 3. Results and Discussion 3.1. Mechanism of Histamine Detection by Electrochemical Biosensor The sensing mechanism of the proposed nanoporous alumina membrane based electrochemical biosensor for histamine sensing assisted by MNP concentration is shown in Figure 2. DMSA modified Fe3O4 MNPs were activated by EDC/NHS and modified with anti-histamine antibody. When the biofunctionalized MNPs conjugated histamine from samples, they were concentrated and separated under magnetic field. Nanoporous alumina membranes were modified by GPMS and immobilized with anti-histamine antibody. BSA was used as a blocking agent to reduce non-specific binding of biomolecules. The membrane was anchored in the PDMS chamber. The MNPs-histamine conjugation was concentrated to the nanoporous membrane and captured in nanopores by the reaction between histamine and the anti-histamine antibody (Figure 2a). MNPs-histamine conjugation caused blocking effect in the nanopores that can be measured by impedance analyzer (Figure 2b). 2.7. Electrochemical Impedance Spectroscopy Measurement 2.7. Electrochemical Impedance Spectroscopy Measurement Electrochemical impedance spectroscopy was recorded by a two-electrodes system across the membrane. The polydimethylsiloxane (PDMS) chamber was fabricated by Sylgard 184 (Dow Corning, Midland, MI, USA) with the assistance of a mold. The prepared nanoporous alumina membrane was integrated into PDMS chamber, which was separated into an upper chamber and lower chamber. Two platinum (Pt) electrodes were inserted into the two chambers as the working electrode and reference electrode, respectively. The PDMS chambers were filled with PBS. The impedance measurement was performed on the electrochemical analyzer Autolab Potentiostat Galvanostat (Metroholm, Zofingen, Switzerland) with 50 mVpp voltage which was controlled by NOVA 1.10 software package (Metroholm, Zofingen, Switzerland). Sensors 2016, 16, 1767 5 of 12 2.7. Electrochemical Impedance Spectroscopy Measurement Electrochemical impedance spectroscopy was recorded by a two-electrodes system across the membrane. The polydimethylsiloxane (PDMS) chamber was fabricated by Sylgard 184 (Dow Corning, Midland, MI, USA) with the assistance of a mold. The prepared nanoporous alumina membrane was integrated into PDMS chamber, which was separated into an upper chamber and lower chamber. Two platinum (Pt) electrodes were inserted into the two chambers as the working electrode and reference electrode, respectively. The PDMS chambers were filled with PBS. The i d f d h l h i l l A l b P i 2.6. Characterization The morphology of the MNPs and antibody modified MNPs were characterized by JEOL-2100F transmission electron microscopy (TEM) equipped with an Oxford Instrument energy-dispersive X-ray (EDX) spectrometry system, operating at 200 kV. Samples (10 µL) were dropped on holey carbon coated 400 mesh copper grids and dried for TEM characterization. The hydrodynamic size and the Zeta potential were measured on a NanoBrook Omni Particle Sizer and Zeta Potential Analyzer (Brookhaven Instruments Corporation, Austin, TX, USA). The scan was conducted three times and the zeta potential values were averaged. Surface morphologies of bare nanoporous alumina membranes and functionalized nanoporous membranes with MNPs conjugation were characterized by scanning 5 of 12 Sensors 2016, 16, 1767 electron microscopy (SEM). The prepared dry samples were coated with gold for 60 s and imaged by a JEOL model JSM-6490 SEM system. 3.1. Mechanism of Histamine Detection by Electrochemical Biosensor p g ( g ) 3 Results and Discussion Two platinum electrodes were immersed in electrolyte solution in the upper and lower chamber for current measurement using an electrochemical impedance analyzer. For nanoporous membranes immobilized with antibody, ions passed through nanopores and large current was detected. The capture of target histamine conjugated MNPs increased the blocking effect in the nanopores leading to the decrease of the electrolyte current and increase in the impedance signals. By recording the impedance spectra across the membranes, histamine was detected. In this system, the measured current was mainly based on electrolyte ion flow in the solution through the nanopores. The main effect of MNPs was to concentrate histamine from samples and to amplify blocking of ion flow in the nanopores Figure 2. Schematic diagram of biofunctionalization of magnetic nanoparticles (MNPs) for histamine concentration to nanoporous alumina membrane (a) with electrochemical biosensing system for detection (b). EDC, N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride; NHS, N- Hydroxysuccinimide; BSA, bovine serum albumin; PDMS, polydimethylsiloxane. Figure 2. Schematic diagram of biofunctionalization of magnetic nanoparticles (MNPs) for histamine concentration to nanoporous alumina membrane (a) with electrochemical biosensing system for detection (b). EDC, N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride; NHS, N-Hydroxysuccinimide; BSA, bovine serum albumin; PDMS, polydimethylsiloxane. Figure 2. Schematic diagram of biofunctionalization of magnetic nanoparticles (MNPs) for histamine concentration to nanoporous alumina membrane (a) with electrochemical biosensing system for detection (b). EDC, N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride; NHS, N- Hydroxysuccinimide; BSA, bovine serum albumin; PDMS, polydimethylsiloxane. Figure 2. Schematic diagram of biofunctionalization of magnetic nanoparticles (MNPs) for histamine concentration to nanoporous alumina membrane (a) with electrochemical biosensing system for detection (b). EDC, N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride; NHS, N-Hydroxysuccinimide; BSA, bovine serum albumin; PDMS, polydimethylsiloxane. Sensors 2016, 16, 1767 Sensors 2016, 16, 1767 6 of 12 6 of 12 3.2. Characterization of Magnetic Nanoparticles and Nanoporous Alumina Membranes 3.2. Characterization of Magnetic Nanoparticles and Nanoporous Alumina Membranes DMSA modified MNPs were used to conjugate histamine molecules and con 3.2. Characterization of Magnetic Nanoparticles and Nanoporous Alumina Membranes a a e i a io of ag e i a opa i e a a opo ou A u i a e a e DMSA modified MNPs were used to conjugate histamine molecules and con DMSA modified MNPs were used to conjugate histamine molecules and concentrate histamine to the antibody modified nanoporous membrane, forming MNPs–histamine–nanoporous membrane sandwich structures in the nanopores. The size and morphology of MNPs and antibody conjugated MNPs were characterized by TEM. 3.1. Mechanism of Histamine Detection by Electrochemical Biosensor p g ( g ) 3 Results and Discussion High resolution TEM (HRTEM) showed that the average diameter of MNPs was about 10 nm dispersed in DI water (Figure 3a). A thin layer of antibody was observed around the MNPs in Figure 3b. Zeta potential measurement showed that the average potential of nanoparticles shifted from −42.7 mV to −26.6 mV after antibody conjugation, which was mainly due to the charges of the conjugated anti-histamine antibody. This further confirmed successful conjugation of antibody on DMSA modified MNPs. The average hydrodynamic size was about 15 nm. The entrance of MNPs into nanopores in the nanoporous alumina membranes was affected by nanoparticle surface interaction and confinement effect [40]. Small sizes decrease the effect and make the entrance possible. SEM was applied to confirm the conjugation between nanoparticles and nanoporous membranes. SEM images showed cross-sectional views of bare nanoporous alumina membranes and nanoporous alumina membranes with histamine-MNPs conjugation. It could be observed that no blocking in the cross-sectional view of nanopores without target capture occurred (Figure 3c). When histamine conjugated MNPs were captured on antibody modified nanoporous alumina membranes, it could be observed that nanoparticle tags were attached on nanopore walls (Figure 3d). The formation of nanoparticle tags could lead to the blockage of ion flow across nanoporous alumina membranes. DMSA modified MNPs were used to conjugate histamine molecules and concentrate histamine to the antibody modified nanoporous membrane, forming MNPs–histamine–nanoporous membrane sandwich structures in the nanopores. The size and morphology of MNPs and antibody conjugated MNPs were characterized by TEM. High resolution TEM (HRTEM) showed that the average diameter of MNPs was about 10 nm dispersed in DI water (Figure 3a). A thin layer of antibody was observed around the MNPs in Figure 3b. Zeta potential measurement showed that the average potential of nanoparticles shifted from −42.7 mV to −26.6 mV after antibody conjugation, which was mainly due to the charges of the conjugated anti-histamine antibody. This further confirmed successful conjugation of antibody on DMSA modified MNPs. The average hydrodynamic size was about 15 nm. The entrance of MNPs into nanopores in the nanoporous alumina membranes was affected by nanoparticle surface interaction and confinement effect [40]. Small sizes decrease the effect and make the entrance possible. SEM was applied to confirm the conjugation between nanoparticles and nanoporous membranes. SEM images showed cross-sectional views of bare nanoporous alumina membranes and nanoporous alumina membranes with histamine-MNPs conjugation. 3.1. Mechanism of Histamine Detection by Electrochemical Biosensor p g ( g ) 3 Results and Discussion It could be observed that no blocking in the cross-sectional view of nanopores without target capture occurred (Figure 3c). When histamine conjugated MNPs were captured on antibody modified nanoporous alumina membranes, it could be observed that nanoparticle tags were attached on nanopore walls (Figure 3d). The formation of nanoparticle tags could lead to the blockage of ion flow across nanoporous alumina membranes. Figure 3. Transmission electron microscopy (TEM) images of magnetic nanoparticles (MNPs) (a) and antibody conjugated MNPs (b); scanning electron microscopy (SEM) images of cross-sectional view of bare nanoporous alumina membranes (c) and antibody functionalized nanoporous alumina membranes after histamine-MNPs conjugation capture (d). Figure 3. Transmission electron microscopy (TEM) images of magnetic nanoparticles (MNPs) (a) and antibody conjugated MNPs (b); scanning electron microscopy (SEM) images of cross-sectional view of bare nanoporous alumina membranes (c) and antibody functionalized nanoporous alumina membranes after histamine-MNPs conjugation capture (d). Figure 3. Transmission electron microscopy (TEM) images of magnetic nanoparticles (MNPs) (a) and antibody conjugated MNPs (b); scanning electron microscopy (SEM) images of cross-sectional view of bare nanoporous alumina membranes (c) and antibody functionalized nanoporous alumina membranes after histamine-MNPs conjugation capture (d). Figure 3. Transmission electron microscopy (TEM) images of magnetic nanoparticles (MNPs) (a) and antibody conjugated MNPs (b); scanning electron microscopy (SEM) images of cross-sectional view of bare nanoporous alumina membranes (c) and antibody functionalized nanoporous alumina membranes after histamine-MNPs conjugation capture (d). 3.4. Histamine Detection without MNPs Histamine detection without MN Histamine detection without MNPs was measured by impedance spectra. Histamine with various concentrations was added into the chamber by a syringe pump and stayed for 30 min to be captured by anti-histamine antibody in the nanoporous alumina membranes. Next, PBS was added into the chamber gently to carry away the nonspecific adsorption of molecules on the nanoporous membranes three times before the impedance measurement. Figure 4a shows the impedance spectra of the biofunctionalized nanoporous alumina membrane and various concentrations of histamine capture without MNPs. Impedance increased after antibody immobilization and various concentrations of histamine capture on nanoporous alumina membranes. However, the impedance spectra had no obvious increase with the histamine concentrations from 10 µM to 100 mM. The relative impedance amplitude change is shown in Figure 4b. Compared to impedance value after antibody immobilization, impedance increased less than about 0.5% even with the large concentration of 100 mM because small histamine molecules did not produce an obvious blocking effect without the nanoparticles. y p p various concentrations was added into the chamber by a syringe pump and stayed for 30 min to be captured by anti-histamine antibody in the nanoporous alumina membranes. Next, PBS was added into the chamber gently to carry away the nonspecific adsorption of molecules on the nanoporous membranes three times before the impedance measurement. Figure 4a shows the impedance spectra of the biofunctionalized nanoporous alumina membrane and various concentrations of histamine capture without MNPs. Impedance increased after antibody immobilization and various concentrations of histamine capture on nanoporous alumina membranes. However, the impedance spectra had no obvious increase with the histamine concentrations from 10 μM to 100 mM. The relative impedance amplitude change is shown in Figure 4b. Compared to impedance value after antibody immobilization, impedance increased less than about 0.5% even with the large concentration of 100 mM because small histamine molecules did not produce an obvious blocking effect without the nanoparticles. Figure 4. (a) Impedance spectra and (b) impedance change rate of various histamine concentrations without magnetic nanoparticles (MNPs). 0 0.2 0.4 0.6 0.8 1 0.01 0.1 1 10 100 Histamine concentrations (mM) Frequency (Hz) Impedance (Ω) Impedance change rate (%) (a) (b) 0 5000 10000 15000 20000 25000 1 10 100 1000 control antibody 10 μM 100 μM 1 mM 10 mM 100 mM Figure 4. (a) Impedance spectra and (b) impedance change rate of various histamine concentrations without magnetic nanoparticles (MNPs). 3.3. Antibody Immobilization on Nanoporous Membrane f h b 3.3. Antibody Immobilization on Nanoporous Membrane Various concentrations of anti-histamine antibody were immobilized on GPMS modified nanoporous alumina membranes and the impedance spectra were recorded to monitor the blocking effect in the nanopores caused by the antibody. During the experiment, PBS was used to gently rinse off unbounded species three times before impedance measuring. Impedance observably increased Various concentrations of anti-histamine antibody were immobilized on GPMS modified nanoporous alumina membranes and the impedance spectra were recorded to monitor the blocking effect in the nanopores caused by the antibody. During the experiment, PBS was used to gently rinse 7 of 12 7 of 12 Sensors 2016, 16, 1767 Se o 2016 16 1767 off unbounded species three times before impedance measuring. Impedance observably increased after antibody was immobilized on the nanoporous alumina membranes, with the greatest impedance increase rate at the concentration of 7 µg/mL. As antibody concentration increased, the impedance increase rate experienced no obvious change. This is because the functionalized sites of membrane and nanopore walls were conjugated with antibody and no more sites for further conjugation. Therefore, the measured impedance amplitude for antibody immobilization on nanoporous membrane reached the maximum. This explained why when the concentration of antibody increased, the impedance amplitude had no further increase. Therefore, the antibody concentration of 7 µg/mL was chosen for later experiment on histamine detection. after antibody was immobilized on the nanoporous alumina membranes, with the greatest impedance increase rate at the concentration of 7 μg/mL. As antibody concentration increased, the impedance increase rate experienced no obvious change. This is because the functionalized sites of membrane and nanopore walls were conjugated with antibody and no more sites for further conjugation. Therefore, the measured impedance amplitude for antibody immobilization on nanoporous membrane reached the maximum. This explained why when the concentration of antibody increased, the impedance amplitude had no further increase. Therefore, the antibody concentration of 7 μg/mL was chosen for later experiment on histamine detection. 3 4 Histamine Detection without MNPs 3.4. Histamine Detection without MNPs Histamine detection without MN (a) (b) Figure 4. (a) Impedance spectra and (b) impedance change rate of various histamine concentrations without magnetic nanoparticles (MNPs). Figure 4. (a) Impedance spectra and (b) impedance change rate of various histamine concentrations without magnetic nanoparticles (MNPs). 3.5. The Effect of MNPs on Histamine Detection 3.5. The Effect of MNPs on Histamine Detection where Rm and Cm represent the resistance and capacitance of the nanoporous alumina membrane, respectively, f is the frequency, and Rel is the electrolyte resistance between the two platinum electrodes. Impedance changes with frequency changes. When the frequency becomes low, impedance change increases. where Rm and Cm represent the resistance and capacitance of the nanoporous alumina membrane, respectively, f is the frequency, and Rel is the electrolyte resistance between the two platinum electrodes. Impedance changes with frequency changes. When the frequency becomes low, impedance change increases. Figure 5. Impedance spectra of various histamine concentrations with MNPs in a nanoporous alumina membrane. 0 10000 20000 30000 40000 1 10 100 1000 control antibody 5 nM his-MNP 50 nM his-MNP 100 nM his-MNP 1 μM his-MNP 10 μM his-MNP 100 μM his-MNP 1 mM his-MNP 4 mM his-MNP 40 mM his-MNP Impedance (Ω) Frequency (Hz) Figure 5. Impedance spectra of various histamine concentrations with MNPs in a nanoporous alumina membrane. Figure 5. Impedance spectra of various histamine concentrations with MNPs in a nanoporous alumina membrane. Figure 5. Impedance spectra of various histamine concentrations with MNPs in a nanoporous alumina membrane. The relative impedance amplitude change rate of different histamine concentrations compared to antibody immobilized nanoporous alumina membranes is shown in Figure 6a. The change rate indicated rather good linearity with impedance increases from 5% to 33.9% for histamine concentrations from 1 μM to 40 mM. The detection sensitivity increased greatly compared to histamine detection by the biosensing system based on nanoporous alumina membrane without MNPs. This is due to the concentration of target histamine by MNPs, which also increased the blocking effect in the nanopores and therefore improved the sensitivity of detection. Impedance change with low concentrations of histamine from 5 nM to 10 μM is shown in Figure 6b. The impedance change rate had linearity with logarithmic histamine concentration shown in the inset figure of Figure 6. The limit of detection (LOD) was calculated by the control signal plus three times the noise signal (standard deviation). The LOD of this biosensor was as low as 3 nM for histamine detection. As the histamine concentrations ranged from tens of micromolars to hundreds of micromolars in rotted food at different spoilage stages, the sensing system showed the potential for sensitive histamine detection for safety support of fish and seafood products [41]. 3.5. The Effect of MNPs on Histamine Detection 3.5. The Effect of MNPs on Histamine Detection In order to increase detection sensitivity of the biosensor, anti-histamine antibody modified MNPs were used to concentrate histamine from samples. Histamine-MNPs conjugation was transferred to antibody modified nanoporous alumina membranes and remained for 30 min in order to make sure that the full reaction of histamine and antibody in nanopores occurred. Impedance spectroscopy was recorded across the nanoporous alumina membrane. Impedance increased obviously as histamine concentrations increased from 1 μM to 40 mM, with the results shown in Figure 5. The maximum impedance change appeared at a low frequency range, which could be described by an equivalent circuit model consisting of resistance in series with parallel connection of resistance and capacitance that was described in our previous study [38]. The impedance calculated In order to increase detection sensitivity of the biosensor, anti-histamine antibody modified MNPs were used to concentrate histamine from samples. Histamine-MNPs conjugation was transferred to antibody modified nanoporous alumina membranes and remained for 30 min in order to make sure that the full reaction of histamine and antibody in nanopores occurred. Impedance spectroscopy was recorded across the nanoporous alumina membrane. Impedance increased obviously as histamine concentrations increased from 1 µM to 40 mM, with the results shown in Figure 5. The maximum impedance change appeared at a low frequency range, which could be described by an equivalent circuit model consisting of resistance in series with parallel connection of resistance and capacitance 8 of 12 Sensors 2016, 16, 1767 that was described in our previous study [38]. The impedance calculated from the model could be expressed by Equation (1). R Sensors 2016, 16, 1767 8 of 12 Z = Rm j2π f RmCm + 1 + Rel, (1) Z = ோ೘ ௝ଶగ௙ோ೘஼೘ାଵ+ ܴ௘௟, (1) (1) (1) where Rm and Cm represent the resistance and capacitance of the nanoporous alumina membrane, respectively, f is the frequency, and Rel is the electrolyte resistance between the two platinum electrodes. Impedance changes with frequency changes. When the frequency becomes low, impedance change increases. where Rm and Cm represent the resistance and capacitance of the nanoporous alumina membrane, respectively, f is the frequency, and Rel is the electrolyte resistance between the two platinum electrodes. Impedance changes with frequency changes. When the frequency becomes low, impedance change increases. 3.5. The Effect of MNPs on Histamine Detection 3.5. The Effect of MNPs on Histamine Detection Histamine concentrations (Log mM) Impedance change rate (%) (a) y = 2.8298ln(x) + 22.964 R² = 0.9783 0 5 10 15 20 25 30 35 40 0.001 0.01 0.1 1 10 100 0 1 2 3 4 5 6 7 8 9 (b) Histamine concentrations (μM) Impedance change rate (%) 1 10 0.1 0.05 0.005 Histamine concentrations (Log μM) Impedance change rate (%) y = 0.908ln(x) + 5.4112 R² = 0.93 0 2 4 6 8 10 0.001 0.01 0.1 1 10 100 Histamine concentrations (Log mM) Impedance change rate (%) (a) y = 2.8298ln(x) + 22.964 R² = 0.9783 0 5 10 15 20 25 30 35 40 0.001 0.01 0.1 1 10 100 Histamine concentrations (Log mM) Impedance change rate (%) (a) y = 2.8298ln(x) + 22.964 R² = 0.9783 0 5 10 15 20 25 30 35 40 0.001 0.01 0.1 1 10 100 ( g ) 0 1 2 3 4 5 6 7 8 9 (b) Histamine concentrations (μM) Impedance change rate (%) 1 10 0.1 0.05 0.005 Histamine concentrations (Log μM) Impedance change rate (%) y = 0.908ln(x) + 5.4112 R² = 0.93 0 2 4 6 8 10 0.001 0.01 0.1 1 10 100 ( g ) 0 1 2 3 4 5 6 7 8 9 (b) Histamine concentrations (μM) Impedance change rate (%) 1 10 0.1 0.05 0.005 Histamine concentrations (Log μM) Impedance change rate (%) y = 0.908ln(x) + 5.4112 R² = 0.93 0 2 4 6 8 10 0.001 0.01 0.1 1 10 100 Histamine concentrations (μM) Histamine concentrations (μM) Figure 6. (a) Linear relationship between impedance change rate versus histamine concentrations from 1 μM to 100 mM; (b) Impedance change with histamine concentrations from 5 nM to 10 μM. Figure 6. (a) Linear relationship between impedance change rate versus histamine concentrations from 1 µM to 100 mM; (b) Impedance change with histamine concentrations from 5 nM to 10 µM. Figure 6. (a) Linear relationship between impedance change rate versus histamine concentrations from 1 μM to 100 mM; (b) Impedance change with histamine concentrations from 5 nM to 10 μM. Figure 7. Comparison of impedance change rate of tryptamine and histamine detection. 0 2 4 6 8 10 12 14 16 18 Impedance change rate (%) Tryptamine Histamine Figure 7. Comparison of impedance change rate of tryptamine and histamine detection. 3.5. The Effect of MNPs on Histamine Detection 3.5. The Effect of MNPs on Histamine Detection 9 of 12 9 of 12 9 of 12 Sensors 2016, 16, 1767 Sensors 2016, 16, 1767 Sensors 2016, 16, 1767 Figure 6. (a) Linear relationship between impedance change rate versus histamine concentrations from 1 μM to 100 mM; (b) Impedance change with histamine concentrations from 5 nM to 10 μM. Histamine concentrations (Log mM) Impedance change rate (%) (a) y = 2.8298ln(x) + 22.964 R² = 0.9783 0 5 10 15 20 25 30 35 40 0.001 0.01 0.1 1 10 100 0 1 2 3 4 5 6 7 8 9 (b) Histamine concentrations (μM) Impedance change rate (%) 1 10 0.1 0.05 0.005 Histamine concentrations (Log μM) Impedance change rate (%) y = 0.908ln(x) + 5.4112 R² = 0.93 0 2 4 6 8 10 0.001 0.01 0.1 1 10 100 Figure 6. (a) Linear relationship between impedance change rate versus histamine concentrations from 1 µM to 100 mM; (b) Impedance change with histamine concentrations from 5 nM to 10 µM. Figure 6. (a) Linear relationship between impedance change rate versus histamine concentrations from 1 μM to 100 mM; (b) Impedance change with histamine concentrations from 5 nM to 10 μM. 3.5. The Effect of MNPs on Histamine Detection 3.5. The Effect of MNPs on Histamine Detection 0 2 4 6 8 10 12 14 16 18 Impedance change rate (%) Tryptamine Histamine Figure 7. Comparison of impedance change rate of tryptamine and histamine detection. Figure 7. Comparison of impedance change rate of tryptamine and histamine detection. Figure 7. Comparison of impedance change rate of tryptamine and histamine detection. Figure 7. Comparison of impedance change rate of tryptamine and histamine detection. 3.5. The Effect of MNPs on Histamine Detection 3.5. The Effect of MNPs on Histamine Detection In order to monitor the specificity of this nanoporous alumina membrane based electrochemical The relative impedance amplitude change rate of different histamine concentrations compared to antibody immobilized nanoporous alumina membranes is shown in Figure 6a. The change rate indicated rather good linearity with impedance increases from 5% to 33.9% for histamine concentrations from 1 µM to 40 mM. The detection sensitivity increased greatly compared to histamine detection by the biosensing system based on nanoporous alumina membrane without MNPs. This is due to the concentration of target histamine by MNPs, which also increased the blocking effect in the nanopores and therefore improved the sensitivity of detection. Impedance change with low concentrations of histamine from 5 nM to 10 µM is shown in Figure 6b. The impedance change rate had linearity with logarithmic histamine concentration shown in the inset figure of Figure 6. The limit of detection (LOD) was calculated by the control signal plus three times the noise signal (standard deviation). The LOD of this biosensor was as low as 3 nM for histamine detection. As the histamine concentrations ranged from tens of micromolars to hundreds of micromolars in rotted food at different spoilage stages, the sensing system showed the potential for sensitive histamine detection for safety support of fish and seafood products [41]. In order to monitor the specificity of this nanoporous alumina membrane based electrochemical biosensor for histamine detection, tryptamine and histamine (100 μM) were measured with the same procedure. As shown in Figure 7, compared to impedance with antibody immobilization, the impedance increased about 1.3% and 15.3% for tryptamine and histamine detection, respectively. The impedance increase of target histamine capture was more than that of non-target tryptamine capture for the same concentration, which demonstrated good specificity of this nanoporous alumina membrane based electrochemical biosensor with MNPs conjugation and signal amplification for histamine detection. In order to monitor the specificity of this nanoporous alumina membrane based electrochemical biosensor for histamine detection, tryptamine and histamine (100 µM) were measured with the same procedure. As shown in Figure 7, compared to impedance with antibody immobilization, the impedance increased about 1.3% and 15.3% for tryptamine and histamine detection, respectively. The impedance increase of target histamine capture was more than that of non-target tryptamine capture for the same concentration, which demonstrated good specificity of this nanoporous alumina membrane based electrochemical biosensor with MNPs conjugation and signal amplification for histamine detection. 3.6. Histamine Determination in Saury 3.6. Histamine Determination in Saury 3.6. Histamine Determination in Saury To evaluate the ability of this electrochemistry biosensor to determine histamine concentrations in fish, histamine concentrations of saury samples that had been in frozen storage for 12 h, 24 h, and 36 h and defrosted at 25 °C were tested. Impedance spectroscopy measured in PBS solution was used To evaluate the ability of this electrochemistry biosensor to determine histamine concentrations in fish, histamine concentrations of saury samples that had been in frozen storage for 12 h, 24 h, and 36 h and defrosted at 25 °C were tested. Impedance spectroscopy measured in PBS solution was used To evaluate the ability of this electrochemistry biosensor to determine histamine concentrations in fish, histamine concentrations of saury samples that had been in frozen storage for 12 h, 24 h, and 10 of 12 Sensors 2016, 16, 1767 36 h and defrosted at 25 ◦C were tested. Impedance spectroscopy measured in PBS solution was used as the reference for comparison. Biofunctionalized MNPs were used to concentrate histamine in the extracts from the three samples, and impedance changes were recorded by the electrochemistry system. The impedance amplitude was increased by about 1.41%, 3.19%, and 8.23% for extracts from samples in frozen storage for 12 h, 24 h, and 36 h, respectively. It was calculated that the histamine concentrations were 0.1 µM, 0.73 µM, and 45.58 µM, for frozen storage in 12 h, 24 h, and 36 h respectively. This demonstrated the potential applicability of this nanoporous alumina membrane based electrochemical biosensor with biofunctional MNPs concentration for histamine determination in fish. 4. Conclusions In summary, a nanoporous alumina membrane based electrochemical biosensor with MNPs has been successfully developed for target histamine concentration and detection. Biofunctionalized MNPs conjugated with anti-histamine antibody were used to concentrate histamine from samples to an anti-histamine antibody functionalized nanoporous alumina membrane and captured in the nanopores. The blocking effect was evaluated by impedance spectra across the nanoporous alumina membrane. By quantifying the impedance change, histamine can be detected with low concentrations and the LOD reached was as low as 3 nM. Compared to histamine detection without MNPs, nanoporous alumina membrane based electrochemical biosensor with MNPs presented high sensitivity for histamine detection. This biosensor can also be successfully applied for histamine determination in saury that was stored in various conditions, showing promising applications for various food quality assessment and safety support. Acknowledgments: This work was supported by Zhejiang Provincial Natural Science Foundation of China (Grant No. LQ16C190002), Research Projects of Education Department of Zhejiang Province (Grant No. Y201534914), the National Natural Science Foundation of China (NSFC) (Grant No. 81471747) and the Center for Membrane and Water Science & Technology (CMWST) in Zhejiang University of Technology. Author Contributions: Weiwei Ye proposed the method, and carried out the experiments and data analysis. Yifan Xu, Lihao Zheng, and Yu Zhang participated in the experiments. Mo Yang evaluated the project. Weiwei Ye, Mo Yang, and Peilong Sun were involved in manuscript writing. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Al Bulushi, I.; Poole, S.; Deeth, H.C.; Dykes, G.A. Biogenic amines in fish: Roles in intoxication, spoilage, and nitrosamine formation—A review. Crit. Rev. Food Sci. Nutr. 2009, 49, 369–377. [CrossRef] [PubMed] 2. Venugopal, V. Biosensors in fish production and quality control. Biosens. Bioelectron. 2001, 17, 147–157. 1. Al Bulushi, I.; Poole, S.; Deeth, H.C.; Dykes, G.A. Biogenic amines in fish: Roles in intoxication, spoilage, and nitrosamine formation—A review. Crit. Rev. Food Sci. Nutr. 2009, 49, 369–377. [CrossRef] [PubMed] . Venugopal, V. 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Nonlinear amplitude dynamics in flagellar beating
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Oriola, D., Gadêlha, H., & Casademunt, J. (2017). Nonlinear amplitude dynamics in flagellar beating. Royal Society Open Science, 4(3). https://doi.org/10.1098/rsos.160698 Oriola, D., Gadêlha, H., & Casademunt, J. (2017). Nonlinear amplitude dynamics in flagellar beating. Royal Society Open Science, 4(3). https://doi.org/10.1098/rsos.160698 Oriola, D., Gadêlha, H., & Casademunt, J. (2017). Nonlinear amplitude dynamics in flagellar beating. Royal Society Open Science, 4(3). https://doi.org/10.1098/rsos.160698 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1098/rsos.160698 Link to publication record on the Bristol Research Portal PDF-document This is the final published version of the article (version of record). It first appeared online via The Royal Society at https://doi.org/10.1098/rsos.160698 . Please refer to any applicable terms of use of the publisher. University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ Author for correspondence: David Oriola e-mail: oriola@mpi-cbg.de; oriola@pks.mpg.de †Present address: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstraße 108, 01307 and Max Planck Institute for the Physics of Complex Systems, Nöthnitzerstraße 38, 01187 Dresden, Germany. 1. Introduction Cilia and flagella play a crucial role in the survival, development, cell feeding and reproduction of micro-organisms [1]. These lash- like appendages follow regular beating patterns which enable cell swimming in inertialess fluids [2]. Bending deformations of the flagellum are driven by the collective action of Electronic supplementary material is available online at https://dx.doi.org/10.6084/m9. figshare.c.3694462. 2017 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. David Oriola1,†, Hermes Gadêlha2,3 and Jaume Casademunt1 David Oriola1,†, Hermes Gadêlha2,3 and Jaume Casademunt1 Keywords: Author for correspondence: David Oriola e-mail: oriola@mpi-cbg.de; oriola@pks.mpg.de Casademunt1 1Departament de Física de la Matèria Condensada, Facultat de Física, Universitat de Barcelona, Avinguda Diagonal 647, 08028 Barcelona, Spain 2Department of Mathematics, University of York, York YO10 5DD, UK 3Wolfson Centre for Mathematical Biology, Mathematical Institute, University of Oxford, Oxford OX2 6GG, UK DO, 0000-0002-8356-7832; HG, 0000-0001-8053-9249 Received: 14 September 2016 Accepted: 2 February 2017 The physical basis of flagellar and ciliary beating is a major problem in biology which is still far from completely understood. The fundamental cytoskeleton structure of cilia and flagella is the axoneme, a cylindrical array of microtubule doublets connected by passive cross-linkers and dynein motor proteins. The complex interplay of these elements leads to the generation of self-organized bending waves. Although many mathematical models have been proposed to understand this process, few attempts have been made to assess the role of dyneins on the nonlinear nature of the axoneme. Here, we investigate the nonlinear dynamics of flagella by considering an axonemal sliding control mechanism for dynein activity. This approach unveils the nonlinear selection of the oscillation amplitudes, which are typically either missed or prescribed in mathematical models. The explicit set of nonlinear equations are derived and solved numerically. Our analysis reveals the spatio-temporal dynamics of dynein populations and flagellum shape for different regimes of motor activity, medium viscosity and flagellum elasticity. Unstable modes saturate via the coupling of dynein kinetics and flagellum shape without the need of invoking a nonlinear axonemal response. Hence, our work reveals a novel mechanism for the saturation of unstable modes in axonemal beating. Subject Category: Biochemistry and biophysics Subject Areas: biophysics/computational physics Keywords: flagellar beating, dynein, spermatozoa, self-organization 2 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Many questions still remain unanswered on how dynein-driven sliding causes the oscillatory bending of cilia and flagella [7]. Over the last half a century, intensive experimental and theoretical work has been done to understand the underlying mechanisms of dynein coordination in axonemal beating. Different mathematical models have been proposed to explain how sliding forces shape the flagellar beat [8–16]. Coordinated beating has been hypothesized considering different mechanisms such as dynein’s activity regulation through local axonemal curvature [9–11,16], owing to the presence of a transverse force (t-force) acting on the axoneme [12] and by shear displacements [13–15]. Other studies also examined the dynamics of flagellar beating by prescribing its internal activity [17,18] or by considering a self-organized mechanism independent of the specific molecular details underlying the collective action of dyneins [13,14,19]. In particular, the latter approach, although general from a physics perspective, does not explicitly incorporate dynein kinetics along the flagellum, which has been shown to be crucial in order to understand experimental observations on sperm flagella [20–22]. Load- accelerated dissociation of dynein motors was proposed as a mechanism for axonemal sliding control, and was successfully used to infer the mechanical properties of motors from bull sperm flagella [15]. In contrast, dynamic curvature regulation has been recently proposed to account for Chlamydomonas flagellar beating [16]. In the previous studies, linearized solutions of the models were fit to experimental data; however, it is not clear that such results still hold at the nonlinear level. Recent studies also investigated the emergence and saturation of unstable modes for different dynein control models [23,24]; however, saturation of such unstable modes was not self-regulated, but achieved via the addition of a nonlinear elastic contribution in the flagellum constitutive relation. Nevertheless, predictions on how dynein activity influences the selection of the beating frequency, amplitude and shape of the flagellum remain elusive. Here, we provide a microscopic bottom-up approach and consider the intrinsic nonlinearities arising from the coupling between dynein activity and flagellar shape, regarding the eukaryotic flagellum as a generalized Euler-elastica filament bundle [25]. This allows a close inspection on the onset of the flagellar bending wave instability, its transient dynamics and later saturation of unstable modes, which is solely driven by the nonlinear interplay between the flagellar shape and dynein kinetics. We first derive the governing nonlinear equations using a load-accelerated feedback mechanism for dynein along the flagellum. 2 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The linear stability analysis is presented, and eigenmode solutions are obtained similarly to references [15,24], to allow analytical progress and pedagogical understanding. The nonlinear dynamics far from the Hopf bifurcation is studied numerically and the resulting flagellar shapes are further analysed using principal component analysis [26,27]. Finally, bending initiation and transient dynamics are studied subject to different initial conditions. ATP-powered dynein motor proteins, which generate sliding forces within the flagellar cytoskeleton, named axoneme [3]. This structure has a characteristic ‘9 + 2’ composition across several eukaryotic organisms, corresponding to nine peripheral microtubule doublets in a cylindrical arrangement surrounding a central pair of microtubules [1]. Additional proteins, such as the radial spokes and nexin cross-linkers, connect the central to the peripheral microtubules and resist free sliding between the microtubule doublets, respectively. Each doublet consists of an A-microtubule in which dyneins are anchored at regular intervals along the length of the doublets, and a B-microtubule, where dynein heads bind in the neighbouring doublet [1,4]. In the presence of ATP, dyneins drive the sliding of neighbouring microtubule doublets, generating forces that can slide doublets apart if cross-linkers are removed [5]. In the presence of cross-linkers, sliding is transformed into bending. Remarkably, this process seems to be carried out in a highly coordinated manner, in such a way that when one team of dyneins in the axoneme is active, the other team remains inactive [6]. This mechanism leads to the propagation of bending undulations along the flagellum, as commonly observed during the movement of spermatozoa [4]. ATP-powered dynein motor proteins, which generate sliding forces within the flagellar cytoskeleton, named axoneme [3]. This structure has a characteristic ‘9 + 2’ composition across several eukaryotic organisms, corresponding to nine peripheral microtubule doublets in a cylindrical arrangement surrounding a central pair of microtubules [1]. Additional proteins, such as the radial spokes and nexin cross-linkers, connect the central to the peripheral microtubules and resist free sliding between the microtubule doublets, respectively. Each doublet consists of an A-microtubule in which dyneins are anchored at regular intervals along the length of the doublets, and a B-microtubule, where dynein heads bind in the neighbouring doublet [1,4]. In the presence of ATP, dyneins drive the sliding of neighbouring microtubule doublets, generating forces that can slide doublets apart if cross-linkers are removed [5]. In the presence of cross-linkers, sliding is transformed into bending. Remarkably, this process seems to be carried out in a highly coordinated manner, in such a way that when one team of dyneins in the axoneme is active, the other team remains inactive [6]. This mechanism leads to the propagation of bending undulations along the flagellum, as commonly observed during the movement of spermatozoa [4]. 2 2. Continuum flagella equations The presence of nexin cross-linkers and dynein motors generates a total force density f(s) along the bundle. Figure1. Schematicviewofthesystem.(a)Two-dimensionalrepresentationofaflagellum,wherethecentreliner(s)andtangentangle φ(s)oftheflagellumareparametrizedbythearclengthparameters.(b)Passive(springs)andactive(dyneinmotors)internalstructures intheaxoneme.Dyneinsinthe(+)and(−)filamentscompeteinatug-of-warandbind/unbindfromfilamentswithratesπ± andε±, respectively. (c) The flagellum as a two-filament bundle: two polar filaments are separated by a small gap of size b ≪L. The presence of nexin cross-linkers and dynein motors generates a total force density f(s) along the bundle. upper and lower filaments, respectively (figure 1c). The shape of the bundle is given at any time by the expression: r(s, t) = r(0, t) +  s 0 (cos φ, sin φ) ds′ (2.1) (2.1) The geometrical constraint of the filament bundle induces an arc length mismatch, (s, t), denoted as sliding displacement: The geometrical constraint of the filament bundle induces an arc length mismatch, (s, t), denoted as sliding displacement: (s, t) =  s 0 (|∂sr−| −|∂sr+|) ds′ = b(φ −φ0), (2.2) (2.2) where φ0 ≡φ(0, t). For simplicity, we have set any arc length incongruity between the two filaments at the base to zero, and we consider the filaments clamped at the base. A similar approach can be used to include basal compliance and other types of boundary conditions at the base (e.g. pivoting or free swimming head). Here we centre our study on the nonlinear action of motors along the flagellum. We aim to study the active and passive forces generated at each point along the arc length of the filament bundle. We define f(s, t) = f(s, t)ˆs as the total internal force density generated at s at time t on the plus- filament owing to the action of active and passive forces (figure 1). By virtue of the action–reaction law, the minus-filament will experience a force density −f at the same point. Next, consider that N dyneins are anchored at each polar filament in a region lc around s, where lc is much smaller than the length of the flagellum L and much larger than the length of the regular intervals at which dyneins are attached along the microtubule doublets. We shall call lc the ‘tug-of-war’ length. We define n±(s, t) as the number of bound dyneins in a region of size lc around s at time t which are anchored in the plus- or minus-filament, respectively. We consider a tug-of-war at each point s along the flagellum with two antagonistic groups of N dyneins. 2. Continuum flagella equations We consider a filament bundle composed of two polar filaments subjected to planar deformations. Each filament is modelled as an inextensible, unshearable, homogeneous elastic rod, for which the bending moment is proportional to the curvature and the Young modulus is E. The filaments are of length L and separated by a constant gap of size b, where b ≪L (figure 1c). We define a material curve describing the shape of the filament bundle centreline as r(s, t). The positions of each polar filament forming the bundle read r± = r ± (b/2)ˆn, with the orientation of the cross section at distance s along its length defined by the normal vector to the centreline ˆn = −sin φ ˆı + cos φ ˆj, being φ ≡φ(s, t) the angle between the tangent vector ˆs ≡∂sr ≡rs and the ˆı direction (taken along the x-axis). The subscripts (+) and (−) refer to the 3 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . f −f f (s) r (s) y o x s = L s = 0 e– p– p+ e+ r+ r– r+ r– r (s) sˆ nˆ f bf b (a) (b) (c) Figure1. Schematicviewofthesystem.(a)Two-dimensionalrepresentationofaflagellum,wherethecentreliner(s)andtangentangle φ(s)oftheflagellumareparametrizedbythearclengthparameters.(b)Passive(springs)andactive(dyneinmotors)internalstructures intheaxoneme.Dyneinsinthe(+)and(−)filamentscompeteinatug-of-warandbind/unbindfromfilamentswithratesπ± andε±, respectively. (c) The flagellum as a two-filament bundle: two polar filaments are separated by a small gap of size b ≪L. The presence of nexin cross-linkers and dynein motors generates a total force density f(s) along the bundle. f −f f (s) r (s) y o x s = L s = 0 e– p– p+ e+ r+ r– r+ r– r (s) sˆ nˆ f bf b (a) (b) (c) 3 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 r+ (a) r– Figure1. Schematicviewofthesystem.(a)Two-dimensionalrepresentationofaflagellum,wherethecentreliner(s)andtangentangle φ(s)oftheflagellumareparametrizedbythearclengthparameters.(b)Passive(springs)andactive(dyneinmotors)internalstructures intheaxoneme.Dyneinsinthe(+)and(−)filamentscompeteinatug-of-warandbind/unbindfromfilamentswithratesπ± andε±, respectively. (c) The flagellum as a two-filament bundle: two polar filaments are separated by a small gap of size b ≪L. 4 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M(s, t) = Ebφs −bF, (2.4) (2.4) where F(s, t) = L s f(s′, t) ds′, provided that ∂sφ± ≈∂sφ for bundles characterized by b ≪L. The combined bending stiffness of the filament bundle is given by Eb = 2EI, where I is the second moment of the area of the external rods. Dynein kinetics is modelled by using a minimal two-state mechanochemical model with states k = 1, 2, corresponding to microtubule bound or unbound dyneins, respectively. Because the sum of bound and unbound motors at s remains constant at all times, we study only the plus and minus bound motor distributions n±(s, t). Dyneins bind with rates π± and unbind with rates ε± (figure 1b). The corresponding bound motor population dynamics reads ∂tn± = π± −ε±. (2.5) (2.5) The binding/unbinding rates are given by π± = π0(N −n±), ε± = ε0n± exp(±F±/fc) where ε0 and π0 are constant rates and fc is the characteristic unbinding force. Motivated by previous studies on the collective action of molecular motors [15,28], we assume an exponential dependence of the unbinding force on the resulting load. By considering that dyneins fulfil a linear velocity–force relationship with stall force f0 and velocity at zero load v0, the loads are given by F±(s, t) = ±f0(1 ∓t/v0). The stall force f0 is defined as the absolute value of the load a motor experiences at stall (t = 0). Substituting the different definitions, the internal force density f(s, t) reads f(s, t) = f0ρ  ¯n −n′t v0  −K, (2.6) (2.6) where ¯n ≡n+ −n−and n′ ≡n+ + n−. For simplicity, we derive the equations governing the tangent angle φ in the limit of small curvature (but possibly large amplitudes) such that tangential forces can be neglected. The derivation for arbitrary large curvature is also presented in the electronic supplementary material. Using resistive force theory in the limit of small curvature, we consider only normal forces along the flagellum obtaining ζ⊥φt = −Msss, where ζ⊥is the normal drag coefficient [8,29]. Combining the last expression with equation (2.4), we have ζ⊥φt = −Ebφssss −bfss. (2.7) (2.7) Hereinafter, we switch to dimensionless quantities while keeping the same notation. 2. Continuum flagella equations The elastic sliding resistance between the two polar filaments exerted by nexin cross-linkers is assumed to be Hookean with an elastic modulus K. Thus, the internal force density f(s, t) reads where φ0 ≡φ(0, t). For simplicity, we have set any arc length incongruity between the two filaments at the base to zero, and we consider the filaments clamped at the base. A similar approach can be used to include basal compliance and other types of boundary conditions at the base (e.g. pivoting or free swimming head). Here we centre our study on the nonlinear action of motors along the flagellum. We aim to study the active and passive forces generated at each point along the arc length of the filament bundle. We define f(s, t) = f(s, t)ˆs as the total internal force density generated at s at time t on the plus- filament owing to the action of active and passive forces (figure 1). By virtue of the action–reaction law, the minus-filament will experience a force density −f at the same point. Next, consider that N dyneins are anchored at each polar filament in a region lc around s, where lc is much smaller than the length of the flagellum L and much larger than the length of the regular intervals at which dyneins are attached along the microtubule doublets. We shall call lc the ‘tug-of-war’ length. We define n±(s, t) as the number of bound dyneins in a region of size lc around s at time t which are anchored in the plus- or minus-filament, respectively. We consider a tug-of-war at each point s along the flagellum with two antagonistic groups of N dyneins. The elastic sliding resistance between the two polar filaments exerted by nexin cross-linkers is assumed to be Hookean with an elastic modulus K. Thus, the internal force density f(s, t) reads (2.3) f(s, t) = ρ(n+F+ + n−F−) −K, (2.3) where ρ ≡l−1 c is the density of tug-of-war units along the flagellum and F±(s, t) is the load per motor each group of dyneins experiences owing to the action of the antagonistic group. The stresses on the filament bundle are given by a resultant contact force N(s, t) and resultant contact moment M(s, t) acting at the point r(s, t). 2. Continuum flagella equations The internal force density, f(s, t), contributes only to the internal moment of the bundle M(s, t) = Mˆk, where ˆk = ˆj × ˆı, such that M(s, t) reads 4 3. Parameter values We present the choice of parameters based on experimental studies on sperm flagella and on the green algae Chlamydomonas. We first discuss the passive properties of a flagellum. The typical length of a human flagellum is L ≃50 µm, and the axonemal diameter is found to be b ≃200 nm [4]. The bending stiffness of the filament bundle has been reported to be Eb ≃0.9 × 10−21 N m2 for sea-urchin sperm [4,25] and Eb ≃1.7 × 10−21 N m2 for bull sperm [15]. On the other hand, the interdoublet elastic resistance from demembranated flagellar axonemes of Chlamydomonas yields an estimated spring constant 2 × 10−3 N m−1 for 1 µm of axoneme [30], thus K ≃2 × 103 N m−2. Finally, typical medium viscosities for sperm flagella range from ζ⊥≃10−3 Pa s in low viscous media to ζ⊥≃1 Pa s in high viscous media [4,18]. spe ge ge o ζ⊥ 0 s o sco s e o ζ⊥ s g sco s e [ , 8] Next, we discuss the mechanochemical parameters associated with axonemal dynein. Axonemal dynein are subdivided into inner and outer arms depending on its position in the axoneme, and can be found in heterodimeric and monomeric forms [22]. For the sake of simplicity, we consider identical force generating dynein motor domains acting along the flagellum. The total number of motor domains in a beating flagellum has been estimated to be ≃105 [31,32]. The stall force has been found in the range f0 ≃1−5 pN [33,34]. Following reference [15], we choose the characteristic unbinding force for dynein such that ¯f = 2. Axonemal dynein is characterized by a low duty ratio estimated to be η ≃0.14 and speeds at zero load in the range v0 ≃5–7 µm s−1 [33,35]. The characteristic time scale of dynein kinetics sets the order of magnitude of the beating frequency in our model. We choose an estimated value of τ0 ≃50 ms [24,35], which corresponds to a frequency of ≃10 Hz, comparable to the case of human sperm [4]. Finally, we need to estimate ρ and N. Considering the length of the human sperm flagellum (L ≃50 µm), we obtain ≃2 × 103 motors µm−1. In our description, we divide the axoneme into two regions with corresponding dynein teams. Therefore, we have ρN ≃103 motors µm−1. 3. Parameter values In order to find ρ, we need to choose a criterion to decide the typical length scale or ‘tug-of-war’ length lc = ρ−1 in our coarse-grained description. The typical length scale in the system is given by lc ∼L/√μa =  Eb/bf0ρN (see Linear stability analysis). Using the previous parameters, we get lc ∼1 µm and therefore ρ ∼1 µm−1 and N ∼103. Hence, we obtain Sp ≃5−20, μ ≃50−100, μa ∼103 and ζ ∼1. The motor activity is studied in a broad range μa ≃(2−6) × 103 because it plays the role of the main control parameter in our study. rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . where η ≡π0/(π0 + ε0) is the duty ratio of the motors and ¯f ≡f0/fc dictates the sensitivity of the unbinding rate on the load. In the electronic supplementary material, we include a table with a summary of all the variables and parameters in the model with their corresponding symbols. where η ≡π0/(π0 + ε0) is the duty ratio of the motors and ¯f ≡f0/fc dictates the sensitivity of the unbinding rate on the load. In the electronic supplementary material, we include a table with a summary of all the variables and parameters in the model with their corresponding symbols. 4 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . We non- dimensionalize the arc length with respect to the length scale L, time with respect to the correlation time of the system τ0 = 1/(ε0 + π0), motor number with respect to N, internal force density with respect to f0ρN and sliding displacement with respect to b. The correlation time defines how fast the motors will respond to a change in load. We also define Sp = L(ζ⊥/Ebτ0)1/4, μ ≡Kb2L2/Eb, μa = bf0ρNL2/Eb and ζ ≡b/v0τ0. The sperm number, Sp, characterizes the relative importance of bending forces to viscous drag. The parameter μ measures the relative importance of the sliding resistance compared with the bending stiffness [25]. On the other hand, the parameter μa denotes the activity of dyneins, measuring the relative importance of motor force generation compared with the bending stiffness of the bundle. Finally, ζ denotes the ratio of the bundle diameter and the characteristic shear induced by the motors. The dimensionless sperm equation in the limit of small curvature reads [2,13,18] Sp4φt = −φssss −μa fss, (2.8) (2.8) Sp4φt = −φssss −μa fss, here, in our case, the dimensionless internal force density f(s, t) takes the form: where, in our case, the dimensionless internal force density f(s, t) takes the form: f(s, t) = ¯n −ζn′t −μ μa  (2.9) (2.9) and  = φ −φ0. Because the flagellar base is clamped, without loss of generality, we set φ0 = 0. Combining equations (2.8) and (2.9), we obtain the nonlinear dynamics for the tangent angle: Sp4φt = −φssss + μφss −μa ¯nss + μaζ[n′ ssφt + 2n′ sφts + n′φtss]. (2.10) (2.10) Sp4φt = −φssss + μφss −μa ¯nss + μaζ[n′ ssφt + 2n′ sφts + n′φtss]. (2.10) In the absence of dynein activity, expression (2.10) reduces to the dynamics of an elastic filament bundle with sliding resistance forces [25]. Note that this expression is obtained considering the sliding mechanism and a linear velocity–force relationship for dyneins, but it is independent of dynein kinetics. On the other hand, the dimensionless form of the bound motor population dynamics n± reads 5 5 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Linear stability analysis In this section, we perform a linear stability analysis of equations (2.10) and (2.11). The non-moving state is characterized by φ = 0 and n± = n0 ≡π0/(π0 + ε0 e ¯f ). This means that the flagellum is aligned with respect to the x-axis, and the number of plus and minus bound motors is constant in space and time. For the linear stability analysis, we consider the perturbed variables around the base state as φ = δφ and n± = n0 + δn±. Introducing the modulation δn ≡δn+ = −δn−around n0 and considering ¯fζφt ≪1, we obtain δnt = −¯τ −1δn + (1 −η)ζ ¯f e¯f n0φt (4.1) δnt = −¯τ −1δn + (1 −η)ζ ¯f e¯f n0φt (4.1) 4 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2.11) ∂tn± = η(1 −n±) −(1 −η)n± exp[¯f(1 ∓ζφt)], (2.11) ∂tn± = η(1 −n±) −(1 −η)n± exp[¯f(1 ∓ζφt)], (4.5) where qj, ˜Φj ∈C. Once ˜φ is known, δ ˜n(s) = δN ˜φ(s) exp(iθ), where δN = |χ′| and θ = arg(χ′). Therefore, the evolution of δn is the same as for φ except for a phase shift θ and an overall change in the amplitude δN, which depends on χ′(σ). This result indicates the presence of a time delay between the action of motors and the response of the flagellum. Time delays commonly arise in systems where molecular motors work collectively [36]. Indeed, the regulation of active forces by the time delay of the curvature was proposed as a mechanism to generate travelling bending waves [9]. In order to find ˜φ(s), we need to impose the four boundary conditions, obtaining a linear system of equations for ˜Φj, j = 1, . . . , 4. The procedure to obtain the set of boundary conditions for a clamped head is detailed in the electronic supplementary material. The boundary conditions read ˜φ(0) = 0, ˜φsss(0) = −¯χ ˜φs(0), ˜φs(1) = 0 and ˜φss(1) = −¯χ ˜φ(1). By setting the determinant of the system to zero, we find the set of complex eigenvalues σn, with the corresponding growth rates λn = Re[σn] and frequencies ωn = Im[σn], which satisfy the boundary conditions, where λn, ωn ∈R. We order the set of different eigenvalues according to its growth rate λn+1 < λn, such that the first one has the largest growth rate λ1. Defining u = (φ, δn)T, the general solution of the system reads u(s, t) =  n An  ˜φn δ ˜nn eλnt eiωnt + c.c, (4.6) (4.6) where An ∈C are free amplitude parameters. For λn < 0, ∀n, solutions decay exponentially to the non- moving state. On the other hand, when λ1 becomes positive, in the range of parameters studied, the system undergoes a Hopf bifurcation and solutions follow an exponential growth, oscillating with frequency ω1. Next, we study the marginal stable solutions, i.e. when the maximum growth rate equals zero (λ1 = 0). For this, we define the critical frequency of oscillation as ωc ≡|ω1|. Travelling waves propagate from tip to base, a feature already reported for the clamped-type boundary condition [13, 14,24]. In figure 2c, the marginal stability curve in phase space is shown. (4.5) Intuitively, as Sp is increased the travelling instability occurs for higher motor activity μa and the critical frequency of oscillation ωc follows a non-monotonic decrease (see electronic supplementary material, figure S1). For low viscosity (Sp = 5), the wave propagation velocity is slightly oscillatory, whereas for high viscosity (Sp = 10), it becomes more uniform (figure 2a,b, bottom panels). These results are in agreement with studies on migrating human sperm, where in the limit of high viscosity waves propagated approximately at constant speed [37]. For high viscosity, curvature tends to increase from base to tip, finally dropping to zero owing to the zero curvature boundary condition at the tail (figure 2d and electronic supplementary material). This modulation is consistent with experimental studies on human sperm, which show viscosity modulation of the bending amplitude [37]. In the latter study, however, the effect is more pronounced possibly owing to external elastic reinforcing structures found along the flagellum of mammalian species, as well as other nonlinear viscoelastic effects. Defining k ≡max |qi|/2π as the characteristic wavenumber, we obtain that it increases almost linearly with Sp (figure 2d, inset). Similar results can be obtained using other definitions for k, for example using the covariance matrix (see Principal component analysis section). and Sp4φt = −φssss + μφss + 2μa[ζn0φtss −δnss], (4.2) (4.2) where ¯τ ≡n0/η. We use the ansatzes φ = ˜φ(s) eσt + c.c and δn = δ ˜n(s) eσt + c.c, where σ is a complex eigenvalue and c.c accounts for complex conjugate. From equation (4.1), we get δ ˜n = χ′(σ) ˜φ, where χ′(σ) is a complex response function: χ′(σ) = ζ ¯fn0(1 −n0) σ 1 + σ ¯τ (4.3) (4.3) Using equation (2.9) and considering f = ˜f(s) eσt + c.c, we obtain ˜f = χ(σ) ˜φ, where χ(σ) is a second complex response function: χ(σ) = 2ζn0  ¯f(1 −n0) σ −σ 2 ¯τ 1 −(σ ¯τ)2 −σ  −μ μa (4.4) (4.4) The latter response functions generalize the work in reference [15] for a complex eigenvalue σ and are equivalent to results presented in reference [24]. With the ansatz ˜φ ∼δ ˜n ∼eiqs in equation (4.2), we obtain the characteristic equation q4 −¯χq2 + ¯σ = 0, where ¯χ ≡μaχ, ¯σ ≡σSp4 and ¯χ, ¯σ ∈C. Solving the characteristic equation, we obtain four possible roots qi, i = 1, . . . , 4 and the eigenfunctions read 6 6 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ˜φ(s) = 4  j=1 ˜Φj eiqjs, (4.5) ˜φ(s) = 4  j=1 ˜Φj eiqjs, (4.5) 5. Nonlinear flagellar dynamics In this section, we study the nonlinear dynamics of the flagellum in the limit of small curvature by numerically solving equations (2.10) and (2.11) using a second-order accurate implicit-explicit numerical scheme (see electronic supplementary material). The unstable modes presented in §4 follow an initial exponential growth and eventually saturate at the steady state owing to the nonlinearities in the system. In figure 3, two different saturated amplitude solutions are shown. Figure 3a (left) corresponds to a case where the system is found close to the Hopf bifurcation, whereas figure 3a (right) corresponds to a regime far from the bifurcation. We note that the marginal solution obtained in the linear stability analysis (figure 2b, top panel) gives a very good estimate of the nonlinear profile close to the bifurcation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0.2 0.4 0.6 0.8 1.0 1 2 3 4 5 6 8 10 12 1 2 3 1 2 3 4 t/T t/T 5 6 –1.0 1 0 1 0 0 f f 0.1 1 2 3 4 5 6 –1.0 0 0.1 Sp = 5 Sp = 10 6 8 10 12 1 2 3 4 5 6 no instability travelling waves Sp |fs| Sp k ×103 (a) (b) ma (a) (b) (c) (d) Figure 2. Linear stability analysis. (a,b) (top panels) Clamped head profile solutions corresponding to the marginal stability case (i.e. λ1 = 0)forSp = 5, 10.Thebeatingcyclesaredividedinto10frames.(Bottompanels)TangentanglekymographswhereT = 2π/ωc is the period. Amplitudes and angles are shown in arbitrary units. (c) Marginal stability curve for the clamped condition. Points (a) and (b) inparameterspacecorrespondtotheprofilesin(a)and(b).(d)CurvaturemodulationsasafunctionofthearclengthforSp = 5(dashed line)andSp = 12(solidline).Inset:wavenumberdefinedask ≡max |qi|/2π asafunctionofSp.μ = 50,ζ = 0.4,η = 0.14, ¯f = 2. t/T 1 0 f 1 2 3 4 5 6 –1.0 0 0.1 Sp = 10 (b) 7 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Nonlinear flagellar dynamics 1 2 3 4 t/T 5 6 –1.0 1 0 0 f 0.1 Sp = 5 (a) 7 hing.org R.Soc. opensci. 4: 160698 6 8 10 12 1 2 3 4 5 6 no instability travelling waves Sp ×103 (a) (b) ma (c) 0 0.2 0.4 0.6 0.8 1.0 1 2 3 4 5 6 8 10 12 1 2 3 |fs| Sp k (d) (c) (d) Figure 2. Linear stability analysis. (a,b) (top panels) Clamped head profile solutions corresponding to the marginal stability case (i.e. λ1 = 0)forSp = 5, 10.Thebeatingcyclesaredividedinto10frames.(Bottompanels)TangentanglekymographswhereT = 2π/ωc is the period. Amplitudes and angles are shown in arbitrary units. (c) Marginal stability curve for the clamped condition. Points (a) and (b) inparameterspacecorrespondtotheprofilesin(a)and(b).(d)CurvaturemodulationsasafunctionofthearclengthforSp = 5(dashed line)andSp = 12(solidline).Inset:wavenumberdefinedask ≡max |qi|/2π asafunctionofSp.μ = 50,ζ = 0.4,η = 0.14, ¯f = 2. point, although it does not provide the magnitude of φ or δn. Frequencies are approximately 10 Hz and maximum amplitudes are found to be small, around ≃4% of the total flagellum length. However, the oscillation amplitude for high motor activity is more than double in respect to the case of low activity (figure 3a). The colour code in figure 3a indicates the value of the semi-difference of plus- and minus- bound motors δn. Plus-bound motors are predominant in regions of positive curvature (φs > 0) along the flagellum and vice versa. Despite the low duty ratio of dynein motors [35], approximately 2% bound dyneins along the flagellum are sufficient to produce micrometre-sized amplitude oscillations. The full flagella dynamics corresponding to figure 3a are provided in the supplementary material, movies S1 and S2. 3 In figure 3b, the time evolution of φ and δn is shown at s = 3 4 for the cases in figure 3a, respectively. As mentioned in §4, the tangent angle φ is delayed with respect to δn, and the time delay is not considerably affected by motor activity. Close to the instability threshold, both signals are very similar, because the system is found near the linear regime; however, far from threshold, both signals greatly differ. For high motor activity, both the tangent angle and the fraction of bound dyneins at certain points along the flagellum exhibit cusp-like oscillations (figure 3b, right). This behaviour is typical of molecular motor assemblies working far from the instability threshold [38]. Despite the signals S in figure 3b 8 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . 5. Nonlinear flagellar dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0.2 0.4 0 0.05 0.10 0.15 |fmax(1/2)| 0 0.2 0.4 2.5 3.0 3.5 100 200 300 400 500 –0.2 0 0.2 –1 0 1 200 300 400 500 –0.1 0 0.1 0 5 –5 ×10–3 ×10–2 0 20 40 0 20 40 –4 –2 0 2 4 –0.02 –0.01 0 0.01 0.02 –0.5 –1.0 0 0.5 1.0 –0.01 0 0.01 t (ms) t (ms) ma= 5600 Y (mm) Y (mm) X (mm) X (mm) ma= 4300 dn (3/4) f (3/4) e e w dn dn (a) (b) (c) ure 3. Nonlinear analysis. (a) Nonlinear flagella steady-state profiles for μa = 4300 (left) and μa = 5600 (right), considering the pectiveeigenmodesasinitialconditions.Thebeatingcyclesaredividedinto10framesasinfigure2a,b(toppanels).(b)φ (solidlines) δn(dashedlines)evaluatedats = 3 4 fortheprofilesin(a),respectively.(c)Maximumabsolutetangentangleevaluatedats = 1 2 and ensionless frequency ω as a function of the relative distance to the bifurcation ε = (μa −μc a)/μc a. Sp = 10, μ = 50, η = 0.14, = 0.4, ¯f = 2, L = 50 µm and τ0 = 50 ms. 0 20 40 –0.5 –1.0 0 0.5 1.0 –0.01 0 0.01 Y (mm) X (mm) ma= 4300 dn (a) 8 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 20 40 –4 –2 0 2 4 –0.02 –0.01 0 0.01 0.02 ma= 5600 Y (mm) X (mm) dn 8 Y (mm) 100 200 300 400 500 –0.2 0 0.2 –1 0 1 ×10–2 t (ms) (m ) dn (3/4) 200 300 400 500 –0.1 0 0.1 0 5 –5 ×10–3 t (ms) f (3/4) (b) 0 0.2 0.4 0 0.05 0.10 0.15 |fmax(1/2)| e (c) 0 0.2 0.4 2.5 3.0 3.5 e w Figure 3. Nonlinear analysis. 5. Nonlinear flagellar dynamics (a) Nonlinear flagella steady-state profiles for μa = 4300 (left) and μa = 5600 (right), considering the respectiveeigenmodesasinitialconditions.Thebeatingcyclesaredividedinto10framesasinfigure2a,b(toppanels).(b)φ (solidlines) andδn(dashedlines)evaluatedats = 3 4 fortheprofilesin(a),respectively.(c)Maximumabsolutetangentangleevaluatedats = 1 2 and dimensionless frequency ω as a function of the relative distance to the bifurcation ε = (μa −μc a)/μc a. Sp = 10, μ = 50, η = 0.14, ζ = 0.4, ¯f = 2, L = 50 µm and τ0 = 50 ms. (right) being nonlinear, they conserve the symmetry S(t + T/2) = −S(t), where T is the period of the signal. This is a consequence of the plus and minus motor populations being identical, a property also found in spontaneous oscillations of motor assemblies [36,38]. Finally, in figure 3c, we study how the amplitude and frequency of the oscillations vary with the relative distance from the bifurcation point ε ≡(μa −μc a)/μc a. For small ε, the maximum absolute value of the tangent angle seems to follow a square- root dependence, characteristic of supercritical Hopf bifurcation; however, in the strongly nonlinear regime the curve deviates from this trend. On the other hand, the beating frequency decreases for increasing activity. The only possibility to increase μa keeping the other dimensionless parameters fixed is by increasing ρN; hence, the larger each dynein team becomes, the larger the activity. Consequently, the necessary time to unbind a sufficient number of dynein motors to drive the instability increases, leading to a lower beating frequency. 5.1. Principal component analysis In this section, we study the obtained nonlinear solutions using principal component analysis [26,27]. This technique treats the flagellar shapes as multi-feature datasets, which can be projected to a lower dimensional space characterized by principal shape modes. Here we analyse the numerically resolved data following reference [26] to study sperm flagella. We discretize our flagella data with time-points ti, i = 1, . . . , p and M = 4 × 103 intervals corresponding to points sj = ( j −1)/M, j = 1, . . . , r along the 9 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 1606 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . flagellum, with r = M + 1. We construct a measurement matrix Φ of size p × r for the tangent angle where Φij = φ(sj, ti). This matrix represents a kymograph of the flagellar beat. We define the r × r covariance matrix as C = (Φ −¯Φ)T(Φ −¯Φ), where ¯Φ = [ ¯φ; . . . ; ¯φ] with all rows equal to ¯φ = [ ¯φ(s1), . . . , ¯φ(sr)], where ¯φ(si) is the mean tangent angle at si. The covariance matrix C is shown for μa = 4300 (figure 4a, left) and μa = 5600 (figure 4a, right). In figure 4a (left), we find negative correlation between tangent angles that are a distance λ/2 apart. Hence, a characteristic wavelength λ can be identified in the system, which manifests as a long-range correlation in the matrix C. On the other hand, strong positive correlations around the main diagonals correspond to short-ranged correlations mainly owing to the bending stiffness of the bundle [26]. The number of local maxima along the diagonals in C decreases from μa = 4300 to μa = 5600, and at the same time λ′ > λ. Hence, an increase in motor activity slightly increases the characteristic wavelength while decreasing the number of local maxima, which is related to the characteristic wavenumber. Employing an eigenvalue decomposition of the covariance matrix, we can obtain the eigenvectors v1, . . . , vr and their corresponding eigenvalues d1, . . . , dr, such that Cvj = djvj. 9 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Φi = ¯φ + r  k=1 Bk(ti)vk, (5.1) (5.1) where Bk are the shape scores computed by a linear least-square fit. In figure 4b (left), the two first eigenvectors v1, v2 are shown for μa = 4300. In figure 4b (right), the flagellar shape at a certain time (thick solid line) is reconstructed (white line) by using a superposition of the two principal shape modes v1, v2 (solid and dashed lines, respectively) and fitting the scores B1, B2. Finally, in figure 4c, we show the shape space trajectories beginning with small amplitude eigenmode solutions. While close to the bifurcation the limit cycle is elliptic (figure 4c, left), far from the bifurcation the limit cycle becomes distorted (figure 4c, right). Elliptic limit cycles were also found experimentally for bull sperm flagella [26]. Hence, as found in §5, motor activity in the nonlinear regime significantly affects the shape of the flagellum when compared with the linear solutions, which only provide good estimates sufficiently close to the Hopf bifurcation. 5.2. Bending initiation and transient dynamics Finally, we study bending initiation and transient dynamics for two different initial conditions, in order to understand the selection of the unstable modes. In figure 5a,b the spatio-temporal transient dynamics are shown for the case of an initial eigenmode solution corresponding to the maximum eigenvalue (figure 5a) and an initial sine perturbation in φ, with equal constant bound motor densities (figure 5b). In case (b), travelling waves initially propagate in both directions and interfere at t = t′ (figure 5b,d and electronic supplementary material, movie S3). However, in the steady state, both the eigenmode and sine cases reach the same steady-state solution, despite the sinusoidal initial condition being a superposition of eigenmodes. This result provides strong evidence that the fastest-growing mode is the one that takes over and saturates in the steady state. In figure 5c, the transient dynamics for case (b) are shown for plus- and minus-bound dynein populations close to the tail (s = 9 10). Both populations decay exponentially with characteristic time ¯τ to n0 and begin oscillating in anti-phase around this value, in a tug-of-war competition. 5.1. Principal component analysis Without loss of generality, we can sort the eigenvalues in descending order d1 ≥· · · ≥dr. We find that the first two eigenvalues capture >99% variance of the data. This fact indicates that our flagellar waves can be suitably described in a two-dimensional shape space, because they can be regarded as single- frequency oscillators. Each flagellar shape Φi = [φ(s1, ti), . . . , φ(sr, ti)] can be expressed now as a linear combination of the eigenvectors vk [26]: 9 6. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s s 0 0.5 1.0 0 0.5 1.0 0 200 400 l¢ / 2 10 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s s 0 0.5 1.0 0 0.5 1.0 –10 0 10 20 30 l / 2 (a) 10 (a) blishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ng.org R.Soc. opensci. 4: 160698 0 0.5 1.0 –0.05 0 0.05 υ1 υ2 s f (b) 0 0.5 1.0 –0.10 –0.05 0 0.05 0.10 s f –5 0 5 –3 0 3 B1 B2 (c) –20 0 20 –10 0 10 B1 B2 B1 Figure 4. Principalcomponentanalysisofflagellar beating. (a)Covariancematrixfor μa = 4300(left)and μa = 5600(right). We can identify characteristic wavelengths λ, λ′ from negative long-range correlations in C. Note λ′ > λ and the number of local maxima decreases when μa is increased. (b) (Left) Two principal shape modes v1, v2 (solid and dashed lines, respectively), corresponding to the two maximum eigenvalues of the covariance matrix in figure 4a (left). (Right) The flagellar shape at time t = 733 ms (thick solid line) is reconstructed (white line) by a superposition of the two principal shape modes v1, v2 in figure 4b (left) fitting the scores B1, B2. (c) Flagellar dynamics in a reduced two-dimensional shape space for μa = 4300 (left) and μa = 5600 (right). Elliptic limit cycles are rescaled to better appreciate the distortion owing to the nonlinear terms. Sp = 10, μ = 50, η = 0.14, ζ = 0.4. a nonlinear axonemal response to account for the saturation of the unstable modes, in contrast to previous studies [23,24]. 6. Discussion In this work, we presented a theoretical framework for planar axonemal beating by formulating a full set of nonlinear equations to test how flagellar amplitude and shape vary with dynein activity. We have shown how the nonlinear coupling of flagellar shape and dynein kinetics in a ‘sliding-controlled’ model provides a novel mechanism for the saturation of unstable modes in flagellar beating. Our study advances understanding of the nonlinear nature of the axoneme, typically studied at the linear level [13–16]. The origin of the bending wave instability can be understood as a consequence of the antagonistic action of dyneins competing along the flagellum [15,39]. The instability is then further stabilized by the nonlinear coupling between dynein activity and flagellum shape, without the need to invoke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s s 0 0.5 1.0 0 0.5 1.0 –10 0 10 20 30 0 0.5 1.0 –0.05 0 0.05 υ1 υ2 –5 0 5 –3 0 3 B1 –20 0 20 –10 0 10 B1 B2 B2 0 0.5 1.0 –0.10 –0.05 0 0.05 0.10 s s s s 0 0.5 1.0 0 0.5 1.0 0 200 400 l / 2 f f l¢ / 2 (a) (b) (c) re 4. Principalcomponentanalysisofflagellar beating. (a)Covariancematrixfor μa = 4300(left)and μa = 5600(right). We can ify characteristic wavelengths λ, λ′ from negative long-range correlations in C. Note λ′ > λ and the number of local maxima ases when μa is increased. (b) (Left) Two principal shape modes v1, v2 (solid and dashed lines, respectively), corresponding to wo maximum eigenvalues of the covariance matrix in figure 4a (left). (Right) The flagellar shape at time t = 733 ms (thick solid is reconstructed (white line) by a superposition of the two principal shape modes v1, v2 in figure 4b (left) fitting the scores B1, B2. agellar dynamics in a reduced two-dimensional shape space for μa = 4300 (left) and μa = 5600 (right). Elliptic limit cycles escaled to better appreciate the distortion owing to the nonlinear terms. Sp = 10, μ = 50, η = 0.14, ζ = 0.4. 10 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . 6. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (b) 11 (a) ing.org R.Soc. opensci. 4: 160698 250 500 750 0 5 10 0 20 40 –1 0 1 n+ n– 0 150 300 450 0.5 mm 20 mm t = 0 t = t¢ t = t¢¢ bound motors (%) t (ms) Y (µm) X (µm) (c) (d) (d) Figure5. Bendinginitiationandtransientdynamics.(a)Tangentanglekymographforaneigenmodeinitialcondition.(b)Tangentangle kymograph for a sinusoidal initial condition for the tangent angle with n+(0) = n−(0) = 0.1. (c) Bound motor time evolution for the plus(solidline)andminus(dashedline)dyneinpopulationsats = 9 10 forthecaseofasinusoidalinitialcondition.Inset:flagellaprofilesat differenttimesinms.(d)Snapshotsoftheflagellarshapeforthesinusoidalinitialconditionuptot = t′′ (whitedashedlineinfigure5b) at equal time intervals (20 ms). At t = t′, wave interference changes the direction of wave propagation. The full movie can be seen in electronicsupplementarymaterial,movieS3.Sp = 5,μ = 100,μa = 2000,η = 0.14,ζ = 0.4,L = 50 µmandτ0 = 50 ms.Arrows indicate the direction of wave propagation. Angular deflections are found to be approximately 0.1 rad in the experimentally relevant activity range μa ≃(2 −6) × 103 and the order of magnitude seems not to be crucially affected by viscosity nor other parameters in the system. Hence, despite of the fact that our description provides an intrinsic mechanism for amplitude saturation, it is only able to generate small deflections, typically an order of magnitude smaller than the ones reported for bull sperm flagella [15]. Other structural constraints, such as line tension, are likely to influence the amplitude saturation, owing to the elastohydrodynamic coupling with motor activity (see electronic supplementary material). The presence of tension on the self-organized beating of flagella was previously investigated at leading nonlinear order; however, deflections were also found to be small [19]. Hence, we conclude that a ‘sliding-controlled’ mechanism may not be sufficient to generate large deflections. Our work adds to other recent studies were the ‘sliding-controlled’ hypothesis seems to lose support as the main mechanism responsible for flagellar beating [16,24]. Basal dynamics and elasticity are also likely to influence the amplitude saturation, and substantial further research is still needed to infer whether sliding-controlled regulation is the responsible mechanism behind flagellar wave generation. 6. Discussion Moreover, the governing equations (equations (2.10) and (2.11)) contain all the nonlinearities in the limit of small curvature, and they are not the result of a power expansion to leading nonlinear order [19]. Far from the Hopf bifurcation, linearized solutions fail to describe the flagellar shape, and nonlinear effects arise in the system solely owing to motor activity. At the nonlinear level, both the tangent angle and dynein population dynamics exhibit relaxation or cusp- like oscillations at some regions along the flagellum. Similar cusp-like shapes for the curvature have also been reported in sea urchin sperm [40]. This phenomenology is characteristic of motor assemblies working in far-from-equilibrium conditions and has been found in other biological systems such as in the spontaneous oscillations of myofibrils [38,41]. Interestingly, despite the low duty ratio of axonemal dynein, a fraction of approximately 2% bound dyneins along the flagellum is sufficient to drive micrometre-sized amplitude oscillations. 11 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 500 750 0 5 10 0 20 40 –1 0 1 250 500 750 0 0.5 1.0 –0.2 0 0.2 250 500 750 0 0.5 1.0 –0.2 0 0.2 f f n+ n– 0 150 300 450 0.5 mm 20 mm t = 0 t = t¢ t = t¢¢ t¢¢ t¢ sine eigenmode bound motors (%) t (ms) t (ms) t (ms) s s Y (µm) X (µm) (a) (b) (c) (d) . Bendinginitiationandtransientdynamics.(a)Tangentanglekymographforaneigenmodeinitialcondition.(b)Tangentangle ph for a sinusoidal initial condition for the tangent angle with n+(0) = n−(0) = 0.1. (c) Bound motor time evolution for the dline)andminus(dashedline)dyneinpopulationsats = 9 10 forthecaseofasinusoidalinitialcondition.Inset:flagellaprofilesat timesinms.(d)Snapshotsoftheflagellarshapeforthesinusoidalinitialconditionuptot = t′′ (whitedashedlineinfigure5b) time intervals (20 ms). At t = t′, wave interference changes the direction of wave propagation. The full movie can be seen in csupplementarymaterial,movieS3.Sp = 5,μ = 100,μa = 2000,η = 0.14,ζ = 0.4,L = 50 µmandτ0 = 50 ms.Arrows he direction of wave propagation. 250 500 750 0 0.5 1.0 –0.2 0 0.2 f t¢¢ t¢ sine t (ms) s (b) 250 500 750 0 0.5 1.0 –0.2 0 0.2 f eigenmode t (ms) s (a) 11 rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . rsos.royalsocietypublishing.org R.Soc. opensci. 4: 160698 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . We have no competing interests. pp p Competing interests. We have no competing interests. Funding. J.C. and D.O. acknowledge financial support from the Ministerio de Economía y Competitividad under projects FIS2010-21924-C02-02 and FIS2013-41144-P, and the Generalitat de Catalunya under projects 2009 SGR 14 and 2014 SGR 878. D.O. also acknowledges a FPU grant from the Spanish Government with award number AP-2010- 2503 and an EMBO Short Term Fellowship with ASTF number 314-2014. H.G. acknowledges support by the Hooke Fellowship, University of Oxford. Funding. J.C. and D.O. acknowledge financial support from the Ministerio de Economía y Competitividad under projects FIS2010-21924-C02-02 and FIS2013-41144-P, and the Generalitat de Catalunya under projects 2009 SGR 14 and 2014 SGR 878. D.O. also acknowledges a FPU grant from the Spanish Government with award number AP-2010- 2503 and an EMBO Short Term Fellowship with ASTF number 314-2014. H.G. acknowledges support by the Hooke Fellowship, University of Oxford. References 7. Brokaw CJ. 2009 Thinking about flagellar oscillation. CellMotil.Cytoskeleton 66, 425–436. (doi:10.1002/cm.20313) 1. Ginger ML, Portman N, McKean PG. 2008 Swimming with protists: perception, motility and flagellum assembly. Nat.Rev.Microbiol. 6, 838–850. (doi:10.1038/nrmicro2009) 1. Ginger ML, Portman N, McKean PG. 2008 Swimming with protists: perception, motility and flagellum assembly. Nat.Rev.Microbiol. 6, 838–850. 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An important aspect which is not studied explicitly in this work is the direction of wave propagation. For simplicity, we used clamped boundary conditions at the head which are known to induce travelling waves which propagate from tip to base in the sliding-controlled model [13,24]. It is beyond the scope of our study to assess the effects of different boundary conditions and the role of basal compliance at the head of the flagellum, which are known to crucially affect wave propagation [15]. This work is also restricted to the case of small curvatures; however, the full nonlinear equations including the presence of tension could, in principle, be numerically solved as in previous studies [18,19] (see electronic supplementary material). Finally, a real flagellum is subject to chemical noise owing to the stochastic binding and unbinding of dynein motors. Recent studies have provided insights into this problem by investigating a noisy oscillator driven by molecular motors. However, their approach was not spatially extended [31]. Our approach could be suitably extended to include chemical noise in the system through equation (2.11) by considering a chemical Langevin equation for the bound dynein populations including multiplicative noise [42]. In particular, it can be easily deduced from our study that by considering a force-independent unbinding rate, fluctuations of bound motors around the base state have mean Nη and variance Nη(1 −η), in agreement with the results in reference [31] where a different model was considered. The possibility to experimentally probe the activity of dyneins inside the axoneme is one of the most exciting future challenges in the study of cilia and flagella. These studies will be of vital importance to validate mathematical models of axonemal beating and the underlying mechanisms coordinating dynein activity and flagellar beating. Data accessibility. Electronic supplementary information for this article includes a supplementary text and three supplementary movies. The data from the supplementary movies are available from the Dryad Digital Repository: http://dx.doi.org/10.5061/dryad.qs65q [43]. Authors’contributions. D.O. carried out analytical work, numerical simulations, data analysis and drafted the manuscript. H.G. and J.C. conceived of the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication. Authors’contributions. D.O. carried out analytical work, numerical simulations, data analysis and drafted the manuscript. H.G. and J.C. conceived of the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication. pp p Competing interests. 6. Discussion Principal component analysis allowed us to reduce the nonlinear dynamics of the flagellum in a two-dimensional shape space, regarding the flagellum as a single-frequency biological oscillator [31]. Note that a two-dimensional description would not hold for multifrequency oscillations, where an additional dimension is required [36]. Interestingly, we found that as activity increases, the characteristic wavenumber of the system slightly decreases. Thus, dynein activity has an opposite effect on wavenumber selection when compared with the medium viscosity (figure 2d, inset). We also showed that the steady-state amplitude is selected by the fastest-growing mode under the influence of competing unstable modes, provided that the initial mode amplitudes are sufficiently small. wavenumber selection when compared with the medium viscosity (figure 2d, inset). 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A Transformation-Based Approach to Implication of GSTE Assertion Graphs
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Hindawi Publishing Corporation Journal of Applied Mathematics Volume 2013, Article ID 709071, 7 pages http://dx.doi.org/10.1155/2013/709071 Hindawi Publishing Corporation Journal of Applied Mathematics Volume 2013, Article ID 709071, 7 pages http://dx.doi.org/10.1155/2013/709071 Hindawi Publishing Corporation Journal of Applied Mathematics Volume 2013, Article ID 709071, 7 pages http://dx.doi.org/10.1155/2013/709071 Hindawi Publishing Corporation Journal of Applied Mathematics Volume 2013, Article ID 709071, 7 pages http://dx.doi.org/10.1155/2013/709071 Guowu Yang,1 William N. N. Hung,2 Xiaoyu Song,3 and Wensheng Guo1 1 School of Computer Science and Engineering, University of Electronic Science and Technology Chengdu, Sichuan 610054, China 2 Synopsys Inc., Mountain View, CA 94043, USA 3 D f ECE P l d S U i i P l d O USA 3 Department of ECE, Portland State University, Portland, Oregon, USA Correspondence should be addressed to Guowu Yang; guowuy@126.com and William N. N. Hung; william hung@alumni.utexas.net Received 8 February 2013; Accepted 13 May 2013 Academic Editor: Guiming Luo Copyright © 2013 Guowu Yang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Generalized symbolic trajectory evaluation (GSTE) is a model checking approach and has successfully demonstrated its powerful capacity in formal verification of VLSI systems. GSTE is an extension of symbolic trajectory evaluation (STE) to the model checking of 𝜔-regular properties. It is an alternative to classical model checking algorithms where properties are specified as finite-state automata. In GSTE, properties are specified as assertion graphs, which are labeled directed graphs where each edge is labeled with two labeling functions: antecedent and consequent. In this paper, we show the complement relation between GSTE assertion graphs and finite-state automata with the expressiveness of regular languages and 𝜔-regular languages. We present an algorithm that transforms a GSTE assertion graph to a finite-state automaton and vice versa. By applying this algorithm, we transform the problem of GSTE assertion graphs implication to the problem of automata language containment. We demonstrate our approach with its application to verification of an FIFO circuit. Guowu Yang,1 William N. N. Hung,2 Xiaoyu Song,3 and Wensheng Guo1 Guowu Yang,1 William N. N. Hung,2 Xiaoyu Song,3 and Wensheng Guo1 1 School of Computer Science and Engineering, University of Electronic Science and Technology Chengdu, Sichuan 610054, China 2 Synopsys Inc., Mountain View, CA 94043, USA 3 Department of ECE Portland State University Portland Oregon USA 1. Introduction Given a finite-state automaton 𝑀= ⟨𝑄, Σ, 𝐸, 𝐿𝐹, 𝑞0, 𝐹⟩, In(𝑞) and Out(𝑞) for 𝑞∈𝑄are called the set of incoming edges of 𝑞and the set of outgoing edges of 𝑞, respectively; that is, In(𝑞) = {(𝑞󸀠, 𝑞) | (𝑞󸀠, 𝑞) ∈𝐸} and Out(𝑞) = {(𝑞, 𝑞󸀠) | (𝑞, 𝑞󸀠) ∈𝐸}. Definition 5. Given a finite-state automaton 𝑀= ⟨𝑄, Σ, 𝐸, 𝐿𝐹, 𝑞0, 𝐹⟩, In(𝑞) and Out(𝑞) for 𝑞∈𝑄are called the set of incoming edges of 𝑞and the set of outgoing edges of 𝑞, respectively; that is, In(𝑞) = {(𝑞󸀠, 𝑞) | (𝑞󸀠, 𝑞) ∈𝐸} and Out(𝑞) = {(𝑞, 𝑞󸀠) | (𝑞, 𝑞󸀠) ∈𝐸}. We apply the proposed theory and algorithm to the GSTE assertion graphs implication problem by transforming this problem to the automata language containment prob- lem. Since, in many cases, assertion graphs are nondeter- ministic, their corresponding finite-state automata are also nondeterministic. This may cause exponential state space blowups in checking automata language containment [16– 22]. We avoid such blowups in determining the implication relation between a nondeterministic assertion graph and its refinement derived following the refinement strategies given in [2], by transforming the implication relation between nondeterministic assertion graphs to the implication relation between deterministic assertion graphs without adding any states. This application is illustrated with a case study for verifying correctness properties of an FIFO circuit.h Definition 6. If for all 𝑞∈𝑄and for all 𝑒, 𝑒󸀠∈Out(𝑞), 𝑒̸= 𝑒󸀠 and 𝐿𝐹(𝑒)∩𝐿𝐹(𝑒󸀠) = 0, 𝑀is called a deterministic finite-state automaton (DFA). Otherwise, 𝑀is called a nondeterministic finite-state automaton (NFA). Definition 6. If for all 𝑞∈𝑄and for all 𝑒, 𝑒󸀠∈Out(𝑞), 𝑒̸= 𝑒󸀠 and 𝐿𝐹(𝑒)∩𝐿𝐹(𝑒󸀠) = 0, 𝑀is called a deterministic finite-state automaton (DFA). Otherwise, 𝑀is called a nondeterministic finite-state automaton (NFA). i Traditionally, an NFA may have a set of initial states. The expressiveness of an NFA with multiple initial states is the same as that of an NFA with a unique initial state. The transformation is easy. Definition 7. An assertion graph 𝐺is a tuple ⟨𝑄, Σ, 𝐸, 𝑞0, ant, cons, 𝐹⟩, where 𝑄is a finite set of states, Σ is a finite alphabet, 𝐸is a set of directed edges over 𝑄, ant : 𝐸󳨃→2Σ and cons : 𝐸󳨃→2Σ are the labeling functions for all edges, 𝑞0 ∈𝑄is the initial state, and 𝐹is a subset of 𝑄and elements of 𝐹are called acceptance states. The remainder of this paper is organized as follows. 1. Introduction In Section 2, we introduce the preliminaries of finite-state automata and GSTE assertion graphs. In Section 3, we present the transformation algorithm and prove its correctness. In Section 4, we propose an application of the transformation algorithm to determine the implication relation between GSTE assertion graphs and demonstrate its effectiveness with a case study. We conclude in Section 5. Definition 8. A finite word (or string) 𝑋= 𝑥1𝑥2 ⋅⋅⋅𝑥𝑛∈Σ∗ is accepted by an assertion graph 𝐺(denoted as 𝑋⊨∗𝐺) if for each finite path 𝜌= 𝑞0𝑞1 ⋅⋅⋅𝑞𝑛with (𝑞𝑖−1, 𝑞𝑖) ∈𝐸(where 1 ≤𝑖≤𝑛) and 𝑞𝑛∈𝐹such that 𝑋⊨ant𝜌⇒𝑋⊨cons𝜌, where 𝑋⊨ant𝜌denotes 𝑥𝑖∈ant((𝑞𝑖−1, 𝑞𝑖)) and 𝑋⊨cons𝜌denotes 𝑥𝑖∈ cons((𝑞𝑖−1, 𝑞𝑖)) for 1 ≤𝑖≤𝑛. An infinite word (or string) 𝑋= 𝑥1𝑥2 ⋅⋅⋅∈Σ𝜔is accepted by 𝐺(denoted as 𝑋⊨𝜔𝐺) if for all infinite path 𝜌= 𝑞0𝑞1 ⋅⋅⋅with (𝑞𝑖−1, 𝑞𝑖) ∈𝐸(where 𝑖≥1) and Inf(𝜌) ∩𝐹̸= 0 such that 𝑋⊨ant𝜌⇒𝑋⊨cons𝜌. The finite language 𝐿∗(𝐺) of 𝐺is 𝐿∗(𝐺) = {𝑋∈Σ∗| 𝑋⊨∗𝐺}. The infinite language 𝐿𝜔(𝐺) of 𝐺is 𝐿𝜔(𝐺) = {𝑋∈Σ𝜔| 𝑋⊨𝜔𝐺}. 1. Introduction For a finite-state automaton over finite words, acceptance is defined according to the last state visited by a run of the automaton; while for a finite-state automaton with B¨uchi acceptance conditions, there is no such “the last state” and acceptance is defined according to the set of states that a run visits infinitely often [16]. Definition 2. A finite word (or string) 𝑋= 𝑥1𝑥2 ⋅⋅⋅𝑥𝑛∈Σ∗is accepted by a finite-state automaton 𝑀(denoted as 𝑋⊨∗𝑀) if there exists a path 𝜌= 𝑞0𝑞1 ⋅⋅⋅𝑞𝑛such that (𝑞𝑖−1, 𝑞𝑖) ∈𝐸, 𝑞𝑛∈𝐹, and 𝑥𝑖∈𝐿𝐹((𝑞𝑖−1, 𝑞𝑖)), denoted as 𝑋⊨𝐿𝐹𝜌. B¨uchi acceptance conditions that accept 𝜔-regular languages. For a finite-state automaton over finite words, acceptance is defined according to the last state visited by a run of the automaton; while for a finite-state automaton with B¨uchi acceptance conditions, there is no such “the last state” and acceptance is defined according to the set of states that a run visits infinitely often [16]. Definition 3. An infinite word (or string) 𝑋= 𝑥1𝑥2 ⋅⋅⋅∈ Σ𝜔is accepted by a finite-state automaton 𝑀(denoted as 𝑋⊨𝜔𝑀) if there exists an infinite path 𝜌= 𝑞0𝑞1 ⋅⋅⋅such that (𝑞𝑖−1, 𝑞𝑖) ∈𝐸, Inf(𝜌) ∩𝐹̸= 0, where Inf(𝜌) is the set of states occurring infinitely often in 𝜌, and 𝑥𝑖∈𝐿𝐹((𝑞𝑖−1, 𝑞𝑖)), denoted as 𝑋⊨𝐿𝐹𝜌. i yt We present a theory on the complement relation between GSTE assertion graphs and finite-state automata and an algorithm which automatically transforms assertion graphs to their equivalent finite-state automata, and vice versa. For an assertion graph 𝐺, we construct a corresponding finite- state automaton 𝑀𝐺such that 𝐿∗(𝐺) = Σ∗−𝐿∗(𝑀𝐺) and 𝐿𝜔(𝐺) = Σ𝜔−𝐿𝜔(𝑀𝐺), the number of states in 𝑀𝐺is only twice as many as that in 𝐺, and a deterministic assertion graph is transformed to a deterministic automaton. On the other hand, for an arbitrary finite-state automaton 𝑀, we construct a corresponding assertion graph 𝐺𝑀such that 𝐿∗(𝐺𝑀) = Σ∗−𝐿∗(𝑀) and 𝐿𝜔(𝐺𝑀) = Σ𝜔−𝐿𝜔(𝑀), the number of states in G𝑀is the same as that in 𝑀, and a deterministic automaton is transformed to a deterministic assertion graph. Definition 4. The finite language 𝐿∗(𝑀) of 𝑀is 𝐿∗(𝑀) = {𝑋∈Σ∗| 𝑋⊨∗𝑀}. The infinite language 𝐿𝜔(𝑀) of 𝑀is 𝐿𝜔(𝑀) = {𝑋∈Σ𝜔| 𝑋⊨𝜔𝑀}. Definition 5. 1. Introduction directed graph where each edge is labeled with two labeling functions: antecedent and consequent [3]. The existing GSTE theory provides an efficient model checking procedure for verifying that a circuit obeys an assertion graph as well as techniques based on abstract interpretation to combat state space explosion [3, 7].i Generalized symbolic trajectory evaluation (GSTE) [1–4] is a model checking approach which was originally developed at Intel and has successfully demonstrated its powerful capacity in formal verification of VLSI systems [1–7]. GSTE is extended from the lattice-based symbolic trajectory evalu- ation (STE) [8, 9] which can handle large industrial designs [10–13]. The STE theory consists of a simple specification language, a simulation-based model checking algorithm, and a powerful quaternary abstraction algorithm. Though STE is very efficient in automatic verification of large-scale industrial hardware designs at both gate and transistor levels, it has a limited specification language which only allows the specification of properties over finite time intervals. How to establish that one specification implies another is a fundamental problem in formal verification. Hu et al. [6, 7] proposed some algorithms to decide whether one assertion graph implies another through building monitor circuits. Yang and Seger [2] verified assertion graphs through manually refining assertion graphs. Yang et al. [14] gave some conditions under which the assertion graphs implication is decided without reachability analysis if two assertion graphs have the same graph structure. Sebastiani et al. [15] gave the complement relation between an assertion graph and a finite- state automaton. An assertion graph with 𝑛states resulted in a nondeterministic automaton with 3 ∗𝑛states.i In GSTE, all 𝜔-regular properties can be expressed and verified with space and time efficiencies comparable with STE. Assertion graphs are introduced in GSTE as an extension of STE specification language and are the key to the usability of GSTE. An assertion graph is a labeled In this paper, we consider both finite-state automata that accept regular languages and finite-state 𝜔-automata with Journal of Applied Mathematics 2 Definition 2. A finite word (or string) 𝑋= 𝑥1𝑥2 ⋅⋅⋅𝑥𝑛∈Σ∗is accepted by a finite-state automaton 𝑀(denoted as 𝑋⊨∗𝑀) if there exists a path 𝜌= 𝑞0𝑞1 ⋅⋅⋅𝑞𝑛such that (𝑞𝑖−1, 𝑞𝑖) ∈𝐸, 𝑞𝑛∈𝐹, and 𝑥𝑖∈𝐿𝐹((𝑞𝑖−1, 𝑞𝑖)), denoted as 𝑋⊨𝐿𝐹𝜌. B¨uchi acceptance conditions that accept 𝜔-regular languages. Theorem 12. 𝐿∗(𝑀𝐺) = Σ∗−𝐿∗(𝐺). Proof. First, we show 𝐿∗(𝑀𝐺) ⊇Σ∗−𝐿∗(𝐺). Suppose 𝑋= 𝑥1𝑥2 ⋅⋅⋅𝑥𝑛∈Σ∗−𝐿∗(𝐺). Then there exists a finite path 𝜌= 𝑞0𝑞1 ⋅⋅⋅𝑞𝑛in 𝐺with (𝑞𝑖−1, 𝑞𝑖) ∈𝐸, 1 ≤𝑖≤𝑛and 𝑞𝑛∈𝐹such that 𝑥𝑖∈ ant((𝑞𝑖−1, 𝑞𝑖)) for 1 ≤𝑖≤𝑛; and ∃𝑙, 1 ≤𝑙≤𝑛, when 1 ≤ℎ< 𝑙, 𝑥ℎ∈ant((𝑞𝑖−1, 𝑞𝑖)) ∩cons((𝑞𝑖−1, 𝑞𝑖)), 𝑥𝑙∈ant((𝑞𝑙−1, 𝑞𝑙)) ∩ ¬cons((𝑞𝑖−1, 𝑞𝑖)). pp 1 2 𝑛 Then there exists a finite path 𝜌= 𝑞0𝑞1 ⋅⋅⋅𝑞𝑛in 𝐺with (𝑞𝑖−1, 𝑞𝑖) ∈𝐸, 1 ≤𝑖≤𝑛and 𝑞𝑛∈𝐹such that 𝑥𝑖∈ ant((𝑞𝑖−1, 𝑞𝑖)) for 1 ≤𝑖≤𝑛; and ∃𝑙, 1 ≤𝑙≤𝑛, when 1 ≤ℎ< 𝑙, 𝑥ℎ∈ant((𝑞𝑖−1, 𝑞𝑖)) ∩cons((𝑞𝑖−1, 𝑞𝑖)), 𝑥𝑙∈ant((𝑞𝑙−1, 𝑞𝑙)) ∩ ¬cons((𝑞𝑖−1, 𝑞𝑖)). 󸀠 pp 1 2 𝑛 Then there exists a finite path 𝜌= 𝑞0𝑞1 ⋅⋅⋅𝑞𝑛in 𝐺with (𝑞𝑖−1, 𝑞𝑖) ∈𝐸, 1 ≤𝑖≤𝑛and 𝑞𝑛∈𝐹such that 𝑥𝑖∈ ant((𝑞𝑖−1, 𝑞𝑖)) for 1 ≤𝑖≤𝑛; and ∃𝑙, 1 ≤𝑙≤𝑛, when 1 ≤ℎ< 𝑙, 𝑥ℎ∈ant((𝑞𝑖−1, 𝑞𝑖)) ∩cons((𝑞𝑖−1, 𝑞𝑖)), 𝑥𝑙∈ant((𝑞𝑙−1, 𝑞𝑙)) ∩ ¬cons((𝑞𝑖−1, 𝑞𝑖)). 󸀠 2. Preliminaries In this section, we give the definitions of finite-state automata and GSTE assertion graphs and of their finite and infinite languages. We also show that the fairness edge constraints for GSTE assertion graphs [1] are equivalent to the fairness state constraints. Remark 9. In [1], an assertion graph 𝐺= ⟨𝑄, Σ, 𝐸, 𝑞0, ant, cons, 𝐹𝑒⟩has a set 𝐹𝑒of acceptance (fair) edges. The final edge of a finite path must be in 𝐹𝑒and for an infinite path; at least one edge in 𝐹𝑒must appear in the path infinitely often. This can be transformed to the above definitions by the following construction. For any 𝑞∈𝑄, if the incoming edges In(𝑞) have both acceptance edges and nonacceptance edges, 𝑞is substituted with 𝑞󸀠and 𝑞󸀠󸀠such that In(𝑞󸀠) = In(𝑞) ∩𝐹𝑒, In(𝑞󸀠󸀠) = In(𝑞) −In(𝑞󸀠); that is, the incoming Definition 1. A finite-state automaton 𝑀is a tuple 𝑀= ⟨𝑄, Σ, 𝐸, 𝐿𝐹, 𝑞0, 𝐹⟩, where 𝑄is a finite set of states, Σ is a finite alphabet, 𝐸is a set of directed edges over 𝑄, 𝐿𝐹: 𝐸󳨃→2Σ is the labeling function for all edges, 𝑞0 ∈𝑄is the initial state, and 𝐹is a subset of 𝑄whose elements are called acceptance states. Journal of Applied Mathematics Journal of Applied Mathematics 3 F F q Suc(q) Suc(q) q󳰀 q󳰀󳰀 Figure 1: Fair edges to fair states. q1 q2 (q1, 0) (q2, 0) (q1, 1) (q2, 1) (q2, 1) Figure 2: Assertion graph to finite-state automaton. (q1, 1) Figure 1: Fair edges to fair states. Figure 2: Assertion graph to finite-state automaton. edges of 𝑞󸀠are all acceptance edges, the incoming edges of 𝑞󸀠󸀠 are nonacceptance edges (See Figure 1). The outgoing edges of 𝑞󸀠and 𝑞󸀠󸀠are the same as the outgoing edges of 𝑞, and the labeling function ant and cons are the same. All such 𝑞󸀠𝑠 are included the acceptance state set 𝐹in the transformed assertion graph. Theorem 11. If 𝐺is a DAG, then 𝑀𝐺is a DFA. Proof. We prove the following two cases. Proof. We prove the following two cases. (i) For any state (𝑞, 1) in 𝑀𝐺and any two outgoing edges ((𝑞, 1), (𝑞1, 1)) and ((𝑞, 1), (𝑞2, 1)), 𝐿𝐹(((𝑞, 1), (𝑞1, 1)))∩ 𝐿𝐹(((𝑞, 1), (𝑞2, 1))) = ant((𝑞, 𝑞1))∩ant((𝑞, 𝑞2)) (the construc- tion of 𝑀𝐺) = 0 (the definition of DAG). Definition 10. Given an assertion graph 𝐺= ⟨𝑄, Σ, 𝐸, 𝑞0, ant, cons, 𝐹⟩, if for all 𝑞∈𝑄, for all 𝑒, 𝑒󸀠∈Out(𝑞) and 𝑒̸= 𝑒󸀠, ant(𝑒)∩ant(𝑒󸀠) = 0, 𝐺is called a deterministic assertion graph (DAG). Otherwise, 𝐺is called a nondeterministic assertion graph (NAG). 𝐺i (ii) For any state (𝑞, 0) in 𝑀𝐺and any two outgoing edges ((𝑞, 0), (𝑞1, 𝑘1)) and ((𝑞, 0), (𝑞2, 𝑘2)). (a) If 𝑞1 = 𝑞2, then 𝑘1 = 0, 𝑘2 = 1, 𝐿𝐹(((𝑞, 0), (𝑞1, 0))) ∩ 𝐿𝐹(((𝑞, 0), (𝑞1, 1))) = ant((𝑞, 𝑞1)) ∩cons((𝑞, 𝑞1)) ∩ ant((𝑞, 𝑞1))∩¬cons((𝑞, 𝑞1)) (the construction of 𝑀𝐺) = 0. For any assertion graph 𝐺= ⟨𝑄, Σ, 𝐸, 𝑞0, ant, cons, 𝐹⟩, we can assume that 𝐺is a nonrestarting assertion graph (i.e., In(𝑞0) = 0); otherwise, we construct a nonrestarting assertion graph 𝐺󸀠= ⟨𝑄󸀠, Σ, 𝐸󸀠, 𝑞0, ant, cons, 𝐹󸀠⟩from 𝐺: 𝑄󸀠= 𝑄∪{𝑞󸀠} (add a new state 𝑞󸀠to 𝑄), In(𝑞󸀠) = {(𝑞, 𝑞󸀠) | (𝑞, 𝑞0) ∈𝐸)}, Out(𝑞󸀠) = {(𝑞󸀠, 𝑞) | (𝑞0, 𝑞) ∈𝐸)}, ant((𝑞󸀠, 𝑞)) = ant((𝑞0, 𝑞)), cons((𝑞󸀠, 𝑞)) = cons((𝑞0, 𝑞)), ant((𝑞, 𝑞󸀠)) = ant((𝑞, 𝑞0)), cons((𝑞, 𝑞󸀠)) = cons((𝑞, 𝑞0)). Basically, we copy the edges and the related labeling functions ant and cons of 𝑞0 to 𝑞󸀠and delete the incoming edges of 𝑞0. The other edges and labeling functions remain the same. (b) If 𝑞1 ̸= 𝑞2, then 𝐿𝐹(((𝑞, 1), (𝑞1, 1))) ∩𝐿𝐹(((𝑞, 1), (𝑞2, 1))) ⊆ant((𝑞, 𝑞1)) ∩ant((𝑞, 𝑞2)) (the construc- tion of 𝑀𝐺) = 0 (the definition of DAG). Therefore, 𝑀𝐺is a DFA. Therefore, 𝑀𝐺is a DFA. Therefore, 𝑀𝐺is a DFA. Theorem 12. 𝐿∗(𝑀𝐺) = Σ∗−𝐿∗(𝐺). 3. Transformations between GSTE Assertion Graphs and Finite-State Automata In this section, we present the transformations between a GSTE assertion graph and a finite-state automaton. Given an assertion graph 𝐺, we build a finite-state automaton 𝑀𝐺 such that 𝐿∗(𝐺) = Σ∗−𝐿∗(𝑀𝐺) and 𝐿𝜔(𝐺) = Σ𝜔− 𝐿𝜔(𝑀𝐺). And if 𝐺is a deterministic assertion graph, then 𝑀𝐺is also a deterministic finite-state automaton. Let 𝑀𝐺= ⟨𝑄󸀠, Σ, 𝐸󸀠, 𝐿𝐹, 𝑞󸀠 0, 𝐹󸀠⟩: 𝑞𝑖1 𝑞𝑖 Let a path 𝜌󸀠of 𝑀𝐺be as follows: Theorem 13. 𝐿𝜔(𝑀𝐺) = Σ𝜔−𝐿𝜔(𝐺). According to the construction of 𝑀𝐺from 𝐺, we know that 𝜌 = 𝑞0𝑞1 ⋅⋅⋅is a path of 𝐺, and Inf(𝜌) ∩𝐹̸= 0; 𝑥𝑖∈ant((𝑞𝑖−1, 𝑞𝑖)) for 1 ≤𝑖, and 𝑥𝑙∈ant((𝑞𝑙−1, 𝑞𝑙)) ∩ ¬cons((𝑞𝑙−1, 𝑞𝑙)), that is, 𝑥𝑙∉cons((𝑞𝑙−1, 𝑞𝑙)).h According to the construction of 𝑀𝐺from 𝐺, we know that 𝜌 = 𝑞0𝑞1 ⋅⋅⋅is a path of 𝐺, and Inf(𝜌) ∩𝐹̸= 0; 𝑥𝑖∈ant((𝑞𝑖−1, 𝑞𝑖)) for 1 ≤𝑖, and 𝑥𝑙∈ant((𝑞𝑙−1, 𝑞𝑙)) ∩ ¬cons((𝑞𝑙−1, 𝑞𝑙)), that is, 𝑥𝑙∉cons((𝑞𝑙−1, 𝑞𝑙)).h A critical difficulty in checking the language containment, 𝐿(𝑀1) ⊆𝐿(𝑀2), is the construction of a complement finite- state automaton 𝑀𝑐 2 of 𝑀2. Complementing a nondetermin- istic automaton with 𝑛states resulted in an automaton with 22𝑂(𝑛) [23]. Some optimized complementing constructions were introduced with 2𝑂(𝑛log 𝑛) [16–22]. However, the com- plexity is still exponential. 𝑞 𝑞 𝑞 𝑞 Therefore, 𝑋⊭𝜔𝐺, that is, 𝑋∈Σ𝜔−𝐿𝜔(𝐺). Given a finite-state automaton 𝑀 = ⟨𝑄, Σ, 𝐸, 𝐿𝐹, 𝑞0, 𝐹⟩, build an assertion graph 𝐺𝑀as 𝐺𝑀= ⟨𝑄, Σ, 𝐸, 𝑞0, ant, cons, 𝐹⟩where ant(𝑒) = 𝐿𝐹(𝑒), cons(𝑒) = 0 for all 𝑒∈𝐸. This construction is the same as the construction proposed in [15]. Although we can transform deterministic assertion graphs to deterministic finite-state automata, in many cases, assertion graphs are nondeterministic. This may cause state space blowups when checking language containment between automata corresponding to these assertion graphs. We observe that in determining the implication relation between an assertion graph and its refinements constructed using the refinement strategies in [2], we can transform the nondeterministic assertion graph 𝐺to deterministic assertion graph 𝐺det by adding only one variable without adding any states and then apply the refinements to 𝐺det. The requirement of our method is that the refinements applied to 𝐺det will not cause nondeterminism. The refinement strate- gies in [2] satisfy our requirement. The language containment of deterministic finite state automata can be checked in polynomial time [19]. Therefore, the state space blowup problem can be avoided in determining the implication relation between a refined assertion graph and an original assertion graph. Theorem 14. 𝐿∗(𝐺𝑀) = Σ∗−𝐿∗(𝑀) and 𝐿𝜔(𝐺𝑀) = Σ𝜔− 𝐿𝜔(𝑀). Proof. Directly from the construction of 𝐺𝑀and the lan- guage definitions in [15]. Theorem 15. If 𝑀is a DFA, then 𝐺𝑀is a DAG. Proof. Directly from the construction of 𝐺𝑀and the deter- ministic definition. Proof. Directly from the construction of 𝐺𝑀and the deter- ministic definition. Journal of Applied Mathematics Journal of Applied Mathematics If 𝑙< 𝑖≤𝑛, 𝑥𝑖∈𝐿𝐹((𝑞𝑖−1, 𝑘𝑖−1), (𝑞𝑖, 𝑘𝑖)) = 𝐿𝐹((𝑞𝑖−1, 1), (𝑞𝑖, 1)) = ant((𝑞𝑖−1, 𝑞𝑖)). Therefore, 𝑋∉𝐿∗(𝐺). If 𝑙< 𝑖≤𝑛, 𝑥𝑖∈𝐿𝐹((𝑞𝑖−1, 𝑘𝑖−1), (𝑞𝑖, 𝑘𝑖)) = 𝐿𝐹((𝑞𝑖−1, 1), (𝑞𝑖, 1)) = ant((𝑞𝑖−1, 𝑞𝑖)). Therefore, 𝑋∉𝐿∗(𝐺). If 𝑙< 𝑖≤𝑛, 𝑥𝑖∈𝐿𝐹((𝑞𝑖−1, 𝑘𝑖−1), (𝑞𝑖, 𝑘𝑖)) = 𝐿𝐹((𝑞𝑖−1, 1), (𝑞𝑖, 1)) = ant((𝑞𝑖−1, 𝑞𝑖)). is, 𝐿∗(𝑀∗𝑐) = Σ∗−𝐿∗(𝑀). Let 𝑀𝜔𝑐be an automaton whose infinite language is the complement of the infinite language of an automaton 𝑀; that is, 𝐿𝜔(𝑀𝜔𝑐) = Σ𝜔−𝐿𝜔(𝑀). Theorem 13. 𝐿𝜔(𝑀𝐺) = Σ𝜔−𝐿𝜔(𝐺). Theorem 13. 𝐿𝜔(𝑀𝐺) = Σ𝜔−𝐿𝜔(𝐺). Theorem 17. 𝐺1⇒∗𝐺2 iff 𝐿∗((𝑀𝐺1)∗𝑐× 𝑀𝐺2) = 0. 𝐺1⇒𝜔𝐺2 iff 𝐿𝜔((𝑀𝐺1)𝜔𝑐× 𝑀𝐺2) = 0. Proof. First, we prove 𝐿𝜔(𝑀𝐺) ⊇Σ𝜔−𝐿𝜔(𝐺). Suppose 𝑋= 𝑥1𝑥2 ⋅⋅⋅∈Σ𝜔−𝐿𝜔(𝐺). Then there exists an infinite path 𝜌= 𝑞0𝑞1 ⋅⋅⋅in 𝐺with (𝑞𝑖−1, 𝑞𝑖) ∈𝐸and Inf(𝜌) ∩𝐹̸= 0 such that 𝑥𝑖∈ant((𝑞𝑖−1, 𝑞𝑖)) for 1 ≤𝑖; and ∃𝑙, 1 ≤𝑙, 𝑥𝑙∉cons((𝑞𝑙−1, 𝑞𝑙)). Let a path 𝜌󸀠of 𝑀𝐺be 𝜌󸀠= (𝑞0, 0)(𝑞1, 0) ⋅⋅⋅(𝑞𝑙−1, 0)(𝑞𝑙, 1)(𝑞𝑙+1, 1) ⋅⋅⋅. This path 𝜌󸀠will visit infinitely often every (𝑞, 1) ∈𝐹󸀠if 𝑞∈Inf(𝜌) ∩𝐹. And 𝑥𝑖∈𝐿𝐹((𝑞𝑖−1, 𝑘𝑖−1), (𝑞𝑖, 𝑘𝑖)), where 𝑘𝑖−1 = 0 if 1 ≤𝑖≤𝑙, 𝑘𝑖= 1 if 𝑙< 𝑖. Thus 𝑋∈𝐿𝜔(𝑀𝐺). Second, we prove 𝐿𝜔(𝑀𝐺) ⊆Σ𝜔−𝐿𝜔(𝐺). Suppose 𝑋= 𝑥1𝑥2 ⋅⋅⋅∈𝐿𝜔(𝑀𝐺).hi 󸀠 Proof. (𝐺1⇒∗𝐺2) ⇔𝐿∗(𝐺1) ⊆𝐿∗(𝐺2) ⇔𝐿∗(𝑀𝐺1) ⊇ 𝐿∗(𝑀𝐺2) ⇔𝐿∗((𝑀𝐺1)∗𝑐× 𝑀𝐺2) = 0. (𝐺1⇒𝜔𝐺2) ⇔ 𝐿𝜔(𝐺1) ⊆ 𝐿𝜔(𝐺2) ⇔ 𝐿𝜔(𝑀𝐺1) ⊇ 𝐿𝜔(𝑀𝐺2) ⇔𝐿𝜔((𝑀𝐺1)𝜔𝑐× 𝑀𝐺2) = 0. Proof. (𝐺1⇒∗𝐺2) ⇔𝐿∗(𝐺1) ⊆𝐿∗(𝐺2) ⇔𝐿∗(𝑀𝐺1) ⊇ 𝐿∗(𝑀𝐺2) ⇔𝐿∗((𝑀𝐺1)∗𝑐× 𝑀𝐺2) = 0. (𝐺1⇒𝜔𝐺2) ⇔ 𝐿𝜔(𝐺1) ⊆ 𝐿𝜔(𝐺2) ⇔ 𝐿𝜔(𝑀𝐺1) ⊇ 𝐿𝜔(𝑀𝐺2) ⇔𝐿𝜔((𝑀𝐺1)𝜔𝑐× 𝑀𝐺2) = 0. In GSTE, because of its aggressive abstraction, abstraction refinement is often necessary [2]. Verifying whether a circuit 𝐶satisfies an assertion graph 𝐺, 𝐶⊨𝐺, using the GSTE engine may produce false negative if 𝐺is too abstract. Thus, it is necessary to refine 𝐺into a refined assertion graph 𝐺ref. If we can verify 𝐶⊨𝐺ref, then we can conclude 𝐶⊨𝐺. But how can we guarantee that (𝐶⊨𝐺ref) ⇒(𝐶⊨𝐺) if the assertion graph is complex? We must establish 𝐺ref ⇒𝐺. We can verify 𝐺ref ⇒𝐺by transforming it to an automata language containment test using Theorem 17.fi 𝑘𝑖−1 = 0 if 1 ≤𝑖≤𝑙, 𝑘𝑖= 1 if 𝑙< 𝑖. Thus 𝑋∈𝐿𝜔(𝑀𝐺). Second, we prove 𝐿𝜔(𝑀𝐺) ⊆Σ𝜔−𝐿𝜔(𝐺). Suppose 𝑋= 𝑥1𝑥2 ⋅⋅⋅∈𝐿𝜔(𝑀𝐺).hi 󸀠 Then there exists an infinite path 𝜌󸀠= (𝑞0, 0)(𝑞1, 0) ⋅⋅⋅ (𝑞𝑙−1, 0)(𝑞𝑙, 1)(𝑞𝑙+1, 1) ⋅⋅⋅in 𝑀𝐺with (𝑞𝑖−1, 𝑞𝑖) ∈ 𝐸and Inf(𝜌󸀠) ∩𝐹󸀠 ̸= 0 such that 𝑥𝑖∈𝐿𝐹((𝑞𝑖−1, 𝑘𝑖−1), (𝑞𝑖, 𝑘𝑖)), where 𝑘𝑖= 0 if 1 ≤𝑖< 𝑙, 𝑘𝑖= 1 if 𝑙≤𝑖≤𝑛. Then there exists an infinite path 𝜌󸀠= (𝑞0, 0)(𝑞1, 0) ⋅⋅⋅ (𝑞𝑙−1, 0)(𝑞𝑙, 1)(𝑞𝑙+1, 1) ⋅⋅⋅in 𝑀𝐺with (𝑞𝑖−1, 𝑞𝑖) ∈ 𝐸and Inf(𝜌󸀠) ∩𝐹󸀠 ̸= 0 such that 𝑥𝑖∈𝐿𝐹((𝑞𝑖−1, 𝑘𝑖−1), (𝑞𝑖, 𝑘𝑖)), where 𝑘𝑖= 0 if 1 ≤𝑖< 𝑙, 𝑘𝑖= 1 if 𝑙≤𝑖≤𝑛. 𝜌󸀠= (𝑞0, 0) (𝑞1, 0) ⋅⋅⋅(𝑞𝑙−0, 0) (𝑞𝑙, 1) ⋅⋅⋅(𝑞𝑛, 1) . (1) 𝜌󸀠= (𝑞0, 0) (𝑞1, 0) ⋅⋅⋅(𝑞𝑙−0, 0) (𝑞𝑙, 1) ⋅⋅⋅(𝑞𝑛, 1) . (1) Then 𝑥𝑖∈𝐿𝐹((𝑞𝑖−1, 𝑘𝑖−1), (𝑞𝑖, 𝑘i)), where 𝑘𝑖= 0 if 1 ≤𝑖< 󸀠 h 𝑙, 𝑘𝑖= 1 if 𝑙≤𝑖≤𝑛; and (𝑞𝑛, 1) ∈𝐹󸀠. Therefore, 𝑋∈𝐿∗(𝑀𝐺). Second, we show 𝐿∗(𝑀𝐺) ⊆Σ∗−𝐿∗(𝐺). Suppose 𝑋= 𝑥1𝑥2 ⋅⋅⋅𝑥𝑛∈𝐿∗(𝑀𝐺). Then there exists a path as follows: h 𝑙, 𝑘𝑖= 1 if 𝑙≤𝑖≤𝑛; and (𝑞𝑛, 1) ∈𝐹󸀠. Therefore, 𝑋∈𝐿∗(𝑀𝐺). Second, we show 𝐿∗(𝑀𝐺) ⊆Σ∗−𝐿∗(𝐺). Suppose 𝑋= 𝑥1𝑥2 ⋅⋅⋅𝑥𝑛∈𝐿∗(𝑀𝐺). Then there exists a path as follows: (1) 𝑄󸀠= (𝑄−{𝑞0}) × {0, 1} ∪{(𝑞0, 0)}, 𝑞󸀠 0 = (𝑞0, 0); (1) 𝑄󸀠= (𝑄−{𝑞0}) × {0, 1} ∪{(𝑞0, 0)}, 𝑞󸀠 0 = (𝑞0, 0); (2) 𝐸󸀠= {((𝑞1, 𝑘1), (𝑞2, 𝑘2)) | (𝑞1, 𝑞2) ∈𝐸; if 𝑘1 = 0, then 𝑘2 ∈{0, 1}; if 𝑘1 = 1, then 𝑘2 = 1} (See Figure 2); (2) 𝐸󸀠= {((𝑞1, 𝑘1), (𝑞2, 𝑘2)) | (𝑞1, 𝑞2) ∈𝐸; if 𝑘1 = 0, then 𝑘2 ∈{0, 1}; if 𝑘1 = 1, then 𝑘2 = 1} (See Figure 2); pp 1 2 𝑛 ( 𝐺) Then there exists a path as follows: 𝜌󸀠= (𝑞0, 0) (𝑞1, 0) ⋅⋅⋅(𝑞𝑙−0, 0) (𝑞𝑙, 1) ⋅⋅⋅(𝑞𝑛, 1) , (2) (3) 𝐿𝐹((𝑞1, 0), (𝑞2, 0)) = ant((𝑞1, 𝑞2)) ∩cons((𝑞1, 𝑞2)), 𝐿𝐹((𝑞1, 0), (𝑞2, 1)) = ant((𝑞1, 𝑞2)) ∩¬cons((𝑞1, 𝑞2)), 𝐿𝐹((𝑞1, 1), (𝑞2, 1)) = ant((𝑞1, 𝑞2)); (4) 𝐹󸀠= {(𝑞, 1) | 𝑞∈𝐹}. such that 𝑥𝑖∈𝐿𝐹((𝑞𝑖−1, 𝑘𝑖−1), (𝑞𝑖, 𝑘𝑖)), where 𝑘𝑖= 0 if 1 ≤𝑖< 𝑙, 𝑘𝑖= 1 if 𝑙≤𝑖≤𝑛; and (𝑞𝑛, 1) ∈𝐹󸀠. such that 𝑥𝑖∈𝐿𝐹((𝑞𝑖−1, 𝑘𝑖−1), (𝑞𝑖, 𝑘𝑖)), where 𝑘𝑖= 0 if 1 ≤𝑖< 𝑙, 𝑘𝑖= 1 if 𝑙≤𝑖≤𝑛; and (𝑞𝑛, 1) ∈𝐹󸀠. such that 𝑥𝑖∈𝐿𝐹((𝑞𝑖−1, 𝑘𝑖−1), (𝑞𝑖, 𝑘𝑖)) 𝑙, 𝑘𝑖= 1 if 𝑙≤𝑖≤𝑛; and (𝑞𝑛, 1) ∈𝐹󸀠. According to the construction of 𝑀𝐺from 𝐺, we know that 𝜌 = 𝑞0𝑞1 ⋅⋅⋅𝑞𝑛is a path of 𝐺; if 1 ≤ 𝑖 < 𝑙, 𝑥𝑖 ∈ 𝐿𝐹((𝑞𝑖−1, 𝑘𝑖−1), (𝑞𝑖, 𝑘𝑖)) = 𝐿𝐹((𝑞𝑖−1, 0), (𝑞𝑖, 0)) = ant((𝑞𝑖−1, 𝑞𝑖)) ∩cons((𝑞𝑖−1, 𝑞𝑖)) ⊆ant((𝑞𝑖−1, 𝑞𝑖)). (4) 𝐹󸀠= {(𝑞, 1) | 𝑞∈𝐹}. Intuitively, 𝑀𝐺is constructed to accept all strings (or words) that are not accepted by 𝐺. Such a string (or word) satisfies the antecedent of each edge traversed while violating the consequent of at least of such edge. 𝑥𝑙∈𝐿𝐹((𝑞𝑙−1, 0), (𝑞𝑙, 1)) = ant((𝑞𝑙−1, 𝑞𝑙)) ∩¬cons((𝑞𝑙−1, 𝑞𝑙)), that is, 𝑥𝑙∈ant((𝑞𝑙−1, 𝑞𝑙)), but 𝑥𝑙∉cons((𝑞𝑙−1, 𝑞𝑙)). 4 4. Application to GSTE Assertion Graphs Implication Figure 3 illustrates the determinization process: there are three outgoing edges from state 𝑝: (𝑝, 𝑞1), (𝑝, 𝑞2), and (𝑝, 𝑞3). Two of these three antecedents ant((𝑝, 𝑞2)) = {3, 4} and ant((𝑝, 𝑞3)) = {3, 5} in Figure 3(a) are overlapped. So, it is a non-deterministic transition. We constrain these two antecedents by adding a variable “det” with a domain of {1, 2} and changing ant((𝑝, 𝑞2)) to {3, 4}×{det = 1} and ant((𝑝, 𝑞3)) to {3, 5} × {det = 2} in Figure 3(b), the determinized assertion graph 𝐺det. For simplification, the consequents and the antecedents which are not needed to be constrained will not be followed by ×det.hii 4.2. Case Study. An FIFO is a common circuit within a microprocessor design. The design requirements of an FIFO can be fairly complex due to the variable lengths, different rates of data throughput, and timing. Therefore, it makes a good practice to verify the FIFO design against these requirements. In general, the behavior of an FIFO must meet the following requirements: (1) correctness of full and empty flags, and (2) enqueued data must be dequeued in the correct order while maintaining uncorrupted data. The FIFO has 0 entry after the reset. If an enqueue only operation occurred, the total number of entries increases by 1. On the other hand, if a dequeue only operation occurred, the total number of entries decreases by 1 when the number of entries is bigger than 0. The empty flag is set when the number of entries is 0 and the full flag is set when contents reach the depth of the FIFO. y The projection of the (finite or infinite) language of 𝐺det is defined as 𝐿∗ proj(𝐺det) = {𝑥1𝑥2 ⋅⋅⋅𝑥𝑛 ∈ Σ∗ | ∃𝑑𝑖∈𝐷(det), s.t. (𝑥1, 𝑑1)(𝑥2, 𝑑2) ⋅⋅⋅(𝑥𝑛, 𝑑𝑛) ∈𝐿∗(𝐺det)}, 𝐿𝜔 proj(𝐺det) = {𝑥1𝑥2 ⋅⋅⋅∈Σ𝜔| ∃𝑑𝑖∈𝐷(det), s.t. (𝑥1, 𝑑1) (𝑥2, 𝑑2) ⋅⋅⋅∈𝐿𝜔(𝐺det)}. Lemma 18. 𝐿∗(𝐺) = 𝐿∗ proj(𝐺𝑑𝑒𝑡), 𝐿𝜔(𝐺) = 𝐿𝜔 proj(𝐺𝑑𝑒𝑡). An assertion graph of a 3-deep FIFO circuit is shown in Figure 4 without determinization variable det.hi Proof. First, suppose 𝑋= 𝑥1𝑥2 ⋅⋅⋅𝑥𝑛∈𝐿∗(𝐺). For any finite path 𝜌= 𝑞0𝑞1 ⋅⋅⋅𝑞𝑛in 𝐺det, it is also a path in 𝐺 according to the construction of 𝐺det (because it does not change the graph structure of the assertion graph 𝐺). If 𝑋⊨ant𝜌, that is, 𝑥𝑖∈ant((𝑞𝑖−1, 𝑞𝑖)) for 1 ≤𝑖≤𝑛, then 𝑋⊨cons𝜌, namely, 𝑥𝑖 ∈ cons((𝑞𝑖−1, 𝑞𝑖)) for 1 ≤ 𝑖 ≤ 𝑛. 4. Application to GSTE Assertion Graphs Implication 4.1. Transforming GSTE Assertion Graphs Implication to Automata Language Containment. The complement relation between an assertion graph and a finite-state automaton can be applied to determine the implication relation between assertion graphs by transforming the GSTE assertion graphs implication to the finite-state automata language contain- ment. 4.1. Transforming GSTE Assertion Graphs Implication to Automata Language Containment. The complement relation between an assertion graph and a finite-state automaton can g pi be applied to determine the implication relation between assertion graphs by transforming the GSTE assertion graphs implication to the finite-state automata language contain- ment. For any given assertion graph 𝐺= ⟨𝑄, Σ, 𝐸, 𝑞0, ant, cons, 𝐹⟩, let 𝐺ref be a refined assertion graph of 𝐺. If 𝐺is a non- deterministic assertion graph, we add an additional variable “det” to constrain the overlapped antecedents of the outgoing edges of every state in 𝐺and the resulting assertion graph is denoted as 𝐺det; that is, 𝐺det = ⟨𝑄, Σ × det, 𝐸, 𝑞0, ant × det, cons × det, 𝐹⟩. The value domain of “det” is from 1 to the maximal number 𝑛that we need to distinguish the overlapped antecedents of the outgoing edges of every state in the assertion graph 𝐺, denoted as 𝐷(det). Definition 16. Given two assertion graphs 𝐺1 and 𝐺2, 𝐺1⇒∗𝐺2 if and only if 𝐿∗(𝐺1) ⊆𝐿∗(𝐺2) and 𝐺1⇒𝜔𝐺2 if and only if 𝐿𝜔(𝐺1) ⊆𝐿𝜔(𝐺2). Definition 16. Given two assertion graphs 𝐺1 and 𝐺2, 𝐺1⇒∗𝐺2 if and only if 𝐿∗(𝐺1) ⊆𝐿∗(𝐺2) and 𝐺1⇒𝜔𝐺2 if and only if 𝐿𝜔(𝐺1) ⊆𝐿𝜔(𝐺2). Let 𝑀∗𝑐be an automaton whose finite language is the complement of the finite language of an automaton 𝑀; that Journal of Applied Mathematics Journal of Applied Mathematics 5 {1, 2}/cons1 {3, 4}/cons2 {3, 5}/cons3 {1, 2}/cons1 {3, 4}&det = 1/cons2 {3, 5}&det = 2/cons3 (a) (b) p p q1 q2 q3 q1 q2 q3 Figure 3: Assertion graph determinization. {1, 2}/cons1 {3, 4}&det = 1/cons2 {3, 5}&det = 2/cons3 (b) p q1 q2 q3 {1, 2}/cons1 {3, 4}/cons2 {3, 5}/cons3 (a) p q1 q2 q3 ⇒(𝐿proj(𝐺ref det) ⊆𝐿proj(𝐺det)) (definition of projection language). {1, 2}/cons1 ⇒(𝐿(𝐺ref) ⊆𝐿(𝐺)) (Lemma 18). fi (a) (b) In the above proof, the language 𝐿(𝐺) can be finite language 𝐿∗(𝐺) or infinite language 𝐿𝜔(𝐺). Thus, Theorem 19 holds for both finite and infinite languages, respectively. Figure 3: Assertion graph determinization. Figure 3: Assertion graph determinization. 4. Application to GSTE Assertion Graphs Implication According to the definition of 𝐺det, there exists 𝑑𝑖∈𝐷(det) such that ant((𝑞𝑖−1, 𝑞𝑖)) × 𝑑𝑖is the antecedent of the edge (𝑞𝑖−1, 𝑞𝑖) in 𝐺det if there is a constraint det = 𝑑𝑖for this antecedent, or a subset of the antecedent of the edge (𝑞𝑖−1, 𝑞𝑖) in 𝐺det if there is no constraint for this antecedent. Therefore, 𝑋det = (𝑥1, 𝑑1)(𝑥2, 𝑑2) ⋅⋅⋅(𝑥𝑛, 𝑑𝑛)⊨ant×det𝜌, and 𝑋det⊨cons×det 𝜌because there is no constraint for the consequents. Thus, 𝑋det ∈ 𝐿∗(𝐺det). According to the definition of the projection language of 𝐺det, 𝑋∈𝐿∗ proj(𝐺det). The top part of the assertion graph specifies the number of filled entries. However, it cannot guarantee whether the enqueued data is corrupted or not. In order to overcome this situation, an enqueued vector of distinct symbolic constants is used at an arbitrary time as shown in the bottom portion of the graph.h The assertion graph shown in Figure 4 without deter- minization variable det has the potential problem of overap- proximation. In order to overcome this problem, a refined assertion graph shown in Figure 5 without determinization variable det is introduced. It unfolds the graph at the states where the precision is lost.i Using the GSTE engine, we can verify the refined asser- tion graph on the FIFO circuit. To conclude that the original assertion graph also holds on the circuit, we must establish that the refined assertion graph implies the original assertion graph. Since both assertion graphs are non-deterministic, we first determinize the original assertion graph. A variable “det” whose domain is {1, 2} is added to determinize the original assertion graph. We add “det = 1” in the antecedents on the edges (𝑖-filled, (𝑖+ 1)-filled) and “det = 2” in the antecedents on the edges (𝑖-filled, 𝑖-ahead), respectively, for 𝑖= 0, 1, 2. And We add “det = 1” in the antecedents on the edges (𝑖-filled, 𝑖-filled) and “det = 2” in the antecedents on the edges (𝑖-filled, (𝑖−1)-ahead), respectively, for 𝑖= 1, 2. The determinized assertion graph is shown in Figure 4. We then refine the assertion graph in Figure 4 by applying the same refinements that refine the original assertion graph to the refined assertion graph and get the determinized and refined assertion graph shown in Figure 5. j Second, suppose that 𝑋= 𝑥1𝑥2 ⋅⋅⋅𝑥𝑛∈𝐿∗ proj(𝐺det). 4. Application to GSTE Assertion Graphs Implication / enq iff deq &det = 1/ !empty & !full enq & !enq/ empty & !full !deq/ !empty & full 2-filled 1-filled deq/ !empty & !full Done 2-ahead Figure 4: Determinized assertion graph for FIFO. Figure 4: Determinized assertion graph for FIFO. Reset/true Init !enq/ empty & !full enq & det = 1/ !empty & !full !enq & !deq & det = 1/ !empty & !full enq & deq & det = 1/ !empty & !full enq & deq & det = 1/ !empty & !full !enq & !deq & det = 1/ !empty & !full enq & !deq & det =1/ !empty & full !deq/ !empty & full enq & !deq & det = 1/ !empty & !full 0-filled 3-filled 2-filled 1-filled !enq & deq/ empty & !full deq/ !empty & !full !enq & deq/ !empty & !full enq & !deq & din = D[9:0] & det = 2/ true enq & !deq & din = D[9:0] & det = 2/ true enq & !deq & din = D[9:0] & det = 2/ true enq & deq & din = D[9:0] & det = 2/ true enq & deq & din = D[9:0] & det = 2/ true Done d / ! 4. Application to GSTE Assertion Graphs Implication Then there exists 𝑑𝑖∈𝐷(det) such that 𝑋det = (𝑥1, 𝑑1) (𝑥2, 𝑑2) ⋅⋅⋅(𝑥𝑛, 𝑑𝑛) ∈𝐿∗(𝐺det), which implies that if (𝑥𝑖, 𝑑𝑖) ∈ ant((𝑞𝑖−1, 𝑞𝑖)) × 𝑑𝑖for 1 ≤ 𝑖 ≤ 𝑛, then (𝑥𝑖, 𝑑𝑖) ∈ cons((𝑞𝑖−1, 𝑞𝑖))×det. And (𝑥𝑖, 𝑑𝑖) ∈ant((𝑞𝑖−1, 𝑞𝑖))×𝑑𝑖implies 𝑥𝑖∈ant((𝑞𝑖−1, 𝑞𝑖)), (𝑥𝑖, 𝑑𝑖) ∈cons((𝑞𝑖−1, 𝑞𝑖)) × det implies 𝑥𝑖∈cons((𝑞𝑖−1, 𝑞𝑖)).h Therefore 𝑋∈𝐿∗(𝐺). h Combining these two cases, we have 𝐿∗(𝐺) = 𝐿∗ proj(𝐺det). Similarly, 𝐿𝜔(𝐺) = 𝐿𝜔 proj(𝐺det) is also true. Theorem 19. (𝐺𝑟𝑒𝑓 𝑑𝑒𝑡⇒𝐺𝑑𝑒𝑡) ⇒(𝐺𝑟𝑒𝑓⇒𝐺). Proof. (𝐺ref det ⇒𝐺det) ⇔(𝐿(𝐺ref det) ⊆𝐿(𝐺det)) (definition of implication). Journal of Applied Mathematics 6 6 Reset/true Init !enq/ empty & !full enq & det = 1/ !empty & !full enq iff deq &det = 1/ !empty & !full enq & !deq &det = 1/ !empty & !full enq iff deq &det = 1/ !empty & !full enq & !deq &det = 1/ !empty & full !deq/ !empty & full 0-filled 1-filled 2-filled 3-filled !enq & deq/ empty & !full enq & !deq & din = D[9:0] & det = 2/ true !enq & deq/ !empty & !full enq & !deq & din = D[9:0] & det = 2/ true deq/ !empty & !full enq & !deq & din = D[9:0] det = 2/ true enq & deq & din = D[9:0] & det = 2/ true enq & deq & din = D[9:0] & det = 2/ true Done 0-ahead 2-ahead 1-ahead deq/ dout = D[9:0] deq/ true deq/ true !deq/ true !deq/ true !deq/ true Figure 4: Determinized assertion graph for FIFO. 4. Application to GSTE Assertion Graphs Implication Reset/true Init !enq/ empty & !full enq & det = 1/ !empty & !full enq iff deq &det = 1/ !empty & !full enq & !deq &det = 1/ !empty & !full enq iff deq &det = 1/ !empty & !full enq & !deq &det = 1/ !empty & full !deq/ !empty & full 0-filled 1-filled 2-filled 3-filled !enq & deq/ empty & !full enq & !deq & din = D[9:0] & det = 2/ true !enq & deq/ !empty & !full enq & !deq & din = D[9:0] & det = 2/ true deq/ !empty & !full enq & !deq & din = D[9:0] det = 2/ true enq & deq & din = D[9:0] & det = 2/ true enq & deq & din = D[9:0] & det = 2/ true Done 0-ahead 2-ahead 1-ahead deq/ dout = D[9:0] deq/ true deq/ true !deq/ true !deq/ true !deq/ true Figure 4: Determinized assertion graph for FIFO. 4. Application to GSTE Assertion Graphs Implication Reset/true Init !enq/ empty & !full enq & det = 1/ !empty & !full !enq & !deq & det = 1/ !empty & !full enq & deq & det = 1/ !empty & !full enq & deq & det = 1/ !empty & !full !enq & !deq & det = 1/ !empty & !full enq & !deq & det =1/ !empty & full !deq/ !empty & full enq & !deq & det = 1/ !empty & !full 0-filled 3-filled 2-filled 1-filled !enq & deq/ empty & !full deq/ !empty & !full !enq & deq/ !empty & !full enq & !deq & din = D[9:0] & det = 2/ true enq & !deq & din = D[9:0] & det = 2/ true enq & !deq & din = D[9:0] & det = 2/ true enq & deq & din = D[9:0] & det = 2/ true enq & deq & din = D[9:0] & det = 2/ true Done deq/ dout = D[9:0] !enq & deq/ true deq/ true deq/ dout = D[9:0] !enq & !deq/ true enq & !deq/ true enq & deq/ true enq & !deq/ true !enq & !deq/ true deq/ true deq/ dout = D[9:0] enq & !deq/ true !enq & !deq/ true !deq/ true !deq/ true 0 ahead, 1 filled 0 ahead, 2 filled 2 ahead, 3 filled 1 ahead, 3 filled 1 ahead, 2 filled 0 ahead, 3 filled Reset/true Init !enq/ empty & !full enq & det = 1/ !empty & !full enq iff deq &det = 1/ !empty & !full enq & !deq &det = 1/ !empty & !full enq iff deq &det = 1/ !empty & !full enq & !deq &det = 1/ !empty & full !deq/ !empty & full 0-filled 1-filled 2-filled 3-filled !enq & deq/ empty & !full enq & !deq & din = D[9:0] & det = 2/ true !enq & deq/ !empty & !full enq & !deq & din = D[9:0] & det = 2/ true deq/ !empty & !full enq & !deq & din = D[9:0] det = 2/ true enq & deq & din = D[9:0] & det = 2/ true enq & deq & din = D[9:0] & det = 2/ true Done 0-ahead 2-ahead 1-ahead deq/ dout = D[9:0] deq/ true deq/ true !deq/ true !deq/ true !deq/ true Figure 4: Determinized assertion graph for FIFO. Journal of Applied Mathematics 7 Journal of Applied Mathematics We select COSPAN [24, 25] as the model checking engine to perform language containment test. We code the automata corresponding to the assertion graphs in Figures 4 and 5 in S/R, the input language of COSPAN, and use COSPAN to verify that the language of the automaton corresponding to the assertion graph in Figure 4 is contained in the language of the assertion graph in Figure 5. COSPAN establishes the language containment using 0.156 megabytes memory and 0.02 CPU seconds on a Pentium IV 2.8 GHz computer. Conference on Correct Hardware Design and Verification Meth- ods (CHARME ’03), pp. 170–184, 2003. Conference on Correct Hardware Design and Verification Meth- ods (CHARME ’03), pp. 170–184, 2003. [8] C. T. Chou, “The mathematical foundation of symbolic trajec- tory evaluation,” in Computer-Aided Verification, vol. 1633, pp. 196–207, 1999. [9] C. J. H. Seger and R. E. Bryant, “Formal verification by symbolic evaluation of partially-ordered trajectories,” Formal Methods in System Design, vol. 6, no. 2, pp. 147–190, 1995. [10] M. Aagaard, R. B. Jones, and C. J. H. Seger, “Combining theorem proving and trajectory evaluation in an industrial environment,” in Design Automation Conference, pp. 538–541, 1998. Acknowledgments This paper is partially supported by the National Natural Sci- ence Foundation of China (no. 60973016 and no. 61272175), and the National Basic Research Program of China (973 Program: no. 2010CB328004). [18] O. Kupferman and M. Y. Vardi, “From complementation to certification,” in Proceedings of the Tools and algorithms for the construction and analysis of systems (TACAS ’04), vol. 2988, pp. 591–606, 2004, Lecture Notes on Computer Science. [19] R. P. Kurshan, “Complementing deterministic B¨uchi automata in polynomial time,” Journal of Computer and System Sciences, vol. 35, no. 1, pp. 59–71, 1987.fi 5. Conclusion [11] P. Bjesse, T. Leonard, and A. Mokkedem, “Finding bugs in an 𝛼 microprocessor using satisfiability solvers,” in Computer-Aided Verification, pp. 454–464, 2001. In this paper, we have established the complement relation between GSTE assertion graphs and finite-state automata with expressiveness of regular languages and 𝜔-regular lan- guages. We present an algorithm that transforms a GSTE assertion graph to a finite-state automaton and vice versa. Using this algorithm, we transform the problem of GSTE assertion graphs implication to the problem of automata language containment. We avoid the exponential state space blowups in checking language containment of non- deterministic finite-state automata when determining the implication relation between a nondeterministic assertion graph and its refinement derived following the refinement strategies given in [2]. This avoidance is achieved by trans- forming the implication relation between nondeterministic assertion graphs to the implication relation between deter- ministic assertion graphs without adding any states. This application has been illustrated with a case study for verifying properties of an FIFO circuit. In this paper, we have established the complement relation between GSTE assertion graphs and finite-state automata with expressiveness of regular languages and 𝜔-regular lan- guages. We present an algorithm that transforms a GSTE assertion graph to a finite-state automaton and vice versa. [12] K. L. Nelson, A. Jain, and R. E. Bryant, “Formal verification of a superscalar execution unit,” in Design Automation Conference, pp. 161–167, 1997. [13] M. Pandey, R. Raimi, D. L. Beatty, and R. E. Bryant, “Formal verification of PowerPC arrays using symbolic trajectory evalu- ation,” in Design Automation Conference, pp. 649–654, 1996. [14] G. Yang, J. Yang, W. N. N. Hung, and X. Song, “Implication of assertion graphs in GSTE,” in Asia South Pacific Design Automation Conference, pp. 1060–1063, 2005. [15] R. Sebastiani, E. Singerman, S. Tonetta, and M. Y. Vardi, “GSTE is partitioned model checking,” in Computer-Aided Verification, vol. 3114, pp. 229–241, Springer, Berlin, Germany, 2004. [16] E. Friedgut, O. Kupferman, and M. Vardi, “B¨uchi complementa- tion made tighter,” in Proceedings of the 2nd International Sym- posium on Automated Technology for Verification and Analysis, pp. 64–78, 2004, Lecture Notes in Computer Science. [17] N. Klarlund, “Progress measures for complementation of 𝜔- automata with applications to temporal logic,” in Proceedings of the 32nd Annual Symposium of Foundations of Computer Science, pp. 358–367, 1991. 4. Application to GSTE Assertion Graphs Implication & d / deq/ 0 ahead, 1 filled 2 ahead, 3 filled 1 ahead, 2 filled Reset/true Init !enq/ empty & !full enq & det = 1/ !empty & !full !enq & !deq & det = 1/ !empty & !full enq & deq & det = 1/ !empty & !full enq & deq & det = 1/ !empty & !full !enq & !deq & det = 1/ !empty & !full enq & !deq & det =1/ !empty & full !deq/ !empty & full enq & !deq & det = 1/ !empty & !full 0-filled 3-filled 2-filled 1-filled !enq & deq/ empty & !full deq/ !empty & !full !enq & deq/ !empty & !full enq & !deq & din = D[9:0] & det = 2/ true enq & !deq & din = D[9:0] & det = 2/ true enq & !deq & din = D[9:0] & det = 2/ true enq & deq & din = D[9:0] & det = 2/ true enq & deq & din = D[9:0] & det = 2/ true Done deq/ dout = D[9:0] !enq & deq/ true deq/ true deq/ dout = D[9:0] !enq & !deq/ true enq & !deq/ true enq & deq/ true enq & !deq/ true !enq & !deq/ true deq/ true !deq/ true deq/ dout = D[9:0] enq & !deq/ true !enq & !deq/ true !deq/ true !deq/ true 0 ahead, 1 filled 0 ahead, 2 filled 2 ahead, 3 filled 1 ahead, 3 filled 1 ahead, 2 filled 0 ahead, 3 filled Figure 5: Determinized and refined assertion graph for FIFO. enq & deq & det = 1/ !empty & !full !enq & !deq & det = 1/ !empty & !full & !d & d 1/ / !enq & !deq & det = 1/ !empty & !full enq & deq & de !empty & !full enq & !d !enq/ empty & !full !deq/ !empty & full enq & !deq & det =1/ !empty & full enq & det = 1/ !empty & !full Reset/true 0-filled Init !enq & deq/ empty & !full !enq & deq/ !empty & !full deq/ !empty & !full 2 ahead, 3 filled Done enq & deq/ true 1 ahead, 3 filled !deq/ true 0 ahead, 3 filled Figure 5: Determinized and refined assertion graph for FIFO. References [20] M. Michel, “Complementation is more difficult with automata on infinite words,” in CNET, Paris, France, 1988. [1] J. Yang and C. J. H. Seger, “Generalized symbolic trajectory evaluation,” Technical Report, 2002. [21] S. Safra, “On the complexity of omega-automata,” in Founda- tions of Computer Science, pp. 319–327, 1988. [2] J. Yang and C. J. H. Seger, “Generalized symbolic trajectory evaluation-abstraction in action,” in FMCAD, vol. 2517, pp. 70– 87, 2002. [22] S. Tasiran, R. Hojati, and R. K. Brayton, “Language containment of non-deterministic omega-automata,” in Proceedings of the IFIP WG 10. 5 Advanced Research Working Conference on Correct Hardware Design and Verification Methods (CHARME ’95), pp. 261–277, Springer, London, UK, 1995. [3] J. Yang and C. J. H. Seger, “Introduction to generalized symbolic trajectory evaluation,” IEEE Transactions on VLSI Systems, vol. 11, no. 3, pp. 345–353, 2003.i [4] J. Yang and C. J. H. Seger, “Compositional specification and model checking in GSTE,” in Computer-Aided Verification, vol. 3114, pp. 216–228, 2004. [23] J. R. B. B¨uchi, “On a decision method in restricted second order arithmetic,” in International Congress For Logic, Methodology and Philosophy of Science, pp. 1–12, 1962. [5] B. Bentley, “High level validation of next generation micro- processors,” in IEEE High Level Design Validation and Test Workshop, 2002. [24] K. Fisler and R. P. Kurshan, “Verifying vhdl designs with cospan,” in Formal Hardware Verification—Methods and Sys- tems in Comparison, pp. 206–247, Springer, London, UK, 1997.i [6] A. J. Hu, J. Casas, and J. Yang, “Efficient generation of monitor circuits for GSTE assertion graphs,” in IEEE/ACM International Conference on Computer-Aided Design, pp. 154–159, 2003. [25] R. P. Kurshan, Computer-Aided Verification of Coordinating Processes, Princeton Series in Computer Science, Princeton University Press, Princeton, NJ, USA, 1994, The automata- theoretic approach. [7] A. J. Hu, J. Casas, and J. Yang, “Reasoning about GSTE assertion graphs,” in Proceedings of the 12th Advanced Research Working
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Calculation of Basalt Fiber Reinforced Concrete Beams for Strength
International journal of multidisciplinary research and analysis
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4,269
1. INTRODUCTION Fiber concrete is a composite material that contains fibers in a cement matrix with an orderly or random distribution. The strength properties of fibrous concrete undoubtedly depend on the type of fiber, fiber geometry, fiber composition, fiber direction and distribution, shape and size of large and fine aggregates. Fiberreinforced concrete improves properties due to fibers. The world's first patent for fiber reinforced concrete structures was awarded to Russian scientist V.P.Nekrasov obtained in 1909 and extensive development of scientific research on fiber concrete, the development of methods for calculating the structures made of it was carried out in the 60s of the twentieth century. The first large-scale application of fiber reinforced concrete in practice began in 1976 with the construction of runways in Russia. However, at that time, this material was not widely used, because the technology of preparation of fiber reinforced concrete and fiber reinforced concrete itself was not perfect [1]. There are many types of fiber concrete products used in various industries. Scientists from Austria, Australia, Belgium, Germany, the Netherlands, Spain, Canada, China, Poland, the United States, France, the Czech Republic, Switzerland, South Africa, Japan and other countries have made significant contributions to the development of fiber concrete, including Shen X., Singha K., Zhang J., Zhang X., Kudyakov K.,NihalL.P., Shiping Li, Yibei Zhang, Wujun Chen and other scholars deserve special mention [2, 3, 4, 5, 6]. The study and analysis of scientific research on the strength, tensile strength, and deformation of flexible fibrous concrete elements based on basalt fibers have shown that to date, experimental and theoretical data in this area have been collected in foreign countries. However, despite the advantages of basalt material and the positive results obtained during many studies, and the practical experience of experimental projects, basalt fiber is not widely used in reinforced-concrete construction [7]. Calculation of Basalt Fiber Reinforced Concrete Beams for Strength Sobirjon Razzakov Jurayevich1, Abdurasul Martazayev Shukirillayevich2 Sobirjon Razzakov Jurayevich1, Abdurasul Martazayev Shukirillayevich2 1 Doctor of Technical Sciences, Professor of Namangan Engineering Construction Institute, 160103, Namangan, Uzbekistan 2 PhD student of department of civil engineering, Namangan Engineering Construction Institute, 160103, Namangan, Uzbekistan 1 Doctor of Technical Sciences, Professor of Namangan Engineering Construction Institute, 160103, Namangan, Uzbekistan 2 PhD student of department of civil engineering, Namangan Engineering Construction Institute, 160103, Namangan, Uzbekistan ABSTRACT: In the scientific research work, theoretical research work on flexible fiber reinforced concrete beams made with the addition of basalt fibers was carried out.The results of theoretical studies show that the values of the ultimate bending moment in fiber reinforced concrete beams were found to be relatively higher than the values of the bending moment in ordinary beams. KEYWORDS: basalt fiber, fiber reinforced concrete, beam, strength, rebar, deflection ABSTRACT: In the scientific research work, theoretical research work on flexible fiber reinforced concrete beams made with the addition of basalt fibers was carried out.The results of theoretical studies show that the values of the ultimate bending moment in fiber reinforced concrete beams were found to be relatively higher than the values of the bending moment in ordinary beams. KEYWORDS: basalt fiber, fiber reinforced concrete, beam, strength, rebar, deflection KEYWORDS: basalt fiber, fiber reinforced concrete, beam, strength, rebar, deflection INTERNATIONAL JOURNAL OF MULTIDISCI ISSN(print): 2643-9840, ISSN(online): 2643-9875 Volume 05 Issue 04 April 2022 DOI: 10.47191/ijmra/v5-i4-06, Impact Factor: 6.072 Page No. 782-787 INTERNATIONAL JOURNAL OF MULTIDISCI ISSN(print): 2643-9840, ISSN(online): 2643-9875 Volume 05 Issue 04 April 2022 DOI: 10.47191/ijmra/v5-i4-06, Impact Factor: 6.072 Page No. 782-787 Calculation of Basalt Fiber Reinforced Concrete Beams for Strength The cross-sectional strength of flexible elements is calculated on the basis of the following condition: M M  (2) The cross-sectional strength of flexible elements is calculated on the basis of the following condition: ult M M  (2) where: M is the bending moment generated by the external load; where: M is the bending moment generated by the external load; Mult –the ultimate bending moment that can be accepted by a section of an element Mult –the ultimate bending moment that can be accepted by a section of an element 0  M 0 ) 5,0 ( ) ( ) 2 ( ) ( 0 ' 0 ' 3                 x h x b R a h A R a x h x h b R M fb s sc fbt ult ) ( ) 2 ( ) ( ) 5,0 ( ' 0 ' 3 0 a h A R a x h x h b R x h x b R M s sc fbt fb ult                (3) 0 ) 5,0 ( ) ( ) 2 ( ) ( 0 ' 0 ' 3                 x h x b R a h A R a x h x h b R M fb s sc fbt ult ) ( ) 2 ( ) ( ) 5,0 ( ' 0 ' 3 0 a h A R a x h x h b R x h x b R s sc fbt fb                (3) 2 f f ) ( ) 2 ( ) ( ) 5,0 ( ' 0 ' 3 0 a h A R a x h x h b R x h x b R M s sc fbt fb ult                (3) (3) In this case, the height of the compression zone is determined by the following formula: In this case, the height of the compression zone is determined by the following formula: IJMRA, Volume 5 Issue 04 April 2022 www.ijmra.in Page 783 0 ) ( ' 3          bx R A R x h b R A R fb s sc fbt s s b R R h b R A R A R x fbt fb fbt s sc s s          ) ( 3 3 ' (4) Figure 1. Calculation of Basalt Fiber Reinforced Concrete Beams for Strength - deformation (stress) in the rebar is determined depending on the height of the compression zone of the fiber concrete; - deformation (stress) in the rebar is determined depending on the height of the compression zone of the fiber concrete; tensile stress in rebar is assumed to be no greater than the calculated resistance Rs in elongation; - the tensile stress in rebar is assumed to be no greater than the calculated resistance Rs in elongati - the compressive stress in the rebar is assumed to be greater than the calculated resistan - the compressive stress in the rebar is assumed to be greater than the calculated resistance Rsс in compression. compressive stress in the rebar is assumed to be greater than the calculated resistance Rsс in compressio The normal cross-sectional strength of reinforced concrete elements is calculated based on the ratio of the relative height of the compressive zone h x 0   to the boundary value of the relative height of the compressive zone in the boundary position of the resulting element when the elongated reinforcement stress is equal to Rs. The value of is determined by the following formula: position of the resulting element when the elongated reinforcement stress is equal to Rs. The value of is determined by the following formula: 2 0 1 fb s R R h x        2 0 1 fb s R R h x        (1) 2 0 1 fb s R R h x        (1) (1) where: - the characteristic of the compressive zone of fiber concrete; 0.8 for heavy fiber concrete up to class В60 and 0.7 for heavy fiber concrete and fine aggregate concrete class B70-B100; where: - the characteristic of the compressive zone of fiber concrete; 0.8 for heavy fiber concrete up to class В60 and 0.7 for heavy fiber concrete and fine aggregate concrete class B70-B100; lue of the acceptable limit relative deformation of the reinforcement according to DR63.13330 s- the calculated value of the acceptable limit relative deformation of the reinforcement according to DR63.13330 2 fb  - Relative deformation of compressive fiber beton at Rfb stress; its value is assumed to be equal to the value given in DR63.13330 for ordinary concrete. 2. RESEARCH METHODOLOGY Calculation of strength under the influence of bending moment and longitudinal force of elements of fiber concrete structures is carried out on the normal section relative to the longitudinal axis.When calculating the load-bearing capacity of working reinforcement elements on the normal cross-section, the limit loads that can be accepted by fiber concrete and reinforcement in the normal section are determined based on the following cases: - it is obtained in the form of residual stresses in which the tensile sresistance of fiber concrete is equal to Rfbt2 or Rfbt3 and evenly distributed in the elongation zone of fiber concrete; - it is obtained in the form of residual stresses in which the tensile sresistance of fiber concrete is equal to Rfbt2 or Rfbt3 and evenly distributed in the elongation zone of fiber concrete; - it is obtained in the form of stress in which the compressive strength of fiber concrete is equal to Rfb and evenly distributed in the compressive zone of fiber concrete; - it is obtained in the form of stress in which the compressive strength of fiber concrete is equal to Rfb and evenly distributed in the compressive zone of fiber concrete; IJMRA, Volume 5 Issue 04 April 2022 Page 782 Page 782 www.ijmra.in Calculation of Basalt Fiber Reinforced Concrete Beams for Strength Calculation of Basalt Fiber Reinforced Concrete Beams for Strength Calculation of Basalt Fiber Reinforced Concrete Beams for Strength In the initial stages of loading on the beams, the amount of stress in the concrete and reinforcement is almost not large, in which case the deformation is in an elastic state. As the load increases, the amount of stress in the elongated zone of the concrete reaches the limit value of elongation. But the concrete does not crack. Plastic deformations occur on the stretched lower surfaces of the concrete, the deformation in these layers being equal to the tensile strength of the concrete. In this case, as the load increases, the elongated zone of the concrete breaks at the end of the deformation index. As a result, cracks are formed and a new condition on the cutting surface of the element. Once the cracks are formed, the reinforcement accepts the tension in the elongation of the cracked cutting surface, and between the cracks, the connection to the concrete is not broken and the concrete continues to work on elongation, giving the reinforcement a little more tension. The process of breaking down reinforced concrete takes place over a very short period of time during the test period. With the onset of the flexibility of the reinforcement, deformation begins, resulting in increased bending, and as the crack increases, the height of the compressed part of the concrete cutting surface decreases. Plastic deformation occurs in the compressed zone of the concrete over the cracks. Deformation begins with the crushing of concrete in the compacted zone. In this case, the compressed zone diagram is close to the appearance of a parabola. The cracks in the elongation zone enlarge, the beam stiffness decreases, and the slope increases rapidly, breaking the beam. The breakdown of normally reinforced reinforced concrete elements begins with elongated reinforcement. When the stress in the reinforcement reaches the leakage limit, the height of the concrete compression zone decreases sharply, which causes the concrete to collapse. In beams with a large number of tensile reinforcement, the collapse begins with the concrete in the compression zone, where the stress in the reinforcement is much smaller than the flow limit. This is definitely the opposite of saving[4-10]. Once the sample beams are loaded, in the initial stages of loading, the concrete and reinforcement work elasticly, with no cracks appearing in them. Calculation of Basalt Fiber Reinforced Concrete Beams for Strength The amount of stresses in concrete and reinforcement is small and inelastic deformations do not develop.As the number of loads increases, the stresses in the concrete in the elongated area of the beams reach their calculated resistance to elongation and the initial cracks appear. From this point on, the qualitatively different stress-strain state begins in the sample beams, and a redistribution of stresses in the concrete and reinforcement is observed. When the first cracks are formed, the concrete almost loses its resistance in the elongated area, and the reinforcement of the concrete is observed only in the areas between the cracks and the reinforcement. Calculation of Basalt Fiber Reinforced Concrete Beams for Strength Diagram of forces and stress diagram in a section normal to the longitudinal axis of a bent fiber-reinforced concrete element of rectangular section with reinforcement, when calculating its strength. (4) Figure 1. Diagram of forces and stress diagram in a section normal to the longitudinal axis of a bent fiber-reinforced concrete element of rectangular section with reinforcement, when calculating its strength. IJMRA, Volume 5 Issue 04 April 2022 Page 783 The experimental results of without basalt fiber reinforced concrete beams and fiber reinforced concrete beams dispersed with basalt fibers were compared with the results of theoretical calculations. The results of theoretical and experimental tests on fiber-reinforced concrete and ordinary reinforced concrete beams reinforced with basalt fibers are shown in Figures 2-4. 3. THE RESEARCH FINDINGS AND DISCUSSION The results of the ultimate moment obtained on the basis of theoretical calculations of ordinary and fiber-reinforced concrete beams are shown in Table 1. The results of the ultimate moment obtained on the basis of theoretical calculations of ordinary and f beams are shown in Table 1. The results of the ultimate moment obtained on the basis of theoretical calculations of ordinary and fiber-reinforced concrete beams are shown in Table 1. The results of the ultimate moment obtained on the basis of theoretical calculations of ordinary and fiber reinforced concrete beams are shown in Table 1. Table 1. The ultimate moment determined by the calculation of the sample beams № Fiber length, mm Fiber dosage,% Rb, МПа Rfbt3, МПа x ult М , кН·м difference 1 - - 25,8 - 13,56 - 2 10 0,1 28,7 1,12 15,29 +12,80 3 10 0,2 29,8 1,18 15,41 +13,70 4 10 0,3 28,3 1,05 15,18 +11,97 5 30 0,1 28,9 1,10 15,27 +12,64 6 30 0,2 30,4 1,27 15,56 +14,79 7 30 0,3 28,1 0,92 14,98 +10,53 The experimental results of without basalt fiber reinforced concrete beams and fiber reinforced concrete beams dispersed with basalt fibers were compared with the results of theoretical calculations. The results of theoretical and experimental tests on fiber-reinforced concrete and ordinary reinforced concrete beams reinforced with basalt fibers are shown in Figures 2-4. IJMRA, Volume 5 Issue 04 April 2022 Page 784 www.ijmra.in tion of Basalt Fiber Reinforced Concrete Beams for Strength Figure 2. Graph of the dependence of the bending moment on the deflection of the I-series sample beams Figure 3. Graph of the dependence of the bending moment on the deflection of the II-series sample beams alculation of Basalt Fiber Reinforced Concrete Beams for Strength Figure 2. Graph of the dependence of the bending moment on the deflection of the I-series sample beams Calculation of Basalt Fiber Reinforced Concrete Beams for Strength Calculation of Basalt Fiber Reinforced Concrete Beams for Strength Calculation of Basalt Fiber Reinforced Concrete Beams for Strength Figure 2. Graph of the dependence of the bending moment on the deflection of the I-series sample beams Figure 2. Graph of the dependence of the bending moment on the deflection of the I-series sample beams Figure 2. Graph of the dependence of the bending moment on the deflection of the I-series sample beams Figure 3. REFERENCES 1) Li, S., Zhang, Y., & Chen, W. (2020). Bending performance of unbondedprestressedbasalt fiber recycled concrete beams. Engineering Structures, 221, 110937. 2) Jin, S., Zhang, X., Zhang, J., & Shen, X. (2016, May). Experimental study on anti-splitting tensile properties of the chopped basalt fiber reinforced concrete. In International Forum on Energy, Environment and Sustainable Development (pp. 282-289). 2) Jin, S., Zhang, X., Zhang, J., & Shen, X. (2016, May). Experimental study on anti-splitting tensile properties of the chopped basalt fiber reinforced concrete. In International Forum on Energy, Environment and Sustainable Development (pp. 282-289). 3) Raj, S., Gopinath, S., &Iyer, N. R. (2013). Compressive behavior of basalt fiber reinforced composite. In Proc. of the Intl. Conf. on Advances in Civil, Structural and Mechanical Engineering, CSM. 3) Raj, S., Gopinath, S., &Iyer, N. R. (2013). Compressive behavior of basalt fiber reinforced composite. In Proc. of the Intl. Conf. on Advances in Civil, Structural and Mechanical Engineering, CSM. 4) Krassowska, J., &Lapko, A. (2013). The influence of steel and basalt fibers on the shear and flexural capacity of reinforced concrete beams. Journal of Civil Engineering and Architecture, 7(7), 789. 4) Krassowska, J., &Lapko, A. (2013). The influence of steel and basalt fibers on the shear and flexural capacity of reinforced concrete beams. Journal of Civil Engineering and Architecture, 7(7), 789. 5) Kizilkanat, A. B., Kabay, N., Akyüncü, V., Chowdhury, S., &Akça, A. H. (2015). Mechanical properties and fracture behavior of basalt and glass fiber reinforced concrete: An experimental study. Construction and Building Materials, 100, 218-224. 5) Kizilkanat, A. B., Kabay, N., Akyüncü, V., Chowdhury, S., &Akça, A. H. (2015). Mechanical properties and fracture behavior of basalt and glass fiber reinforced concrete: An experimental study. Construction and Building Materials, 100, 218-224. 6) Alsayed, S. H. (1993). Flexural deflection of reinforced fibrous concrete beams. Structural Journal, 90(1), 72-76. 6) Alsayed, S. H. (1993). Flexural deflection of reinforced fibrous concrete beams. Structural Journal, 90(1), 72-76. 7) Scholar, P. G. (2017). Flexural Behaviour of Basalt Chopped Strands Fibre Reinforced Concrete Beams. International Journal of Engineering Science, 5497. 8) Gvazdeva А.А.,(2017)“Fiber reinforced concrete structures and precast products with non-steel fibers” Design rules, DR297.1325800.2017 9) [9]RAZZAKOV S. J. Research of stress-strain state of single-storey buildings with internal partitions under static pulling load of the upper belt of a structure //Structural Mechanics of Engineering Constructions and Buildings. – 2016. – №. 6. – С. 14-19. 10) Razzakov S. 4. CONCLUSIONS 1. According to the results of theoretical calculations, the value of the ultimate moment in the beams without the addition of basalt fibers was 13.56 kN·m. The value of breaking moment in flexible fiber reinforced concrete beams made by adding 0.1% of basalt fibers with a length of 10 mm was 15.29 kN·m. The value of ultimate moment increased by 12.80% compared to ordinary reinforced concrete beams. 2. The value of ultimate moment in flexible fiber reinforced concrete beams made by adding basalt fibers with a length of 10 mm in the amount of 0.2 and 0.3% was 15.41 kN·m and 15.18 kN·m, respectively. 3. In fiber reinforced concrete beams with a content of 0.1% and a length of 30 mm, the value of the ultimate moment is 15.27 kN·m, the value of the ultimate moment is 15.56 kN·m, the amount of fibers is 0.3%, the value of the ultimate moment was 14.98 kN·m. It was found that the values of the ultimate moment increased by 10-15% compared to the values of the ultimate moment in ordinary beams. 3. In fiber reinforced concrete beams with a content of 0.1% and a length of 30 mm, the value of the ultimate moment is 15.27 kN·m, the value of the ultimate moment is 15.56 kN·m, the amount of fibers is 0.3%, the value of the ultimate moment was 14.98 kN·m. It was found that the values of the ultimate moment increased by 10-15% compared to the values of the ultimate moment in ordinary beams. 3. THE RESEARCH FINDINGS AND DISCUSSION Graph of the dependence of the bending moment on the deflection of the II-series sample beams Figure 4. Graph of the dependence of the bending moment on the deflection of the III-series sample beams The results of experimental and theoretical studies show that the values of the ultimate bending moment ed concrete beams are relatively high than the values of the bending moment in ordinary beams re 2. Graph of the dependence of the bending moment on the deflection of the I-series sample beams Figure 3. Graph of the dependence of the bending moment on the deflection of the II-series sample beams Figure 4. Graph of the dependence of the bending moment on the deflection of the III-series sample beams The results of experimental and theoretical studies show that the values of the ultimate bending moment in Figure 4. Graph of the dependence of the bending moment on the deflection of the III-series sample beams . Graph of the dependence of the bending moment on the deflection of the III-series sample beams The results of experimental and theoretical studies show that the values of the ultimate bending moment in fiber reinforced concrete beams are relatively high than the values of the bending moment in ordinary beams. The results of experimental and theoretical studies show that the values of the ultimate bending moment in fibe reinforced concrete beams are relatively high than the values of the bending moment in ordinary beams. IJMRA, Volume 5 Issue 04 April 2022 Page 785 www.ijmra.in REFERENCES Zh., Zhuraev B. G., Zhuraev E. S. U. Stability of the walls of individual residential houses with a wooden frame. - 2018. - T. 14. - No. 5. 11) Razzakov S. Zh., Kholmirzaev S. A., Ugli B. M. Calculation of the forces of cracking of compressed reinforced concrete elements in a dry hot climate // Symbol of Science. – 2015. – no. 3. - S. 57-60. 12) Razzakov S. Zh., Ilyina L. V., Kholmirzaev S. A. Temperature deformations of concrete in a dry hot climate // Proceedings of the Novosibirsk State University of Architecture and Civil Engineering (Sibstrin). - 2018. - T. 21. - No. 3. - S. 22-30. 13) Razzakov S. Zh., Kholmirzae S. A. Influence of frame reinforcement on the stress-strain state of a two-story building made of low-strength materials. – 2017. – no. 4. - S. 43-49. 14) Razzakov S. J. et al. Stretching curved wooden frame-type elements “Sinch” //European science review. – 2017. – no. 1- 2. – S. 223-225. 15) Abdurakhmonov S. E., Martazaev A. Sh., Eshonjonov Zh. B. Cracks in reinforced concrete products during their manufacture in a non-stationary climate // Bulletin of Science and Creativity. – 2017. – no. 2. - S. 6-8. 16) Nasriddinov M. M., Martazaev A. Sh., Vakkasov Kh. S. Crack resistance and strength of inclined sections of bent concrete elements on porous aggregates from loess-like loams and TPP ash // Symbol of Science. – 2016. – no. 1-2. - S. 85-87. 17) Abdurakhmonov S. E. et al. Crack formation and water separation of concrete mixture in reinforced concrete products during manufacture in areas with a hot climate // Bulletin of Science and Creativity. – 2018. – no. 2. - S. 35-37. 18) Martazaev A. Sh., Eshonjonov Zh. B. Issues of calculation of bending elements along inclined sections // Bulletin of Science and Creativity. – 2017. – no. 2 (14). - S. 123-126. 18) Martazaev A. Sh., Eshonjonov Zh. B. Issues of calculation of bending elements along inclined sections // Bulletin of Science and Creativity. – 2017. – no. 2 (14). - S. 123-126. IJMRA, Volume 5 Issue 04 April 2022 Page 786 www.ijmra.in 22) I. Kh. – 2017. – no. 3. - S. 350-352. Calculation of Basalt Fiber Reinforced Concrete Beams for Strength 19) Juraevich R. S., Shukirillayevich M. A. Mechanical properties of basalt fiber concrete. 20) Juraevich R. S., Shukirillayevich M. A. The Effect of the Length and Amount of Basalt Fiber on the Properties of Concrete //Design Engineering. - 2021. - S. 11076-11084. 21) Shukurillaeich M. A. et al. CHECKING THE BEARING CAPACITY OF BENDING REINFORCED CONCRETE PRODUCTS ON AN INCLINED SECTION //ScienceTime. – 2018. – no. 6 (54). - S. 42-44. 22) I. Kh. – 2017. – no. 3. - S. 350-352. ) 23) Martazaev A. Sh., Fozilov O. K., Nosirzhonov N. R. Significance of calculations of static and dynamic impact of land dams // Innovative science. – 2016. – no. 5-2 (17). - S. 132-133. 23) Martazaev A. Sh., Fozilov O. K., Nosirzhonov N. R. Significance of calculations of static and dynamic impact of land dams // Innovative science. – 2016. – no. 5-2 (17). - S. 132-133. There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and building upon the work for non-commercial use, provided the original work is properly cited. There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and building upon the work for non-commercial use, provided the original work is properly cited. IJMRA, Volume 5 Issue 04 April 2022 IJMRA, Volume 5 Issue 04 April 2022 Page 787 Page 787 www.ijmra.in Calculation of Basalt Fiber Reinforced Concrete Beams for Strength Calculation of Basalt Fiber Reinforced Concrete Beams for Strength IJMRA, Volume 5 Issue 04 April 2022 Page 788 Page 788 www.ijmra.in
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RPA1 Gene Mutation
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Qeios · Definition, February 2, 2020 Open Peer Review on Qeios RPA1 Gene Mutation National Cancer Institute National Cancer Institute Qeios ID: M4LWSY · https://doi.org/10.32388/M4LWSY Source National Cancer Institute. RPA1 Gene Mutation. NCI Thesaurus. Code C132007. National Cancer Institute. RPA1 Gene Mutation. NCI Thesaurus. Code C132007. A change in the nucleotide sequence of the RPA1 gene. Qeios ID: M4LWSY · https://doi.org/10.32388/M4LWSY 1/1
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DA SALA OU DA COZINHA: QUE HORAS ELA VOLTA? - ESTUDO DE RECEPÇÃO COM DOMÉSTICAS E PATROAS: REFLEXÕES SOBRE PERCURSOS METODOLÓGICOS
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1Doutorando em Educação pela Universidade Federal de Sergipe (PPGED/UFS). E-mail: ewertton.nunes@gmail.com. 2Doutoranda em Sociologia pela Universidade Federal de Sergipe (PPGS/UFS). E-mail: ernabarros@gmail.com 3Doutora em Mídia, Comunicação e Cultura pela Universitat Autònoma de Barcelona, mestra em Antropologia pela Universidade Federal de Sergipe e jornalista, graduada pela Universidade Metodista de São Paulo. E-mail: danielledenoronha@gmail.com. -ESTUDIO DE RECEPCIÓN CON DOMÉSTICAS YEMPLEADORA: REFLEXIONES SOBRE PERCURSOS METODOLÓGICOS Everton de Almeida Nunes1 Erna Barros2 Danielle Parfentieff de Noronha3 10.21665/2318-3888.v7n13p180-202 Audiovisual. Que Horas Ela Volta? Recepção. Metodologia. Teoria Fundamentada em Dados. O presente artigo propõe discutir alguns dos aspectos conceituais da Teoria Fundamentada em Dados (Grounded Theory) e apresentar a aplicabilidade deste procedimento metodológico na análise de recepção, utilizando como exemplo a pesquisa de mestrado intitulada Da Sala ou Da Cozinha: Que horas ela volta? - estudo de recepção com domésticas e patroas. A dissertação foi realizada no curso de Comunicação Social da Universidade Federal de Sergipe (UFS), entre 2015 a 2018, e tomou como ponto de partida o filme brasileiro “Que Horas Ela Volta? (2015), dirigido por Anna Muylaert. Neste texto buscamos apresentar como tal abordagem teórica, junto aos Estudos da Recepção, pode contribuir na compreensão dos significados construídos pelo recorte de audiência a ser analisado, sobretudo por ser uma metodologia com raízes no interacionismo simbólico. Palavras-chave: Audiovisual. Que Horas Ela Volta? Recepção. Metodologia. Teoria Fundamentada em Dados. 181 Artigos The present article proposes to discuss some of the conceptual aspects of Grounded Theory and to present the applicability of this methodological procedure in the reception analysis carried out in the master's research entitled The Room or The Kitchen: The Second Mother - reception study with domestic servants and female employers. The research was conducted in the Communication college of the Universidade Federal de Sergipe (UFS), between 2015 and 2018, and took as a starting point the Brazilian film "The Second Mother" (2015), directed by Anna Muylaert. In this text, we would like to present how such a theoretical approach, combined to the Reception Studies, can contribute to understanding of meanings built by the audience cut off to be analyzed, especially since it is a methodology related to the symbolic interactionism. Keywords: Audiovisual. The Second Mother. Reception. Methodology. Grounded Theory. El presente artículo propone discutir algunos de los aspectos conceptuales de la Teoría Fundamentada en Datos (Grounded Theory) y presentar la aplicabilidad de este procedimiento metodológico en el análisis de recepción utilizando como ejemplo la investigación de maestría titulada De la Sala o De la Cocina: ¿Qué horas ella vuelve? - estudio de recepción con domésticas y empleadoras. La investigación fue realizada en el curso de Comunicación Social de la Universidade Federal de Sergipe (UFS), entre 2015 y 2018, y tomó como punto de partida la película brasileña "¿Qué Horas Ella Vuelve?" (2015), dirigida por Anna Muylaert. En este texto buscamos presentar como tal abordaje teórico, junto a los Estudios de la Recepción, puede contribuir en la comprensión de los significados construidos por el recorte de audiencia a ser analizado, sobre todo por ser una metodología con raíces en el interaccionismo simbólico. Palabras clave: Audiovisual. ¿Qué Horas Ella Vuelve? Recepción. Metodología. Teoría Fundamentada en Datos. Audiovisual. ¿Qué Horas Ella Vuelve? Recepción. Metodología. Teoría Fundamentada en Datos. 5 “Que Horas Ela Volta?” estreou nos cinemas brasileiros em 27 de agosto de 2015. Também foi lançado na Europa e América do Norte, conquistando diversos prêmios internacionais, com o título em inglês “The Second Mother". Além disso, vale mencionar que o Ministério da Cultura, em setembro de 2015, escolheu o filme para representar o Brasil na disputa pelo Oscar de melhor filme estrangeiro da edição de 2016, mas não houve a indicação ao prêmio. Segundo o jornal Correio do Povo, em dezembro de 2016, a obra cinematográfica foi eleita uma das cinco melhores produções estrangeiras pela organização estadunidense National Board of Review e, no mesmo mês, foi eleito o melhor filme do ano pela Associação Brasileira dos Críticos de Cinema (Abraccine). Ainda em 2016 foi indicado pelos Estados Unidos Satellite Awards e Estados Unidos Critics' Choice Awards como melhor filme Estrangeiro. V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 ABSTRACT 182 Everton Nunes • Erna Barros • Danielle Noronha 182 4 Dissertação de mestrado produzida e defendida por Everton de Almeida Nunes. 5 ação de mestrado produzida e defendida por Everton de Almeida Nunes. Neste artigo, de perspectiva teórico-metodológica, apresentaremos alguns aspectos conceituais da Teoria Fundamentada em Dados (TFD) e refletiremos sobre o seu uso como ferramenta para a análise de recepção filmica, a partir do percurso metodológico desenvolvido para a dissertação de mestrado Da Sala ou Da Cozinha: Que horas ela volta? - estudo de recepção com domésticas e patroas4. O objetivo é pensar como tal abordagem teórica, junto aos Estudos da Recepção, pode ser um caminho possível para compreensão dos significados construídos pelo recorte de audiência a ser analisado, sobretudo por ser uma metodologia com raízes no interacionismo simbólico. O filme escolhido para ánalise, “Que Horas Ela Volta?”5, se apresentou como um importante objeto de análise da recepção por tensionar duas realidades distintas a partir um ponto de vista – “da cozinha” – que normalmente ocupa pouco espaço nas obras audiovisuais produzidas no país (e em outros lugares do globo)6. O filme, escrito e dirigido por Anna Muylaert, conta a vida da pernambucana Val (interpretada pela atriz Regina Casé), que deixa a filha Jéssica no Nordeste para trabalhar em São Paulo como babá de Fabinho, único filho dos patrões Bárbara e Carlos. Apesar do filme iniciar com os primeiros anos de Val como Babá de Fabinho, o enredo mostra principalmente a realidade da empregada doméstica 11 anos após a sua chegada ao Sudeste. A narrativa passa por uma atualização nos contextos socioeconômico e culturais, dialogando com uma realidade brasileira bastante recente. O principal ponto de virada na obra acontece com a chegada da filha de Val a São Paulo. A garota também 5 “Que Horas Ela Volta?” estreou nos cinemas brasileiros em 27 de agosto de 2015. Também foi lançado na Europa e América do Norte, conquistando diversos prêmios internacionais, com o título em inglês “The Second Mother". Além disso, vale mencionar que o Ministério da Cultura, em setembro de 2015, escolheu o filme para representar o Brasil na disputa pelo Oscar de melhor filme estrangeiro da edição de 2016, mas não houve a indicação ao prêmio. Segundo o jornal Correio do Povo, em dezembro de 2016, a obra cinematográfica foi eleita uma das cinco melhores produções estrangeiras pela organização estadunidense National Board of Review e, no mesmo mês, foi eleito o melhor filme do ano pela Associação Brasileira dos Críticos de Cinema (Abraccine). Ainda em 2016 foi indicado pelos Estados Unidos Satellite Awards e Estados Unidos Critics' Choice Awards como melhor filme Estrangeiro. g 6 Partimos da premissa de que grande parte da população não se reconhece nas representações simplificadoras tipificadas (e nos padrões sociais) mais usuais elaboradas nos campos hegemônicos da produção cultural. Desta forma, consideramos que uma grande porcentagem de pessoas faz parte de uma esfera subalterna de representação, que endossa e auxilia a manutenção de relações de poder em diferentes níveis sociais. V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 Introdução V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 Introdução V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 Introdução 183 183 Artigos deixa um filho no Nordeste, porém, vai a São Paulo prestar vestibular para arquitetura. No período que antecede o vestibular, Jéssica fica hospedada na casa dos patrões de sua mãe, e a partir daí muitos conflitos surgem com a sua presença, sobretudo pela forma como a garota enxerga o mundo e pelas relações de poder estabelecidas na casa. Neste sentido, o filme nos possibilita refletir sobre as relações que interseccionam (CRENSHAW, 1989; LUGONES, 2008) diversos marcadores sociais da diferença, como gênero, classe social e trabalho, região e geração. As obras audiovisuais são compreendidas como narrativas míticas, no sentido que Barthes (2009) deu ao mito, isto é, como parte de um sistema de comunicação que desta forma, opera simbolicamente pela linguagem. O mito não é apenas o que foi dito, mas também o que foi entendido. Não é apenas um objeto, um conceito ou uma ideia, é um modo de significação. Além disso, partimos do pressuposto que todo filme é um trabalho de ficção, no sentido que sempre se trata de uma representação, que inclui a mise-en- scène e os diversos processos de escolha, recorte e montagem. Neste sentido, entendemos o cinema como um texto narrativo, passível de interpretação e investigação, que como linguagem é uma ferramenta ideológica e política, capaz de através de suas representações suscitar imaginários e reformular memórias, individuais e coletivas (DE NORONHA, 2013). Este artigo está divivido em cinco partes. Na primeira, apresentamos algumas considerações sobre os estudos da recepção que nortearam a pesquisa, apresentando também seus desdobramentos e significados. Em seguida, trazemos uma análise entre a relação do interacionismo simbólico e os estudos sobre a recepção fílmica. Na terceira parte, passamos a expor os principais elementos da teoria fundamentada em dados e sua aplicação para compreensão da interpretação de produtos audiovisuais para, na quarta seção, apresentar as aplicações metodológicas e os caminhos utilizados na referida pesquisa, que contou com entrevistas com a diretora do filme e com as espectadoras, no nosso caso, domésticas e patroas. Por último, encerramos com as considerações finais do artigo. V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 184 Everton Nunes • Erna Barros • Danielle Noronha 184 Os estudos de recepção ganham força devido às mudanças no olhar acerca dos processos comunicativos dos meios massivos. Após a fase da sublimação dos meios de comunicação impositivos, consolida-se a ênfase da audiência liderando o processo comunicativo. É importante salientar que a relação entre o público e os veículos midiáticos era objeto de investigação desde o início do século XX. Porém, para se chegar às premissas mais atuais sobre a audiência, seu entendimento passou por várias formas de concepção, dentre elas, as perspectivas de receptor-manipulado, receptor- persuadido, receptor-reificado, o receptor-consumidor, dentre outras definições. Seja qual tenha sido a concepção, o que se observa desde então é a tentativa de investigar o sujeito da recepção e o seu processo interpretativo no contexto social. Segundo descreve Sousa (1995), alguns estudiosos percebem como “inócuo e desnecessário indagar hoje sobre o sujeito da recepção, enquanto outros entendem que a questão da recepção é temática já exaurida”. Entretanto, com o reconhecimento das mediações culturais e com o surgimento de novas tecnologias, mídias e formas de subjetivação, restaura-se o interesse acerca do receptor, e emerge a necessidade de revisitar conceitos essenciais que dinamizam sua compreensão. Para Ruótolo (1998) não existe uma teoria geral que explique o receptor desde o momento que decide se expor a um conteúdo da comunicação até as suas últimas consequências comportamentais. Dentro dessa perspectiva científica, descreve Ferreira (2015) que os estudos de recepção são conduzidos pelo processo subjetivo de decodificação das mensagens dos meios pelos receptores. Para Immacolata (apud BARBERO, 2009), a recepção é o momento privilegiado da produção de sentido, contestando a concepção reprodutivista de que a comunicação se restringe aos meios, e é realizada através das mediações, ou seja, da cultura. A audiência nesse contexto deve ser compreendida de maneira distinta do que acontecia até os anos 1970. Até este período, os estudos da comunicação massiva, que vez ou outra ponderava sobre o receptor, eram guiados pela ideia de que a recepção representava apenas um lugar de chegada (das mensagens dos meios) e nunca um lugar de partida, isto é, também de produção de sentido. V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 1. Breves considerações sobre os estudos da recepção V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 1. Breves considerações sobre os estudos da recepção 185 Artigos Com uma proposta mais compreensiva dos processos de comunicação, o entendimento da ação social no contexto do receptor passou a incorporar, nos estudos interpretativos, como elemento essencial e mediador das interpretações dos meios de comunicação e dos seus produtos. A noção de que o sentido ultrapassa as formulações textuais fez com que a recepção deixasse de ser entendida como um processo direto e automático para outro subjetivo. As novas investigações apontam uma dimensão “multissemântica” que nasce do relacionamento entre a audiência e os meios de comunicação. O que percebeu Ferreira (2015) foi que dentro de uma complexa possibilidade de intervenção da audiência sobre os meios existem predisposições para seleção, interpretação, aceitação (ou não) das sugestões dos conteúdos com subsequente mudança (ou não) de atitudes, opiniões e comportamentos. O receptor, então, apontado como elemento central da comunicação, é considerado “ativo” e para compreender os processos de trocas que efetiva com os meios de comunicação e seus produtos é preciso levar em conta os processos de interação com ambiente social e cultural no qual vive. Neste sentido, a leitura das imagens é sempre histórica e cultural e depende do conhecimento pré-existente do público. Sua compreensão passa pelo processo de verbalização e empalavramento (DUCH & CHILLÓN, 2012) da realidade representada, que, em contextos específicos, é promotora de significados simbólicos e conceituais, conotados e denotados. Sendo assim, para entender o fluxo de interação com os sujeitos e compreender respostas emitidas pelos receptores, Antônio Ruótolo (1998) construiu algumas considerações: Sendo assim, para entender o fluxo de interação com os sujeitos e compreender respostas emitidas pelos receptores, Antônio Ruótolo (1998) construiu algumas considerações: As respostas dos receptores aos meios de comunicação podem ser classificadas nos seguintes grupos: respostas de exposição, respostas de recepção, respostas atitudinais e respostas comportamentais (...). As análises teóricas dos receptores podem ser realizadas dentro desses quatro grupos de respostas (...) cada teoria elege e privilegia um grupo de respostas como sendo o eixo analítico dos receptores. Não existe uma teoria geral que explique o receptor desde o momento que decide expor-se a um conteúdo de comunicação até suas últimas consequências comportamentais. Todas as análises tendem a ser perspectivas teóricas de médio alcance enfatizando apenas um grupo de respostas [...] (RUÓTOLO 1998, p. 160). Dessa forma, é possível refletir acerca das práticas sociais e culturais que emergem do produto fílmico em questão (Que Horas Ela Volta?), traçando as especificidades das V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 186 Everton Nunes • Erna Barros • Danielle Noronha 186 diferentes compreensões dos papéis desempenhados pelos personagens que representam as duas figuras icônicas deste estudo: a empregadora e a trabalhadora doméstica. Para tanto, observaremos as experiências relacionadas ao campo da recepção fílmica. A experiência de se assistir a um filme demanda ponderarmos sobre a percepção da relação interativa entre espectador e obra apreciada, ou seja, é necessário considerar o aspecto comunicativo receptivo. Stam (2000) diz que a história do cinema não é apenas a história dos filmes e cineastas, ela é também a história dos vários significados que os públicos têm atribuído aos filmes. O autor desenvolveu um grande interesse na compreensão do papel do espectador na teoria do cinema, muito influenciado pelas chamadas teorias da recepção na literatura associadas ao Reader response theory, de Stanley Fish e Norman Holland, e também pelos escritos sobre a recepção estética de Hans Robert Jauss e Wolfgang Iser na Escola de Konstanz na Alemanha. Tais estudos compreendem que os receptores se tornam coprodutores do produto cultural por meio do rompimento dos significados preestabelecidos. Segundo Stam (2008), todas as esferas que formam o receptor como sujeito cultural; tais como gênero, sexualidade, classe, raça, nação são fundamentais para o fenômeno coprodutivo. A natureza histórica e socialmente condicionada da espectatorialidade é reconhecida nos estudos de recepção como algo imprescindível para entender o processo cinematográfico. O filme é o lugar onde interagem autor e receptor e de modo algum está fechado em si mesmo. Pelo contrário, esse ambiente é recheado de fissuras, janelas, e é dada ao espectador a tarefa de cobri-las de sentido: Ao ver um filme, o receptor identifica certas indicações que o incitam a executar numerosas atividades de inferência, que vão desde a atividade obrigatória e rapidíssima de perceber o movimento aparente, passando pelo processo mais ‘penetrável do ponto de vista cognitivo’, de construir, digamos, vínculos entre as cenas, até ao processo ainda mais aberto de atribuir significados abstratos ao filme. Na maioria dos casos o espectador aplica estruturas de conhecimento às indicações que reconhece dentro do filme (BORDWELL, 1991, p. 3). V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 187 Artigos A utilização das imagens (fixas e em movimento) no campo das ciências humanas tem servido como forma de disseminação de pensamentos. Talvez, isso aconteça porque os processos de recepção de um filme fazem parte de um ato dinâmico, que são também interligados à concepção que os espectadores possuem sobre o mundo e sobre eles mesmos. 7 Vale ressaltar que, atualmente, ao fazermos uma análise sobre a recepção, também já não é possível pensar apenas na ideia da individualidade que o espectador tem com a obra através de seu relacionamento com a tela na sala escura, já que a experiência de ver um filme alcança as mais diversas proporções: os filmes também são exibidos na televisão, no computador ou no celular. A experiência que antes era individual pode ser compartilhada, seja com pessoas, com a presença de uma segunda tela nas mãos, seja dividindo espaço com outra atividade (DE NORONHA, 2013). Segundo Souza (2014), um filme também é aquilo que fazemos dele, o que sentimos e como interpretamos ao que assistimos, seja na sala escura, seja ao abrigo de nossas casas7. Para Gomes (2005), as teorias da recepção no campo cinematográfico têm recebido, nos dias que correm, merecido destaque após um longo período de ostracismo no qual o espectador era visto apenas e, tão somente, como um mero dado empírico. Mascarello (2004) aponta que essas mudanças de postura com relação ao espectador começaram a acontecer após os anos 1980. Ele afirma que naquele período se passa a examinar a relação entre o texto fílmico e audiência em termos de suas manifestações pontuais e historicizadas, contemplando-se, assim, a diversidade encontrada (extra textualmente) nos momentos da produção e da recepção. O campo de análise de recepção das pesquisas cinematográficas deve considerar a relação interativa entre espectador e obra, de forma a ter em vista que, em todas as esferas contemporâneas da produção audiovisual, os processos explícitos e sugestivos da imagem se caracterizam como instrumentos possuidores de um caráter bastante simbólico e representativo. Essa representação trata principalmente do enfoque dado à análise no receptor, através de uma “compreensão da relação que se estabelece entre a mídia, o cinema e o espectador e que se dá a partir das distintas posições ocupadas pelo sujeito na estrutura e na cena social representada” (ESCOSTEGUY, 2004, p. 130). Conforme nos lembra Stam (2000, p. 305): V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 188 Everton Nunes • Erna Barros • Danielle Noronha Se, por um lado, o cinema é mimese e representação, por outro é também enunciado, um ato de interlocução contextualizada entre produtores e receptores socialmente localizados. Não basta dizer que a arte é construída. Temos de perguntar: construída para quem e em conjunção com quais ideologias e discursos? Neste sentido, a arte é uma representação não tanto em um sentido mimético quanto político, da delegação da voz. Se, por um lado, o cinema é mimese e representação, por outro é também enunciado, um ato de interlocução contextualizada entre produtores e receptores socialmente localizados. Não basta dizer que a arte é construída. Temos de perguntar: construída para quem e em conjunção com quais ideologias e discursos? Neste sentido, a arte é uma representação não tanto em um sentido mimético quanto político, da delegação da voz. Nesse sentido, o desafio a ser enfrentado é observar, conforme aponta Bordwell (1991), que nos filmes os significados não são encontrados na obra, mas construídos. O espectador da obra ao interpretá-la, dá-lhe significação através de uma série de fatores (sociais, culturais, históricos, etc.) e, principalmente, do contexto em que se deu sua experiência cinematográfica. Desta forma, a interpretação de uma obra audiovisual pode ser compreendida como o resultado daquilo que o autor buscou construir – a partir de referências, estereótipos e sentidos já negociados e muitas vezes naturalizados dentro dos contextos sociais determinados – e as vivências prévias relacionadas ao cotidiano prático e aos processos imaginativos e subjetivos dos espectadores. Observar o sujeito como centro de reflexão da linguagem audiovisual é se aproximar da experiência fílmica examinando as maneiras pelas quais o espectador assiste a um filme. O que está em jogo não é apenas o sentido do texto (nem mesmo em seus efeitos ideológicos ou sociais), nem apenas os comportamentos de consumo (nem mesmo no contexto de práticas sociais mais amplas), mas as condições de inteligibilidade de todos os atos que convergem e resultam no consumo de uma película (CASETTI, 2007). É importante que os estudos analíticos da cinematografia possam dar conta da discussão da pluralidade existente nas narrativas fílmicas por meio da relação entre o público consumidor e as obras, investigando a interpretação destas obras e a apropriação/uso dos discursos que delas emanam. Para Alencar (2007), o cinema possibilita o encontro entre pessoas, amplia o mundo de cada um, mostra na tela o que é familiar e o que é desconhecido e estimula o aprender. Para ele “o cinema aguça a percepção, a torna mais ágil o raciocínio na medida em que, para entendermos o conteúdo de um filme, precisamos concatenar todos os recursos da linguagem fílmica, utilizados no desenrolar do espetáculo e que evoluem com rapidez” (ALENCAR, 2007, p. 137). V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 189 Artigos Analisar as informações que circunscrevem o cinema de representação é trabalhar com elementos de diferentes linguagens. Levando em conta que o espectador age emocionalmente sobre o filme, aderindo mais ou menos a ele, projetando nele sua subjetividade (AUMONT, 2012, p. 106). É necessário observar que condições variáveis definem esse espectador e modificam sua compreensão, e também sua resposta ao filme. O espectador das salas escuras, segundo Xavier (1983), “é o sujeito [...] e tudo se passa dentro de si, na sua coenestesia psíquica”. A interação sensorial, psíquica, cultural e social em relação ao filme permite ao espectador certa consciência de si mesmo, consciência esta que começa a se estruturar mesmo antes de ver o filme, na escolha – mesmo que influenciada – em assisti-lo. Ver um filme é um ato dinâmico que implica uma reflexão (ainda que embrionária) de que os discursos e representações sociais possivelmente existentes no filme se identificam com seus próprios discursos. É justamente nesse caminho de discursos emergentes das representações apresentadas pelo filme “Que Horas Ela Volta?” que se buscou acessar resultados que trouxessem uma percepção ampliada sobre o objeto em questão. Desta forma, antes de adentrarmos na metodologia utilizada na pesquisa, faz- se necessário apresentar as principais teorias que tiveram como foco de interesse a recepção e a interpretação dos sujeitos frente aos meios e produtos da comunicação. Os Estudos de Recepção, ou da Interpretação das Audiências, a partir da década de 1980, começaram a conquistar importante lugar devido ao amplo número de pesquisas realizadas, sobretudo, na Europa, EUA e América Latina. Segundo Maigret (2010), naquele período a forma de olhar os meios de comunicação passou por mudanças, foram desvelados grupos cognitivos que passaram a registrar as relações de força entre os grupos sociais. A produção de “mitos” midiáticos não significa deformação da verdade, enganação, ilusão, manipulação, mas naturalização do mundo social por imposição de um sistema de conotação a favor dos dominantes, e, em concordância com a sociologia empírica, fortalecimento das opiniões (MAIGRET, 2010, p.24). V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 190 Everton Nunes • Erna Barros • Danielle Noronha 190 O autor acredita que a transferência do foco da produção para a recepção possibilitou que os meios de comunicação deixassem de ser domínio reservado dos “especialistas” da comunicação, daqueles que eram conhecedores dos processos de produção e de recepção. Uma outra dinâmica aflorava com objetivos que implicaram na ativação de saberes sobre o mundo social, as mediações, a família, as identidades de gênero, os meios urbanos, a nação etc. Nessa perspectiva surgem formas de estudar o comportamento humano considerando sua interação mediante comunicações simbólicas, elas observam que os significados das ações podem ser mantidos, modificados ou dados pelos atores. O Interacionismo Simbólico8, de acordo com o pensamento de Coulon (1995), trouxe para o sujeito social um lugar teórico. Ele passou a agir como intérprete do mundo e, com isto, as ciências sociais ganharam métodos de pesquisa que privilegiam o ponto de vista destes sujeitos. Tais abordagens interacionistas têm como objetivo elucidar as significações que os próprios sujeitos praticam para construir seu mundo social. Embora o Interacionismo Simbólico se configure uma perspectiva teórica adequada à condução de investigações qualitativas, os pressupostos nele contidos são também compatíveis com métodos quantitativos. Desta maneira, epistemologicamente serve para pesquisas que se caracterizem pela adoção de múltiplos métodos (BENZIES & ALLEN, 2001). A ampla possibilidade metodológica tende a contribuir para aumentar a profundidade e a amplitude de conhecimento acerca do fenômeno estudado. Kanter (1972) e Hall (1987) garantem a utilidade e importância do Interacionismo Simbólico no estudo da vida social. 8 Os problemas sociais decorrentes da industrialização e urbanização do início do século XX na sociedade ocidental constituíram o contexto que serviu como uma das fontes de estímulo aos estudiosos da época para desenvolver uma perspectiva teórica distinta, voltada ao estudo sistemático do comportamento social humano. As contribuições teóricas que vieram dar origem a esta perspectiva, conhecida como interacionismo simbólico, são advindas das obras de clássicos da Escola de Chicago como Charles Cooley, John Dewey, William Thomas e, sobretudo, George Mead, considerado como o principal precursor e inspirador do movimento interacionista (HAGUETTE, 1995; JOAS, 1999; TREZZA, 2002; ÁLVARO, GARRIDO, SCHWEIGER & TORREGROSA, 2007). 9 Esse referencial foi desenvolvido por Barney Glaser e Anselm Strauss, no início da década de 1960, sociólogos que desfrutavam de conhecimentos inerentes à tradição em pesquisa na Universidade de Chicago e influência do IS e do pragmatismo. Assim, originou-se a TFD, cuja sistematização técnica e procedimentos de análise capacitam o pesquisador para desenvolver teorias sociológicas sobre o mundo da vida dos indivíduos, uma vez que alcança significação, compatibilidade entre teoria e observação, capacidade de generalização e reprodutibilidade, precisão, rigor e verificação. Para eles, esse caminho pode apresentar uma percepção dos indivíduos como dotados da utilização do seu raciocínio e poder de V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 191 191 Artigos simbolização, assim estes indivíduos podem interpretar e se adaptar às circunstâncias, dependendo do modo como eles mesmos venham a definir a situação. simbolização, assim estes indivíduos podem interpretar e se adaptar às circunstâncias, dependendo do modo como eles mesmos venham a definir a situação. Nessa perspectiva, a abordagem da pesquisa em questão, baseada no filme “Que Hora Ela Volta?”, possui raízes no Interacionismo Simbólico e na Teoria Fundamentada em Dados (TFD), uma vez que estas bases possibilitam a compreensão do modo como os indivíduos interpretam os objetos e as pessoas com as quais interagem, além de apontar como tal processo de interpretação conduz o comportamento individual em situações específicas. Acredita-se que a TFD tenha a possibilidade de ser utilizada para estudos de diversos fenômenos, sobretudo, os de natureza exploratória e indutiva no desenvolvimento de teorias “conceitualmente densas”, ou seja, aquelas com muitas relações conceituais (GLASER & STRAUSS, 1967, p. 31-32). Para entender o caminho metodológico aplicado na pesquisa de recepção realizada a partir do filme “Que Horas Ela Volta?”, e compreender a construção de sentidos por parte do público alvo da pesquisa (domésticas e patroas), foi necessário antes entender alguns fundamentos metodológicos da Teoria Fundamentada em Dados9 (Grounded Theory). Esta abordagem de pesquisa qualitativa, a partir dos procedimentos e técnicas utilizados, possibilita ao pesquisador descobrir teorias, conceitos e hipóteses. É uma proposta baseada nos dados coletados em campo e se difere de outros métodos por não utilizar dados predeterminados e pode ser aplicada em diversos campos do conhecimento. De acordo com Charmaz (2009, p. 21), a TFD foi pautada por “noções da atividade humana, dos processos emergentes, das significações sociais e subjetivas, das práticas da solução de problemas, e do estudo irrestrito da ação”. V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 192 Everton Nunes • Erna Barros • Danielle Noronha 192 A TFD serve às investigações em ciências da comunicação na medida em que ajuda a entender a realidade de acordo com o conhecimento da percepção/significado que os contextos ou objetos representam para os sujeitos. A intenção é revelar um modelo conceitual que dê clareza ao fenômeno a ser investigado e de alguma forma venha também possibilitar que o investigador possa desenvolver e relacionar conceitos. Charmaz (2006) assegura que essa teoria se constitui num método de condução da pesquisa qualitativa que tem como foco o desenvolvimento de estruturas teóricas construídas a partir da análise indutiva ou dedutiva das informações. A ideia central desse procedimento é que o resultado da investigação seja substancialmente fundamentado nos dados que são recolhidos pelo pesquisador em contato com o objeto dele. A partir de categorias conceituais é possível alcançar explicações acerca do fenômeno investigado, seja para organizar, seja para interpretar os eventos. Os propositores da TFD, Glaser e Strauss (CHARMAZ, 2006; GLASER, 1998), ressaltam a pluralidade do procedimento. Ferreira e Felizola (2012) afirmam que a TFD permite o desenvolvimento de um trabalho sequenciado com simultaneidade e continuadas inserções de ideias. Para esses pesquisadores, os procedimentos da sua prática contemplam: 1). Envolvimento simultâneo na coleta dos dados e análise; 2). Diante da explanação acima realizada sobre os fundamentos da TFD, a pesquisa intitulada Da Sala ou Da Cozinha: Que Horas Ela Volta? - estudo de recepção com domésticas e patroas, ao qual este artigo se reporta, encontrou pressupostos metodológicos que a ajudaram a compreender de maneira mais aprofundada o objeto em análise. A aplicabilidade de suas técnicas foi essencial, como assevera Fielding e Lee (1998), para proporcionar fundamentação lógica à teoria empiricamente construída, legitimando assim a investigação qualitativa proposta. Nesta seção vamos apresentar os percursos metodológicos vivenciados durante a pesquisa. Num primeiro momento, vamos discorrer sobre o processo de realização das entrevistas com as informantes – mulheres que já trabalharam como domésticas e mulheres que já contrataram este serviço – e, em seguida, com a diretora do filme analisado, Anna Muylaert, destacando as intenções e formas da cineasta compreender a temática abordada na obra. Vale ressaltar que o filme, por dar voz a outras perspectivas e formas de representação, possibilita que grupos que normalmente são silenciados e/ou estereotipados (Biroli, 2011) nas obras audiovisuais que fazem parte da esfera hegemônica de produção e circulação criem identificações com suas realidades, além de auxiliar que (pelo menos parte) dos espectadores re(pensem) seus papéis sociais e formas de relacionar-se com o outro. Por se tratar de um produto cultural – resultado de uma forma de entender o contexto e o mundo em que se vive – os filmes falam muito da cultura na qual está inserido e podem ser utilizados como ferramentas críticas questionadoras e de transformação social. A construção de códigos analíticos e categorias surgidas dos dados e não da lógica preconcebida de hipóteses dedutivas; 3) O uso do método comparativo constante que envolve todas as fases de investigação; 4) O avanço do desenvolvimento teórico durante cada passo da coleta de dados; 5) A escrita de memorandos para elaborar categorias, especificar suas propriedades e relacionamentos, além de ir ao longo da investigação identificando lacunas de conhecimentos; 6) Saturação amostral de dados que garante todas as peças do “quebra-cabeça” da investigação e por fim, 7) A condução da revisão de literatura que acontece somente após o desenvolvimento de uma análise independente dos seus dados por parte do pesquisador (FERREIRA & FELIZOLA, 2012, p. 9). 1). Envolvimento simultâneo na coleta dos dados e análise; 2). A construção de códigos analíticos e categorias surgidas dos dados e não da lógica preconcebida de hipóteses dedutivas; 3) O uso do método comparativo constante que envolve todas as fases de investigação; 4) O avanço do desenvolvimento teórico durante cada passo da coleta de dados; 5) A escrita de memorandos para elaborar categorias, especificar suas propriedades e relacionamentos, além de ir ao longo da investigação identificando lacunas de conhecimentos; 6) Saturação amostral de dados que garante todas as peças do “quebra-cabeça” da investigação e por fim, 7) A condução da revisão de literatura que acontece somente após o desenvolvimento de uma análise independente dos seus dados por parte do pesquisador (FERREIRA & FELIZOLA, 2012, p. 9). A utilização da TFD requer envolvimento com o objeto de estudo, o que acaba por demandar ao pesquisador grande disponibilidade de tempo e criatividade. Por isso, é fundamental o domínio dos preceitos da Teoria Fundamentada, ampliar a capacidade dedutiva e indutiva, assim como a sensibilidade teórica. A respeito da sensibilidade teórica, Santos e Nóbrega (2002) define como a qualidade do investigador de perceber e compreender o significado dos dados e às habilidades de discernir entre o que é ou V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 193 193 Artigos não relevante ao seu estudo. não relevante ao seu estudo. V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 Everton Nunes • Erna Barros • Danielle Noronha 194 A pesquisa Da Sala ou Da Cozinha: Que Horas Ela Volta? - estudo de recepção com domésticas e patroas foi realizada em duas instâncias a partir do início das investigações em 2015. A primeira etapa aconteceu em três Centros de Referência da Assistência Social - CRAS, localizados no município de Aracaju-SE. A escolha por esse público se deu pela relevância dos serviços oferecidos à comunidade, espaço físico adequado para a aplicação da pesquisa e pelo diferencial dos projetos desenvolvidos com mulheres pelo Serviço de Proteção e Atendimento Integral à Família - PAIF. A primeira etapa é a sensibilização dos técnicos das unidades socioassistencias (assistentes sociais, psicólogos e educadores sociais) para obtenção de informações primárias sobre as mulheres que integram os projetos, tais como: idade, profissão, atividades desenvolvidas etc. Este primeiro passo foi fundamental para o recorte de público necessário ao estudo, ou seja, mulheres que já tiveram contato com o trabalho doméstico. Após ter sido realizada a seleção das participantes, foi promovida uma sessão do filme “Que Horas Ela Volta?”. Para não haver contaminação da amostra não foram realizadas atividades posteriormente à apreciação da obra cinematográfica. Outro encontro era marcado uma semana após a exibição do filme com a finalidade de trocar impressões sobre o mesmo. Estas impressões eram materializadas em forma de cartazes, textos, poemas etc. Porém, antes do segundo encontro, foram realizadas entrevistas em profundidade com as participantes da pesquisa. A proposta é que as conversas fossem efetuadas na residência ou trabalho dessas mulheres (empregadas domésticas) para que houvesse contato com o universo sociocultural no qual estão inseridas. As entrevistas em profundidade serviram para apreender a construção individual de sentidos acerca do que foi assistido, assim como para perceber as eventuais alterações de análises que pudessem aparecer no confronto de impressões do grupo focal durante o segundo encontro. A constante análise comparativa dos dados é uma característica da Teoria fundamentada (GLASSER & STRAUSS, 1967, p. 8), em que a cada etapa de coleta são codificadas e analisadas as informações obtidas com as fontes. Da primeira fase da pesquisa foram extraídos incidentes contidos nos dados coletados, V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 195 195 Artigos para isto foi utilizado um procedimento de codificação aberta. Segundo a perspectiva de Strauss e Corbin (1990), a codificação aberta é um processo analítico que possibilita que os conceitos sejam identificados e desenvolvidos em relação às suas propriedades e dimensões. As informações coletadas junto às mulheres entrevistadas na primeira fase da pesquisa foram examinadas e comparadas a fim de conceituar e categorizar os dados para que gerassem uma lista de códigos e categorias. As categorias nascem das perguntas e comparações em busca de similaridade e diferenças entre cada incidente, evento ou situação: “O que é isto? O que representa?” (STRAUSS & CORBIN, 1990, p. 63). Na segunda etapa da pesquisa foram entrevistadas mulheres que já tinham contratado serviços domésticos de outras mulheres. Esta categoria, intitulada na pesquisa como “patroas”, foi submetiva a entrevistas em profundidade. Este novo momento da pesquisa suscitou um redirecionamento nas técnicas utilizadas para que fosse possível aprofundar o conhecimento sobre o universo estudado. A obtenção de informação junto a este público foi estimulada até que se chegasse à “saturação teórica dos dados”, a fim de obter variedade de dimensões (TRINIDAD; CARRERA; SORIANO, 2006, p. 25). Vale ressaltar que todas as etapas foram registradas por meio de recursos audiovisuais, posteriormente transcritos e codificados segundo os princípios da “Tríade Problemática” da metodologia, definição de Bryant e Charmaz (2007) para as três características diferenciais da TFD: a amostra teórica; a constante comparação dos dados às categorias teóricas; e a focalização do desenvolvimento de teoria via saturação teórica de categorias substantivas. As fases de codificação (inicial, focalizada e teórica) foram percorridas para levantamento dos significados tácitos atribuídos pelas domésticas e patroas ao filme por elas assistido; foram vasculhados os conceitos sensibilizantes que, como define Blumer (1954), sugerem caminhos sobre “o que olhar”. Acredita-se que, desta forma, foi possível alcançar a categoria principal (core category), elemento fundamental para a construção da teoria. V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 Everton Nunes • Erna Barros • Danielle Noronha 196 10 A entrevista foi concedida a Everton de Almeida Nunes em 25 de outubro de 2016. V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 4.2. Entrevista com a diretora do filme Anna Muylaert 197 Artigos sobre o mal que essas relações laborais domésticas causam aos empregados e empregadas e aos patrões e patroas, além das crianças que ficam no meio de toda esta dinâmica. Para ela, a narrativa é uma forma de falar sobre educação. Quando questionada sobre as motivações ideológicas transpostas para o drama, a cineasta reconheceu que a obra traz muito de um olhar particular sobre o problema, uma proposta crítica ao modo escravagista presente nos dias atuais: [...] Eu tenho uma influência da cultura oriental através do budismo e do taoísmo, onde se ter um serviçal fazendo todo o seu trabalho privado é considerado algo muito negativo para o patrão. Existe uma história zen que o discípulo se aproxima do mestre e pergunta ‘mestre o que é o zen?’ E o mestre diz: o zen é comer o arroz e lavar a tigela. Acho que o filme fala também sobre isso, sobre a necessidade de cada um fazer o próprio serviço particular. Mas fala também de muitas outras coisas. Acho que o filme serve ainda para que se valorize a figura da mãe e a sua importância na educação das crianças (Anna Muylaert). Quando indagada sobre os possíveis discursos políticos contidos na produção audiovisual, a diretora compartilhou que a realidade social do ano de 1996 a motivou a escrever o roteiro. Ela relembrou que naquele ano teve um filho e percebeu que amigas de classe social equivalente a dela entregavam os filhos aos cuidados de babás. “Eu não queria fazer isso... Queria fazer todo o trabalho da mãe. Percebi que quanto mais eu fazia, mais forte eu ficava. Eu senti que a maternidade poderia ser uma grande chance de crescimento”, descreveu Muylaert. Quando indagada sobre os possíveis discursos políticos contidos na produção audiovisual, a diretora compartilhou que a realidade social do ano de 1996 a motivou a escrever o roteiro. Ela relembrou que naquele ano teve um filho e percebeu que amigas de classe social equivalente a dela entregavam os filhos aos cuidados de babás. “Eu não queria fazer isso... Queria fazer todo o trabalho da mãe. Percebi que quanto mais eu fazia, mais forte eu ficava. Eu senti que a maternidade poderia ser uma grande chance de crescimento”, descreveu Muylaert. Ao descrever de forma objetiva as personagens femininas em “Que Horas Ela Volta?”, a criadora ressalta as seguintes características: [...] Val é uma guerreira, mas é submissa. Entrevista com a diretora do filme Anna Muylaert Por meio de entrevista semiestruturada, concedida exclusivamente para a pesquisa, a diretora do longa-metragem brasileiro explicou as intenções discursivas presentes na narrativa fílmica10. As respostas concedidas podem ajudar a compreender as principais intenções do emissor e auxiliar na análise dos usos feitos pela audiência sobre o filme, entendendo os encontros e desencontros refletidos na interpretação do texto fílmico. O contato com a realizadora aconteceu através da página do filme na rede social Facebook. Uma entrevista contendo 12 perguntas abertas foi encaminhada e integralmente respondida pela diretora. O conteúdo devolvido possibilitou o levantamento de um panorama inicial sobre os discursos da criadora, transformados em texto/imagem em “Que Horas Ela Volta?”. O objetivo principal do questionário foi ampliar a compreensão sobre as intenções da criadora da obra, entender o universo simbólico proposto para o filme e, assim, traçar um paralelo entre a enunciação e a construção de sentidos pelo público da pesquisa. A respeito do universo discursivo (textual e imagético) presente na concepção do filme, Muylaert afirma que a pretensão foi mostrar a história de uma empregada doméstica do ponto de vista da cozinha e não da sala. A diretora explicou que a câmera também toma esse partido, estando sempre na cozinha, olhando por uma fresta para a sala. Para ela, a chegada de Jéssica significa o rompimento dessa barreira: [...] Até a chegada da Jéssica que rompe essa barreira. Vemos o ponto de vista da empregada e a parte da cena principal que lhe cabe ver. É quase como um camarim e o palco, ela pode estar no camarim, mas não no palco. A empregada aqui não é mera figurante que entra e sai de cena para servir. Ela é a protagonista, o centro da história e o ponto de vista de onde vemos os patrões. Portanto, ela é a saúde, a força, a trabalhadora, enquanto seu patrão vive sentado, preguiçoso, sem fazer nada. Isso é uma pequena mudança de ponto de vista, mas que causa bastante impacto é geral reflexão (Anna Muylaert). Muylaert assevera que a obra foi criada para gerar debate sobre o separatismo social que acontece no Brasil dentro dos lares de classe média e classe alta – que mantêm a empregada não como uma funcionária comum, mas como uma cidadã de segunda classe, um resquício da escravidão. A criadora da obra ficcional garantiu querer falar ISSN 2318-3888 4.2. Entrevista com a diretora do filme Anna Muylaert Se acha inferior aos outros. Representa o brasil antigo; Barbara também é uma guerreira, mas se acha superior aos outros, ela representa a arrogância e a ignorância da elite; Jéssica é guerreira, mas não se acha nem inferior nem superior a ninguém, representa a força da nova juventude brasileira (Anna Muylaert). Sobre a audiência, Muylaert falou do retorno que obteve por parte da crítica especializada. A diretora explicou que leu centenas de escritos publicados em diversos países e definiu as críticas como sendo ótimas análises, sobretudo as estadunidenses e V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 198 Everton Nunes • Erna Barros • Danielle Noronha 198 as alemãs por serem mais longas, investigativas intelectuais. Para ela, algumas exceções traziam análises ruins sobre o filme. as alemãs por serem mais longas, investigativas intelectuais. Para ela, algumas exceções traziam análises ruins sobre o filme. A respeito das expectativas quanto à recepção das representações sociais materializadas no roteiro, Muylaert disse ser importante conceber um produto para que as empregadas se vissem e debatessem, acredita que os aspectos culturais contidos em “Que horas Ela Volta?” podem ter contribuído para a boa aceitação do filme. [...] Esse modus operandi dentro de casa, do modo carinhoso e promiscuo como acontece aqui, é um jeito muito brasileiro de lidar com a coisa. Eu queria mostrar como o brasileiro ainda tem a mente muito escravagista e nem se dá conta disso. Eu queria que as pessoas vissem o filme e percebessem o que estão fazendo, quanto desrespeito, quanta violência pode estar contida em pequenos gestos cotidianos (Anna Muylaert). A PEC das Domésticas (PEC 66/2012) foi outro fator que, segundo a cineasta, influenciou na formatação final do roteiro do longa-metragem. A nova realidade para o trabalhador e trabalhadora doméstica no país, de acordo com o posicionamento da diretora, estava em consonância com as discussões que o filme propunha. Para ela, se o filme tivesse sido concretizado 20 anos atrás, período da sua primeira versão, a personagem Val não tivesse coragem de pedir demissão e fazer outra coisa da vida. Por último, a idealizadora do filme expôs o seu ponto de vista acerca do cinema idealizado por mulheres. Quando questionada sobre a ideia de haver uma forma feminina de construir discursos, afirma que embora seja um assunto complexo e longo, há particularidades que diferenciam o cinema feito por mulheres11. 11 Vale ressaltar que grande parte da equipe do filme “Que Horas Elas Volta” foi ocupada por mulheres, como o caso da direção de fotografia (Barbara Alvarez) e da montagem (Karen Harley). Entrevista com a diretora do filme Anna Muylaert [...] Acho que todas as narrativas que estão interessadas em heróis que vão para a Guerra, para a floresta, caçar, lutar estão mais ligadas à perspectiva masculina. Quando se faz histórias mais intimistas, mais sentimentais ou críticas, acho que estão ligadas mais à visão feminina – embora nem sempre isso tenha a ver com o gênero do autor. Um homem pode sim ser feminino ao construir suas histórias – como Almodóvar, por exemplo – e uma mulher pode também ser masculina ao fazer seus filmes, como Katrin Bigelow por exemplo. Acho que tem a ver com um aspecto da alma (Anna Muylaert). As informações obtidas com a aplicação da entrevista se constituem como elementos a serem confrontados no aprofundamento do objeto desta pesquisa; e se apresentam como 11 Vale ressaltar que grande parte da equipe do filme “Que Horas Elas Volta” foi ocupada por mulheres, como o caso da direção de fotografia (Barbara Alvarez) e da montagem (Karen Harley). V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 199 Artigos possibilidade analítica ao problema por ela suscitado, como por exemplo, como acontece a construção de sentidos por parte de domésticas e patroas, diante das relações laborais representadas no filme “Que Horas Ela Volta?”. Considerações finais 12 Para fins de conhecimento, os resultados completos da pesquisa estão disponíveis no acervo de dissertações da Universidade Federal de Sergipe – UFS no Programa de Pós-graduação em Comunicação. V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 Diante do que foi apresentado, é possível perceber que a utilização da Teoria Fundamentada possibilitou para a pesquisa Da Sala ou Da Cozinha: Que Horas Ela Volta? - estudo de recepção com domésticas e patroas12 que os critérios de amostra teórica fossem evidenciados. Por intermédio do procedimento metodológico e as técnicas que ele sugere, foi possível ter a percepção dos padrões recorrentes nas entrevistas que foram realizadas de maneira individual (domésticas e patroas) e também com a realização do grupo focal (domésticas), relacionando com a entrevista realizada com a diretora da obra que nos apresenta elementos extra fílmicos e nos possibilita ter contato com as intenções da autora. Utilizando-se do pressuposto de que os universos socioculturais de patroas e empregadas entram em confronto na prática social do trabalho doméstico, bem como na interpretação e vivencia do filme analisado, o método comparativo foi aplicado no desenvolvimento de códigos analíticos, ou seja, para comparar os incidentes que estão nos dados levantados no processo de estudo. Desta etapa, pretendeu-se desenvolver uma estrutura substantiva do fenômeno que desse sustentação a formulações conceituais pertinentes à pesquisa. A aplicação da Teoria Fundamentada no estudo de recepção em destaque neste trabalho foi determinante para o desenvolvimento de um percurso conceitual sistemático e lógico, cujos princípios se pautam na verificação dedutiva. E foi também essencial não somente como metodologia, mas na formulação de uma Grounded Theory acerca dos significados atribuídos por patroas e empregadas ao filme “Que horas Ela Volta?”, 12 Para fins de conhecimento, os resultados completos da pesquisa estão disponíveis no acervo de dissertações da Universidade Federal de Sergipe – UFS no Programa de Pós-graduação em Comunicação. V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 200 200 Everton Nunes • Erna Barros • Danielle Noronha oportunizando maior entendimento do universo social e psicossocial dos sujeitos envolvidos. Referências oportunizando maior entendimento do universo social e psicossocial dos sujeitos envolvidos. Referências ALENCAR, S.E.P. O cinema na sala de aula: uma aprendizagem dialógica da disciplina história. Dissert. mestrado. Fac. de Educação. Univ. Federal do Ceará. Fortaleza/CE. 2007 AUMONT, J. A Imagem. Campinas: Papirus, 2012. AUMONT, J. A Imagem. Campinas: Papirus, 2012. BARBERO, M. J. Uma aventura epistemológica. Matrizes, v. 2, n. 2, 2009-b. Entrevista concedida à Maria Immacolata Vassalo de Lopes. BENZIES, K. M., & ALLEN, M. N. Symbolic interactionism as a theoretical perspective for multiple method research. Journal of advanced nursing, 2001. BIROLI, F. Mídia, tipificação e exercícios de poder: a reprodução dos estereótipos no discurso jornalístico. Revista Brasileira de Ciência Política, nº 6. Brasília: 71-98, 2011. BLUMER, H. What is wrong with social theory? American Sociological Review, 1954. BLUMER, H. What is wrong with social theory? American Sociological Review, 1954. BORDWELL, D. Making meaning: inference and rhetoric in the interpretation of cinema. USA: Harvard University Press, 1991. BRYANT, A., & CHARMAZ, K. Grounded Theory in Historical Perspective: An Epistemological Account. In A. Bryant & K. Charmaz (Eds.), The SAGE Handbook of Grounded Theory. London: Sage, 2007. CASETTI, F. Cómo analizar un film. Buenos Aires: Paidós, 2007. CASETTI, F. Cómo analizar un film. Buenos Aires: Paidós, 2007. CHARMAZ K. A construção da teoria fundamentada: guia prático para análise qualitativa. Porto Alegre: Artmed, 2009. _____________. Constructing Grounded Theory. A Practical Guide Through Qualitative Analysis. London: Sage Publications, 2006. FERREIRA, R. M. C. Telenovelas Brasileiras e Portuguesas: padrões de audiência e consumo. Aracaju: Edise, 2015. FERREIRA, R. M. C. Telenovelas Brasileiras e Portuguesas: padrões de audiência e consumo. Aracaju: Edise, 2015. FIELDING, N.; LEE, R. Computer Analysis and Qualitative Research, London: Sage. 1998 GLASER, B. G.; STRAUSS, A. L. The discovery of grounded theory: strategies for qualitative research. London: Aldine Transaction, 1967. GOMES, I. Efeito e recepção: a interpretação do processo receptivo em duas tradições de investigação sobre os media. Rio de Janeiro: E-papers, 2005. HALL, P. M. Interactionism and the study of social organization. The sociological quarterly, 1987. DUCH, L.; CHILLÓN, A. Un ser de mediaciones: Antropología de la comunicación, Vol 1. Barcelona: Herder, 2012. DUCH, L.; CHILLÓN, A. Un ser de mediaciones: Antropología de la comunicación, Vol 1. Barcelona: Herder, 2012. ESCOSTEGUY, A C. Notas para um estado da arte sobre os estudos brasileiros da recepção nos anos 90. Porto Alegre: Editora Sulina, 2004. COULON, A. A Escola de Chicago. Campinas: Papirus, 1995. CRENSHAW, K. Demarginalizing the Intersection of Race and Sex: A Black Feminist Critique of Antidiscrimination Doctrine, Feminist Theory and Antiracist Politics. University of Chicago Legal Forum, 14: 139-67, 1989. V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888 201 201 Artigos DE NORONHA, D. P. Cinema, memória e ditadura civil-militar: representações sobre as juventudes em O que é isso, companheiro? e Batismo de Sangue. Tesina de máster: Universidade Federal de Sergipe, 2013. HALL, P. M. Interactionism and the study of social organization. The sociological quarterly, 1987. LUGONES, M. “Colonialidad y género”. Tabula Rasa, nº 9: 75-101, 2008. STRAUSS, Anselm L.; CORBIN, Juliet. Basics of Qualitative Research: Grounded Theory, Procedures and Techniques. Newbury: SAGE, 1990. TRINIDAD, A., CARRERO, V. e SORIANO, R. M. Teoría Fundamentada “Grounded Theory”: La Construcción de la teoria através del análisis interpretacional, Madrid: CSI Cuadernos Metodológicos, nº 37, 2006. TRINIDAD, A., CARRERO, V. e SORIANO, R. M. Teoría Fundamentada “Grounded Theory”: La Construcción de la teoria através del análisis interpretacional, Madrid: CSI Cuadernos Metodológicos, nº 37, 2006. MAIGRET, E. Sociologia da comunicação e das mídias. São Paulo: Senac, 20 MAIGRET, E. Sociologia da comunicação e das mídias. São Paulo: Senac, 2010. MASCARELLO, F. Os estudos culturais e a recepção cinematográfica: um mapeamento crítico. Revista EcoPós, v. 7, n.2. 2004. RUÓTOLO, A. C., Audiência e recepção: perspectivas, Comunicação e Sociedade. São Bernardo do Campo: UMESP, 1998. UE HORAS ELA VOLTA?. Direção: Anna Muylaert. Pandora Filmes. Brasil, 2015. SANTOS SR, NÓBREGA MML. A Grounded Theory como alternativa metodológica para pesquisa em enfermagem. Rev Bras Enferm. setembro/outubro; 5(55), p. 575-9, 2002. SOUZA, M. L. R.. Modos de Ver e Viver o Cinema: etnografia da recepção fílmica e seus desafios. Revista Brasileira de Estudos de Cinema e Audiovisual, ano 3, ed 5, 2014. SOUSA, M. W. Recepção e comunicação: a busca do sujeito. In SOUSA, Mauro Wilton (org). Sujeito, o Lado Oculto do Receptor. São Paulo: Brasiliense, 1995. STAM, Robert. Introdução à Teoria do Cinema. Ed. Papirus, 2000. 202 202 Everton Nunes • Erna Barros • Danielle Noronha ________, Robert. Multiculturalismo Tropical. São Paulo: Ed. Edusp, 2008. STRAUSS, Anselm L.; CORBIN, Juliet. Basics of Qualitative Research: Grounded Theory, Procedures and Techniques. Newbury: SAGE, 1990. XAVIER, I. A experiência do cinema. Rio de Janeiro: Graal, 1983. Recebido: 30.04.2019 Aprovado: 19.08.2019 Recebido: 30.04.2019 Aprovado: 19.08.2019 KANTER, R. M. Symbolic interactionism and politics in systemic perspective. Sociological inquiry, 1972. KANTER, R. M. Symbolic interactionism and politics in systemic perspective. Sociological inquiry, 1972. V.7 • N.13 • p. 180 – 202 • Jan-Jun/2019 ISSN 2318-3888
https://openalex.org/W4306315765
https://zenodo.org/records/7199731/files/21(116-120).pdf
Kirghiz, Kyrgyz
null
HISOR TOG' TIZMASIDAGI QIZIL DО'LANANING (CRATAEGUS TURKESTANICA POJARK.) TABIIY KО'PAYISHI VA О'RMONCHILIKDAGI AHAMIYATI
Zenodo (CERN European Organization for Nuclear Research)
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HISOR TOG‘ TIZMASIDAGI QIZIL DО‘LANANING (CRATAEGUS TURKESTANICA POJARK.) TABIIY KО‘PAYISHI VA О‘RMONCHILIKDAGI AHAMIYATI H X F ll i h Annotatsiya. Ushbu maqolada doʻlana tabiiy tarqalgan hududlarni aniqlash, turkum tarkibidagi turlar bioxilmaxilligi va doʻlanazorlarning tabiiy tiklanish koʻrsatkichlari boʻyicha tadqiqotlar natijalari keltirib oʻtilgan. Oʻrganish natijasiga koʻra, 1 gektardagi 5 yoshgacha bо‘lgan yosh kо‘chatlar miqdori 3580 donadan 4290 donagacha ekanligi aniqlandi. Maydonchalardagi kо‘chatlarning о‘rtacha soni Mingchinor bо‘limida 42 tani tashkil etgan bо‘lsa, Matmon bо‘limida 36 donani, Siyob bо‘limida esa 35 donani tashkil etdi. Bu esa, xoʻjalikdagi doʻlanazorlarda qoʻriqlash ishlari amalga oshirish zarurligini bildiradi. Kalit so‘zlar: Hisor tog‘ tizmasi, sariq do‘lana (Crataegus pontica C.Koch), qizil doʻlana (Crataegus turkestanica Pojark.), tabiiy tiklanish, ko‘chat, o‘rmon, bioxilmaxillig, tajriba maydoni. ЕСТЕСТВЕННОЕ ВОСПРОИЗВОДСТВО БОЯРЫШНИКА КРАСНОГО (CRATAEGUS TURKESTANICA POJARK.) В ГОРНОМ МАССИВЕ ХИСАР И ЕГО ЗНАЧЕНИЕ В ЛЕСНОМ ХОЗЯЙСТВЕ ЕСТЕСТВЕННОЕ ВОСПРОИЗВОДСТВО БОЯРЫШНИКА КРАСНОГО (CRATAEGUS TURKESTANICA POJARK.) В ГОРНОМ МАССИВЕ ХИСАР И ЕГО ЗНАЧЕНИЕ В ЛЕСНОМ ХОЗЯЙСТВЕ Аннотация. В данной статье представлены результаты исследований по выявлению ареалов естественного распространения боярышника, биоразнообразия видов в сериях и показателей естественного восстановления боярышниковых насаждений. По результатам исследования насаждения боярышника количество молодых сеянцев в возрасте до 5 лет на гектар составляет от 3580 до 4290 шт. Среднее количество саженцев на полях составило 42 в Мингчиноре, 36 в Матмоне и 35 в Сиобе. Это означает, что необходимо проводить защитные работы на полях боярышника в хозяйстве. Ключевые слова: Хисарский хребет, боярышник желтый (Crataegus pontica C. Koch), боярышник красный (Crataegus turkestanica Pojark.), естественное возобновление, сеянец, лес, биоразнообразие, опытный участок. II International scientific and practical conference "SUSTAINABLE FORESTRY" 14-15 October 2022 | scientists.uz II International scientific and practical conference "SUSTAINABLE FORESTRY" 14-15 October 2022 | scientists.uz HISOR TOG‘ TIZMASIDAGI QIZIL DО‘LANANING (CRATAEGUS TURKESTANICA POJARK.) TABIIY KО‘PAYISHI VA О‘RMONCHILIKDAGI AHAMIYATI Hamroyev Xusen Fatullayivech Oʻrmonchilik kafedrasi dotsenti, TDAU husen.hamroyev@mail.ru Rayimov Behruz Neʼmat oʻgʻli rayimovbehruz94@gmail.com Tayanch doktoranti, TDAU https://doi.org/10.5281/zenodo.7199731 Olingan natijalar quyidagicha formulalar yordamida tahlil etildi. ц р р у NATURAL REPRODUCTION OF RED HAWTHORN (CRATAEGUS TURKESTANICA POJARK.) IN THE HISAR MOUNTAIN RANGE AND ITS IMPORTANCE IN FORESTRY NATURAL REPRODUCTION OF RED HAWTHORN (CRATAEGUS TURKESTANICA POJARK.) IN THE HISAR MOUNTAIN RANGE AND ITS IMPORTANCE IN FORESTRY Abstract. In this article, the results of research on the identification of hawthorn natural distribution areas, the biodiversity of the species in the series and the indicators of natural recovery of hawthorn groves are presented. According to the results of the study, the number of young seedlings under 5 years old per hectare is from 3580 to 4290 units. The average number of seedlings in the fields was 42 in Mingchinor, 36 in Matmon, and 35 in Siyob. This means that it is necessary to carry out protection works in hawthorn fields on the farm. 116 II International scientific and practical conference "SUSTAINABLE FORESTRY" 14-15 October 2022 | scientists.uz 117 II International scientific and practical conference "SUSTAINABLE FORESTRY" 14-15 October 2022 | scientists.uz  = n N M M – maydonchalardagi daraxtlar о‘rtacha soni, dona. N – maydonchalardagi daraxtlar soni, dona. n – maydonchalar soni, dona. n – maydonchalar soni, dona. 1 − =  n C ∆ - standart chegaralanish, dona. n m  =  m – о‘rtacha arifmetik xato. % 100 * N V  = V – variatsiya koeffitsiyenti. n V P = R – hisobning aniqligi S M M га 10000 * = m – о‘rtacha arifmetik xato. Mga – 1 gektardagi daraxtlar soni, dona. S – umumiy maydon, ga S – umumiy maydon, ga n k Г 100 * = G – daraxtlarning mavjud bо‘lish koeffitsiyenti, %. G – daraxtlarning mavjud bо‘lish koeffitsiyenti, %. k – daraxtlari mavjud bо‘lgan maydonchalar soni, dona. га M КГ 2  = *100 га M КГ 2  = *100 KG – gomogenlik koeffitsiyenti. KG – gomogenlik koeffitsiyenti. ∆2 – dispersiY. II International scientific and practical conference "SUSTAINABLE FORESTRY" 14-15 October 2022 | scientists.uz Keywords: Hisar mountain range, yellow hawthorn (Crataegus pontica C. Koch), red hawthorn (Crataegus turkestanica Pojark.), natural regeneration, seedling, forest, biodiversity, experimental area. Kirish. О‘zbekiston о‘rmonlari rang-barang, bebaho daraxtlar, ozuqabop va shifobaxsh maxsulotlarga boy. Grek yong‘og‘i, handon pista, shirin bodom, olma, olcha, dо‘lana, zirk, na’matak kabi mevalilar shular jumlasidandir. Bizning о‘rmonlarimiz о‘simlik dunyosi ham benihoya boy bо‘lib, bu yerlarda 68 xil daraxt, 320 xil buta, 134 xil chala buta, 2953 xildan ziyod giyoh turlari mavjud. О‘rmonlar biologik rang-barangligini saqlashda biz insonlarning rolimiz juda katta. [4] Respublikaning tog‘li – tog‘ oldi mintaqalaridagi о‘rmonlar suv oqimini boshqaruvchi sifatida axamiyati katta, ular tuproqni yuvilish va о‘pirilishidan ximoya qiladi. Talofat keltiruvchi jala oqimining xosil bо‘lishining oldini oladi. CHо‘lli mintaqa о‘rmonlari qumlarning xarakati va ularning vohaga kelishi uchun tо‘siq xisoblanadi. Sug‘oriladigan erlarda о‘rmon daraxtlari qishloq xо‘jaliklari ekinlariga tabiat zararli omillarining salbiy ta’sirlarini kamaytiradi, ularning xosildorligini oshirishga yordam beradi. Shu sabablarni inobatga olgan holda do‘lana turkum vakillarini tabiiy tarqalish areallari o‘rganilmoqda. Dо‘lana ra’noguldoshlar oilasiga mansub daraxt yoki buta hisoblanib, balandligi butalariniki 0,5 - 4 metrga, daraxtlariniki esa 10 - 15 metrgacha bо‘ladi. Tanasining yо‘g‘onligi turiga qarab har xil о‘zi sershox, Xitoy, Ispaniya, Italiya, Jazoir, Amerikada 890 turi bor. MDH da 50 turi uchraydi. Shundan 10 turi О‘zbekistonda о‘sadi. [2] Hisor togʻ tizmasidagi doʻlanazorlar togʻli hududlarida asosan, Siyob, Mingchinor, Matmon oʻrmonchilik boʻlimlarida keng tarqalgan. Oʻrmonchilik boʻlimlari boʻyicha tahlil etilganda, Matmon oʻrmonchilik boʻlimida sariq doʻlana (Crataegus pontica C.Koch), Mingchinor oʻrmonchilik boʻlimida Turkiston yoki qizil doʻlana (Crataegus turkestanica Pojark.), Siyob oʻrmonchilik boʻlimida esa sariq va qizil doʻlana turlari tarqalgan [5]. Oʻrganish natijasida mevalarining oʼlchami diametr boʻyicha 1,6-1,9 sm, uzunligi boʻyicha 1,3-1,5 sm, meva ogʻirligi 2,2-3,2 gr, urugʻ ogʻirligi 0,6-0,8 g, hamda magʻiz chiqish koʻrsatkichi 71,3-75,9% ni tashkil etdi.[3]. О‘zbekiston Respublikasi Prezidentining 2017 yil 11 maydagi PQ2966-sonli “О‘zbekiston Respublikasi О‘rmon xо‘jaligi davlat qо‘mitasi faoliyatini tashkil etish tо‘g‘risida” gi qarorida hamda о‘rmonchilikka tegishli boshqa meyoriy-xuquqiy xujjatlarda belgilab berilgan vazifalarni amalga oshirishda ushbu dissertatsiya ishi tadqiqotlari muayyan darajada xizmat qiladi. [1] Tadqiqot uslubi: Tajriba ishlari Oʻzbekiston hududining janubida joylashgan Hisor togʻ tizmasida olib borildi. Doʻlananing tavsifi, biologik, ekologik xususiyatlari va tabiiy tarqalish areali adabiy manbalarni tahlil etilgan holda oʻrganiladi. Bunda ushbu daraxtning balandligi, diametri, vegetativ va generativ organlarining tuzilishi boʻyicha maʼlumotlar keltirib oʻtiladi. Shu bilan birga uning tashqi muhit omillariga munosabati oʻrganilgan holda daraxtning ekologik xususiyatlariga tavsif beriladi. Hisor tog‘ tizmasida tabiiy tarqalgan qizil do‘lana (Crataegus turkestanica Pojark.) turing maydonlari aniqlanadi. Olingan natijalar quyidagicha formulalar yordamida tahlil etildi. II International scientific and practical conference "SUSTAINABLE FORESTRY" 14-15 October 2022 | scientists.uz Yuqoridagi jadval ma’lumotlariga asosan Kitob о‘rmon xо‘jaligining Mingchinor va Matmon bо‘limlaridagi qizil dо‘lanazorlarining tabiiy tiklanish darajasi qoniqarli. Lekin ushbu maydonlardagi qizil dо‘lanazorlarni antropogen ta’sirlardan himoyalash kerak bо‘ladi. Xulosa. Olib borilgan kuzatuvlar tahlili shuni koʻrsatadiki, Hisor tog‘ tizmasida joylashgan Kitob davlat oʻrmon xoʻjaligi hududida qizil do‘lana turining tabiiy tiklanish ko‘rsatkichlari kuzatilib, hozirgi holati o‘rganildi. Bunda bo‘limlardagi do‘lanazorlarning holat Bо‘limlar bо‘yicha 1 gektardagi 5 yoshgacha bо‘lgan yosh kо‘chatlar miqdori 3580 donadan 4290 donagacha ekanligi aniqlandi; Maydonchalardagi kо‘chatlarning о‘rtacha soni Mingchinor bо‘limida 42 tani tashkil etgan bо‘lsa, Matmon bо‘limida 36 donani, Siyob bо‘limida esa 35 donani tashkil etdi. Bu ko‘rsatkichlarni inobatga olgan holda tog‘ning do‘lan o‘sa oladigan balandliklarida do‘lana ko‘chatlarini ekish, tabiiy o‘sib turgan do‘lanazorlarni saqlab qolish va asrab avaylash talab etiladi. Bu hududlarda qo‘shimcha o‘rmonchilik tadbirlarini amalga oshirishni talab etadi. ∆2 – dispersiY. Tadqiqot natijalari: Tadqiqot uslubiga kо‘ra tajriba maydonchalarining о‘lchami о‘rtacha 10x10 m2 ni tashkil etib, ular turli ekspozitsiyalarda joylashgan. Tajriba uchun 5-10 yoshgacha bо‘lgan sariq va qizil dо‘lanalarning kо‘chatlari tanlab olindi va ularni sanab chiqish orqali hududdagi dо‘lanazorlarning tabiiy tiklanish darajasi tahlil etildi. Shu bilan birga xо‘jalikdagi dо‘lanazorlar tо‘liqligi ekspozitsiyalar bо‘yicha turlicha bо‘lib, shimoliy ekspozitsiyada yuqori (0,8), janubiy ekspozitsiyalarda esa eroziyaning hamda xо‘jalik faoliyatining ta’siri natijasida tо‘liqlik 0,6 ni tashkil etdi. 118 1-jadval Kitob о‘rmon xо‘jaligi hududidagi dо‘lanazorlarning joylashgan о‘rni № Dо‘lanazor joylashgan о‘rmonchilik bо‘limi Tо‘liqligi Maydoni Turi 1 Matmon 0,6 560 Sariq dо‘lana II International scientific and practical conference "SUSTAINABLE FORESTRY" 14-15 October 2022 | scientists.uz 2 Siyob 0,7 320 Sariq va qizil dо‘lana 3 Mingchinor 0,8 654 Qizil dо‘lana 2 Siyob 0,7 320 Sariq va qizil dо‘lana 3 Mingchinor 0,8 654 Qizil dо‘lana Xо‘jalikdagi dо‘lanazorlarining tabiiy tiklanish darajasini tahlil etish uchun 3 ta bо‘limdan 10 tadan (jami 30 ta) tajriba maydonchalari о‘rnatildi va ulardagi 5-10 yoshlik kо‘chatlar miqdori hisoblanib chiqildi. Unga tajriba maydonchalarida kо‘chatlari miqdori umuman yо‘q bо‘lgan holatlari kuzatilmadi. j Xо‘jalik dо‘lanazorlaridagi qizil dо‘lana yosh nihollarning miqdori, dona № Bо‘limlar nomi Mingchinor Siyob Matmon 1 45 34 35 2 40 36 41 3 42 30 42 4 42 30 35 5 38 30 38 6 50 32 35 7 44 34 41 8 48 40 30 9 40 42 35 10 40 50 31 Xо‘jalik dо‘lanazorlaridagi qizil dо‘lana yosh nihollarning miqdori, dona Bо‘limlar nomi Tajriba maydonidagi yosh nihollar miqdoridan kelib chiqqan holda hududdagi tabiiy tiklanish darajasi tahlil etilganda quyidagilar aniqlandi: Bо‘limlar bо‘yicha 1 gektardagi 5 yoshgacha bо‘lgan yosh kо‘chatlar miqdori 3580 donadan 4290 donagacha ekanligi aniqlandi; Maydonchalardagi kо‘chatlarning о‘rtacha soni Mingchinor bо‘limida 42 tani tashkil etgan bо‘lsa, Matmon bо‘limida 36 donani, Siyob bо‘limida esa 35 donani tashkil etdi. Bizning dasturimiz bо‘yicha va Pobedenskiy bergan tavsiyalar bо‘yicha KG (gomogenlik koeffitsiyenti) ning qiymati 1 dan kichik bо‘lsa tabiiy tiklanish juda yaxshi, 1 ga yaqin bо‘lsa qoniqarli va 1 dan yuqori bо‘lsa qoniqarsiz deb topilishi qayd etib о‘tilgan. j Kitob о‘rmon xо‘jaligidagi qizil dо‘lanazorlarining bо‘limlar bо‘yicha tabiiy tiklanishi darajasini baholash jadvali Bо‘limlar nomi О‘rtacha kо‘rsatkich M m Satatistik tahlil Δ n S Mga KG Mingchinor 42,9 ± 1,28 3,8 10 100 4290 0,3825 Siyob 35,8 ± 2,16 6,5 10 100 3580 1,3091 Matmon 36,3 ± 1,38 4,1 10 100 3630 0,5241 Kitob о‘rmon xо‘jaligidagi qizil dо‘lanazorlarining bо‘limlar bо‘yicha tabiiy tiklanishi darajasini baholash jadvali 119 REFERENCES 1. Oʻzbekiston Respublikasi Prezidentining 2017-yil 11-maydagi “Oʻzbekiston respublikasi oʻrmon xoʻjaligi davlat qoʻmitasini tashkil etish toʻgʻrisida” PF-5041 sonli farmoni. 2. Qayimov A.Q., Berdiyev E.T., Xamroyev X.F., Turdiyev S.A. Dendrologiya (darslik). Toshkent. Fan va texnologiya, 2015 y. 360 b. 2. Qayimov A.Q., Berdiyev E.T., Xamroyev X.F., Turdiyev S.A. Dendrologiya (darslik). Toshkent. Fan va texnologiya, 2015 y. 360 b. 3. Khamroyev Kh.F., Rayimov B.N. Indicators Of Natural Recovery Of Hawthorn In Southern Uzbekistan. International Journal of Progressive Sciences and Technologies (IJPSAT) ISSN: 2509-0119 2021 Vol. 25 No. 2 March 2021, pp. 266-270 3. Khamroyev Kh.F., Rayimov B.N. Indicators Of Natural Recovery Of Hawthorn In Southern Uzbekistan. International Journal of Progressive Sciences and Technologies (IJPSAT) ISSN: 2509-0119 2021 Vol. 25 No. 2 March 2021, pp. 266-270 4. Пояркова А.И. Crataegus L – Боярышник II флора Узбекистана, Том-III Ташкент. 1955 г. Ст-68-82. 5. Шайматов О.А., Холиқов Д.М. Китоб ўрмон хўжалигидаги сариқ ва қизил дўлана турларининг тарқалиши. // Жанубий Оролбўйи биологик хилма-хиллигини сақлаш, қайта тиклаш ва муҳофаза қилишнинг экологик масалалари номли халқаро илмий-амалий конференция материаллари. Нукус 2018. 285-286 б. 120
https://openalex.org/W3127352272
http://jkd.stikesdirgahayusamarinda.ac.id/index.php/jkd/article/download/71/34
Indonesian
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GAMBARAN PEGETAHUAN REMAJA PUTRI TENTANG DISMINORE PADA MAHASISWA TINGKAT I PROGRAM STUDI DIPLOMA III KEPERAWATAN STIKES DIRGAHAY SAMARINDA
Jurnal Keperawatan ( Samarinda)/Jurnal Keperawatan Dirgahayu
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GAMBARAN PENGETAHUAN REMAJA PUTRI TENTANG DISMINORE PADA MAHASISWA TINGKAT I PROGRAM STUDI DIPLOMA III KEPERAWATAN STIKES DIRGAHAYU SAMARINDA Endang Wiwiek Program Studi Diploma III Keperawatan STIKES Dirgahayu Samarinda Jalan Pasundan No.21 Telp (0541) 748335, Fax. (0541)748335 email: endangwiwiekpurnama@gmail.com ABSTRAK Rasa nyeri saat haid merupakan keluhan ginekologi yang paling umum dan banyak dialami oleh wanita. Rasa nyeri saat haid tidak diketahui secara pasti kaitannya dengan penyebabnya, namun beberapa faktor dapat mempengaruhi yaitu ketidak seimbangan hormon dan faktor psikologis. Walaupun umumnya tidak berbahaya, namun sering kali dirasa mengganggu bagi wanita yang mengalaminya. Derajat nyeri dan kadar gangguan tentu tidak sama untuk setiap wanita. Ada yang masih bisa beraktifitas, ada pula yang tidak bisa beraktifitas karena nyeri. Dismenore ditandai dengan nyeri perut bagian bawah, mual, muntah, maupun gejala lainnya. Derajat nyeri tiap individu berbeda tergantung dari nyeri, persepsi, maupun pengalaman individu. Tujuan dari penelitian ini yaitu untuk memperoleh pengetahuan mahasiswi STIKES Dirgahayu Samarinda mengenai dismenore. Jenis penelitian ini adalah penelitian deskriptif. Teknik pengambilan sampel menggunakan teknik total sampling dari kriteria inklusi. Populasi dalam penelitian ini adalah mahasiswi tingkat I. Jumlah responden yang didapat yaitu sebanyak 50 responden. Metode yang dipakai dalam penelitian ini yaitu dengan menyebarkan kuesioner. Hasil penelitian ini menggambarkan bahwa tingkat pengetahuan termasuk dalam kategori baik 40 responden (80%), mengetahui tanda dan gejala sebanyak 39 responden (78%), penanganan dismenore sebanyak 32 responden (64%). Kata kunci : dismenore, tanda dan gejala, penanganan Jurnal Keperawatan Dirgahayu Volume 1, Nomor 2, Oktober 2019 Jurnal Keperawatan Dirgahayu Volume 1, Nomor 2, Oktober 2019 EISSN: 2685-3086 PENDAHULUAN Berdasarkan latar belakang di atas dan menurut survey awal yang sudah dilakukan peneliti ternyata masih kurangnya pengetahuan remaja putri tentang dismenore, maka penulis tertarik melakukan penelitian mengenai tingkat pengetahuan remaja putri tentang tentang dismenore pada mahasiswi STIKES Dirgahayu Samarinda Program Studi Diploma III Keperawatan Tingkat I Tahun 2018 Rasa nyeri saat haid merupakan keluhan ginekologi yang paling umum dan banyak dialami oleh wanita. rasa nyeri saat haid tidak diketahui secara pasti kaitannya dengan penyebabnya, namun beberapa faktor dapat mempengaruhi yaitu ketidak seimbangan hormon dan faktor psikologis. Kesehatan reproduksi adalah suatu keadaan sejahtera fisik, mental dan sosial secara utuh, tidak semata- mata bebas dari penyakit atau kecacatan dalam semua hal yang berkaitan dengan sistem reproduksi (Azwar, 2001). EISSN: 2685-3086 EISSN: 2685-3086 ABSTRACT A sense of pain during menstruation is the most common and many gynecological complaints experienced by women. A sense of pain during menstruation is not known with certainty the relation to the cause, but several factors can influence namely hormonal imbalance and psychological factors. Although it is generally harmless, it is often felt annoying for women who experience it. The degree of pain and the level of the disorder are certainly not the same for every woman. Some are still able to do activities, some are not able to indulge in pain. Dysmenorrhea is characterized by lower abdominal pain, nausea, vomiting, and other symptoms. The degree of pain of each individual is different depending on pain, perception, and individual experience. The purpose of this study is to obtain an overview of the knowledge of STIKES Dirgahayu Samarinda students regarding dysmenorrhea. This type of research was descriptive research. Sampling technique used the total sampling technique from the inclusion criteria. The population in this study were first-level students. The number of respondents obtained was 50 respondents. The method used in this study is by distributing questionnaires. The results of this study illustrate that the level of knowledge included in the good category of 40 respondents (80%), knowing signs and symptoms as many as 39 respondents (78%), handling dysmenorrhea as many as 32 respondents (64%). Keywords: dysmenorrhea, signs and symptoms, treatment 1 METODE PENELITIAN Penelitian ini adalah penelitian deskriptif dimana peneliti menguraikan atau memberi gambaran atau deskripsi tentang suatu keadaan secara objektif mengenai pengetahuan mahasiswa tingkat I tentang dismenorea. Penelitian ini adalah penelitian deskriptif dimana peneliti menguraikan atau memberi gambaran atau deskripsi tentang suatu keadaan secara objektif mengenai pengetahuan mahasiswa tingkat I tentang dismenorea (Notoadmodjo. 2005). Rancangan dalam penelitian ini untuk mengetahui pengetahuan remaja putri tentang dismenore. Setiap bulan, secara periodik, seorang wanita normal akan mengalami peristiwa reproduksi, yaitu menstruasi meluruhnya jaringan endometrium karena tidak adanya telur matang yang dibuahi oleh sperma peristiwa itu begitu wajar dan alami sehingga dapat dipastikan bahwa semua wanita normal pasti akan mengalami proses itu. walaupun begitu kenyataannya banyak wanita yang mengalami masalah menstruasi, di antaranya adalah nyeri haid (Dismenore). Walaupun umumnya tidak berbahaya, namun sering kali dirasa mengganggu bagi wanita yang mengalaminya. Derajat nyeri dan kadar gangguan tentu tidak sama untuk setiap wanita. Dismenorea adalah kekakuan atau kekejangan di bagian bawah perut yang terjadi pada waktu menjelang atau selama menstruasi, yang memaksa wanita untuk beristirahat atau berakibat pada menurunnya kinerja dan kurangnya aktifitas sehari-hari. Ternyata di Indonesia, lebih banyak perempuan yang mengalami dismenorea tidak melaporkan atau berkunjung ke dokter. Rasa malu ke dokter dan kecenderungan meremehkan penyakit sering membuat data penderita tidak dapat dipastikan secara mutlak. Boleh dikatakan 90% perempuan pernah mengalami dismenore (Anurogo, 2011). Jurnal Keperawatan Dirgahayu Volume 1, Nomor 2, Oktober 2019 Jurnal Keperawatan Dirgahayu Volume 1, Nomor 2, Oktober 2019 EISSN: 2685-3086 (2010), gejala dismenore adalah nyeri pada perut yang timbul sebelumnya, bersamaan dengan permulaan haid dan berlangsungbeberapa jam namun bisa sampai bertahan hingga beberapa hari. Berdasarkan hasil penelitian dari 50 responden 39 orang mengetahui tanda dan gejala.(78%) Tabel 5.1.3 Karakteristik Penanganan dismenore Responden Variabel Frekuensi Persen Total Persen Tahu Tidak tahu 32 18 64 36 50 100 Tabel 5.1.3 Karakteristik Penanganan dismenore Kesimpulan Berdasarkan analisis data dan intepretasinya, maka dapat ditarik kesimpulan tingkat pengetahuan siswa tentang dismenore adalah sebagai berikut: 1. Tingkat pengetahuan remaja putri mahasiswi Program Studi Diploma III Keperawatan Tingkat I STIKES Dirgahayu Samarinda tentang pengertian dismenore dalam kategori baik sebanyak 40 orang (80%). 2. Tingkat pengetahuan remaja putri mahasiswi Program Studi Diploma III Keperawatan Tingkat I STIKES Dirgahayu Samarinda tentang gejala dismenore dalam kategori cukup sebanyak 39 orang (78%). Pembahasan 1. Pengetahuan remaja putri tentang pengertian dismenore Pengetahuan remaja putri tentang pengertian dismenore memiliki beberapa tingkatan yang salah satunya adalah tahu yang merupakan kemampuan mengenali atau mengingat materi yang telah didapat sebelumnya,sebagian besar responden sudah sesuai dengan teori Dari hasil penelitian didapatkan responden yang mengetahui tentang pengertian dismenore sebanyak 40 responden (80%). Responden sebagian besar memiliki pengetahuan yang baik mengenai pengertian dismenore. Hal ini dapat disebabkan responden pernah mendapat materi tentang dismenore. Jurnal Keperawatan Dirgahayu Volume 1, Nomor 2, Oktober 2019 Jurnal Keperawatan Dirgahayu Volume 1, Nomor 2, Oktober 2019 EISSN: 2685-3086 Hasil Penelitian Tabel 5.1.1 Karakteristik Pengertian dismenore Responden Variabel Frekue nsi Persen Total % Tahu Tidak tahu 40 10 80 10 50 100 Pada penelitian ini dari 50 responden mengetahui pengertian dismenore ada 40 orang (80%). Tabel 5.1.2 Karakteristik Tanda dan gejala dismenore Responden Variabel Frekuensi % Total % Tahu Tidak tahu 39 11 578 22 50 11100 Tabel 5.1.2 Karakteristik Tanda dan gejala dismenore 2 3. Sikap remaja putri tentang penanganan saat dismenorea. Tingkat pengetahuan tentang penanganan dismenore pada remaja putri dari hasil penelitian didapatkan 32 responden (64 %) responden mengetahui tentang penanganan, dan 18 responden (36%) yang tidak tahu tentang penanganan dismenore. Pengetahuan responden tentang penanganan dismenorea dalam kategori cukiup. Arti cukup dapat diterjemahkan bahwa lebih dari 50% responden mempunyai pengetahuan yang cukup sebelum melakukan penanganan dismenore. Berdasarkan hasil penelitian dari 50 responden 32 orang (64%) mengetahui cara penanganan dismenore 2. Bagi Institusi Tempat Penelitian 2. Bagi Institusi Tempat Penelitian 3. Hasil penelitian ini diharapkan dapat menjadi masukan bagi pihak kampus, untuk melakukan penanganan yang tepat agar dismenore yang dirasakan remaja putri tidak mengganggu aktivitas kuliah. Jacoeb. 2006. “Dismenorea aspek patofisiologi dan pena talaksanaan”. Jakarta: 4. Bagi Peneliti Berikutnya Kasdu D. 2005. “Solusi Problem Wanita Dewasa Cet. I”. Jakarta : Puspa Swara.Kelly T. 2007. 5. Hasil penelitian ini dapat menjadi informasi untuk peneliti selanjutnya yang melakukan penelitian terkait agar dapat mengembangkan variabel penelitian, sehingga dapat mengkaji hal-hal yang belum dimunculkan atau belum dibahas dalam penelitian. Laila, N.N. 2011. Buku Pintar Menstruasi. Buku Biru: Yogyakarta Lakhsmi, A. Saraswathi, P.Ramamchandran. 2011. Prevalence of Pre-Menstruasion Syndrome and Dysmenorrhoea among Female Medical Students and Its Associationwith College Absenteeism: International Jurnal of Biological & Medical Research Vol 2(4): 1011 -1016 Benson R. 2008. “Buku saku Obstetri dan Ginekologi”. Jakarta: EGC 1. Bagi Perkembangan Ilmu Keperawatan :Hasil dari penelitian ini diharapkan dapat dijadikan sebagai sumber informasi dalam upaya pengembangan ilmu keperawatan khususnya dalam hal gambaran kejadian dismenore karena dismenore merupakan gangguan reproduksi. Perawat diharapkan agar dapat melakukan edukasi terkait upaya penanganan dismenore yang baik dan benar. kategori cukup sebanyak 32 orang (64,2%). kategori cukup sebanyak 32 orang (64,2%). Pengukurannya”.Yogyakarta; Pustaka Pelajar. -----------2007. “Validitas d Reliabilitas”. Yogyakarta Pustaka Pelajar. Pengukurannya”.Yogyakarta; Pustaka Pelajar. -----------2007. “Validitas d Reliabilitas”. Yogyakarta Pustaka Pelajar. Jurnal Keperawatan Dirgahayu Volume 1, Nomor 2, Oktober 2019 EISSN: 2685-3086 2. Pengetahuan remaja putri tentang tanda dan gejala dismenore Tingkat pengetahuan tentang tanda dan gejala dismenore pada remaja putri dari hasil penelitian didapatkan 39 responden (78 %) responden mengetahui tentang tanda dan gejala, dan hanya 11 responden (22 %) yang tidak tahu tanda dan gejala.Gejala dismenorea dapat disertai dengan rasa mual, muntah, diare dan kram perut. Menurut Winkjosatro 3. Tingkat pengetahuan remaja putri mahasiswi Program Studi Diploma III Keperawatan Tingkat I STIKES Dirgahayu Samarinda tentang penanganan dismenore dalam 3 Saran Berdasarkan kesimpulan hasil penelitian, maka disarankan sebagai berikut : Benson R. 2008. “Buku saku Obstetri dan Ginekologi”. Jakarta: EGC Dewi, N. S. (2012). Biologi reproduksi. Yogyakarta: Pustaka Rihama Defi Nafiroh, dkk, Gambaran Pengetahuan Remaja.tentang dismenore pada siswa putri di MTS NU Mranggen Kabupaten Demak. Jurnal Ilmiah Kebidanan, Vol. 4 No. 1 Edisi Desember 2013, hlm. 157-166 Hiday at A. 2009. “Metode Penelitian Kebidanan dan Teknik Analisis Data Edisi2”. Jakarta: Salemba Medika 2. Bagi Institusi Tempat Penelitian DAFTAR PUSTAKA Arikunto S. 2010. “Prosedur Penelitian Suatu Pendekatan Praktik. Jakarta : Rineka Cipta. Anurogo, D. Wulandari, A. (2011). Cara Jitu Menangani Nyeri Haid.Yogyakarta: Andi Offset. Llewellyn D. 2001. “Dasar-Dasar Obstetri dan Ginekologi”. Jakarta: Hipokrates Azwar S. 2005. “Sikap Manusia, Teori Dan 4 4 Sugiyono. 2007. “Statistika Untuk Penelitian”. Bandung : Alfabeta. Notoatmodjo S. 2010. “Metodologi Penelitian Kesehatan”. Jakarta : Rineka Cipta. Sukarni K, I & Wahyu, P. (2013). Buku ajar keperawatan maternitas. Yogyakarta: Nuha Medika -----------. 2012. ”Promosi Kesehatan dan Perilaku”. Jakarta : Rineka Cipta. Jurnal Keperawatan Dirgahayu Volume 1, Nomor 2, Oktober 2019 EISSN: 2685-3086 Soekanto S. 2004. “Sosiologi Keluarga Tentang Ikhwal Keluarga, Remaja, dan Anak”. Jakarta : Rineka Cipta. Manuaba, Ida Bagus Gde, (2012). Memahami KesehatanReproduksi Wanita. Jakarta: Arean. Morgan G. 2009. “Obstetri dan Ginekologi: Panduan Praktik”. Jakarta: EGC Sugiyono. 2007. “Statistika Untuk Penelitian”. Bandung : Alfabeta. Sulistina, D. 2009. “Hubungan antara Pengetahuan Menstruasi dengan Perilaku Kesehatan Nursalam. 2008. “Konsep dan Penerapan Metodologi Penelitian Ilmu Keperawatan, . Jakarta : Salemba Medika. Taufiqurrahman M. A. 2008. “Pengantar Metologi Penelitian untuk Ilmu Kesehatan”. Surakarta: LPP UNS Prawiroharjo, 2011. “Ilmu Kebidanan”. Jakarta: Yayasan Bina Pustaka Sarwono Prawirohardjo Tia M, Faktor-Faktor Yang Berhubungan Dengan Kejadian Dismenore Jurnal Kesmas Indonesia, Volume 8 No 1, Januari 2016, Hal 40-48 Proverawati,A. dan Misaroh, S. (2009).Menarche (MenstruasiPertama Penuh Makna). Yogyakarta:Muha Medika Walgito B. 2003. “Psikologi Sosial (Suatu Pengantar)”. Yogjakarta: Andi Offset Sarwono S. 2007. “Psikologi Remaja”. Jakarta : PT. Raja Grafindo Persada. Wiknjosastro H. 2009. “Ilmu Kandungan”. Jakarta: Yayasan Bina Pustaka Sarwono Prawirohardjo. Saxena T, Kumari R, Khurana S, dan Rawat M. Effectivenes of dance therapy on primary dysmenorrhea in young females. Guru Drone Journal of Pharmacy & Research. 2014; 2(3):11-16. Yatim F. 2001. “Haid Tidak Wajar dan MenopauseEd. 1”. Jakarta : Pustaka Populer Obor. 5
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Four Centuries of Change in Northeastern United States Forests
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Introduction The land use history of the northeastern United States is well documented but its ecological consequences remain poorly understood, especially at a regional scale [1–4]. For more than 10,000 years native people cleared modest areas along waterways and seasonal settlements and managed some upland areas through sporadic understory burning [5]. Even so, the region was overwhelmingly forested and chiefly governed by non-anthropo- genic disturbances and successional dynamics until around 1650, when two centuries of logging and agricultural clearing were initiated that removed more than half of the forest cover and cut over almost all of the rest. Outside of the far north and rugged mountainous regions, the northeast became a predominantly humanized agrarian landscape. Forest cover reached its nadir in the mid nineteenth century, after whichagricultural expansion to the Midwest and eastern industrialization resulted in widespread farm abandonment, population concentration and, in turn, a century of natural reforestation and forest growth [6,7]. The emerging forest supported new wood-based industries and natural processes including forest succession interrupted by damage from severe storms such as the powerful 1938 Hurricane, which was compounded by subsequent salvage logging on massive scales [6,8,9]. The modern landscape appears to have recently reached its apex of reforestation and the region is again experiencing a net loss of forest cover. While agricultural land cover continues to Given the long and tumultuous history of landscape change, an important ecological question emerges: How similar is the composition of today’s forests compared to those existing prior to European colonization? This question has relevance far beyond the northeastern U.S. Conversion of natural ecosystems for agriculture and developed uses are a hallmark of human civilization. Indeed, more than one-third of the land surface of the earth is currently under agricultural use and more than half of the human population lives in urban regions [12]. Throughout human history shifts in economic forces have resulted in land abandonment followed by natural reforestation, as happened in the northeastern U.S. [13], in Central America after the Mayan collapse [14], in parts of the Ecuadorian Amazon [15] and western Europe, and elsewhere [16]. Large-scale Reforestation and natural restoration of forest regions are also major goals for modern conservation. Therefore the question looms: how do regional ecosystems recover from such massive scales of anthropogenic disturbance? Are the secondary ecosystems qualitatively different in terms of composition, function, and services? Four Centuries of Change in Northeastern United States Forests Jonathan R. Thompson1*, Dunbar N. Carpenter2, Charles V. Cogbill3, David R. Foster3 Jonathan R. Thompson1*, Dunbar N. Carpenter2, Charles V. Cogbill3, David R. Foster3 1 Smithsonian Conservation Biology Institute, Smithsonian Institution Front Royal, Virginia, United States of America, 2 Department of Forest and Wildlife Ecology, U i it f Wi i M di Wi i U it d St t f A i 3 H d F t H d U i it P t h M h tt U it d St t f A i 1 Smithsonian Conservation Biology Institute, Smithsonian Institution Front Royal, Virginia, United States of America, 2 Department of Forest and Wildlife Ecology, University of Wisconsin, Madison, Wisconsin, United States of America, 3 Harvard Forest, Harvard University, Petersham, Massachusetts, United States of America Abstract The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: thompsonjr@si.edu decline throughout the region, land cover transitions to developed uses now override reforestation [10]. As of 2010, approximately 80 percent of the region was forested, though less than one-percent of old-growth forest remains intact [11]. Abstract The northeastern United States is a predominately-forested region that, like most of the eastern U.S., has undergone a 400- year history of intense logging, land clearance for agriculture, and natural reforestation. This setting affords the opportunity to address a major ecological question: How similar are today’s forests to those existing prior to European colonization? Working throughout a nine-state region spanning Maine to Pennsylvania, we assembled a comprehensive database of archival land-survey records describing the forests at the time of European colonization. We compared these records to modern forest inventory data and described: (1) the magnitude and attributes of forest compositional change, (2) the geography of change, and (3) the relationships between change and environmental factors and historical land use. We found that with few exceptions, notably the American chestnut, the same taxa that made up the pre-colonial forest still comprise the forest today, despite ample opportunities for species invasion and loss. Nonetheless, there have been dramatic shifts in the relative abundance of forest taxa. The magnitude of change is spatially clustered at local scales (,125 km) but exhibits little evidence of regional-scale gradients. Compositional change is most strongly associated with the historical extent of agricultural clearing. Throughout the region, there has been a broad ecological shift away from late successional taxa, such as beech and hemlock, in favor of early- and mid-successional taxa, such as red maple and poplar. Additionally, the modern forest composition is more homogeneous and less coupled to local climatic controls. n JR, Carpenter DN, Cogbill CV, Foster DR (2013) Four Centuries of Change in Northeastern United States Forests. PLoS ONE 8(9): e7 pone.0072540 Carpenter DN, Cogbill CV, Foster DR (2013) Four Centuries of Change in Northeastern United States Forests. PLoS ONE 8(9): e72540 0072540 Editor: Ben Bond-Lamberty, DOE Pacific Northwest National Laboratory, United States of America Editor: Ben Bond-Lamberty, DOE Pacific Northwest National Laboratory, United States of America Received May 21, 2013; Accepted July 10, 2013; Published September 4, 2013 Received May 21, 2013; Accepted July 10, 2013; Published September 4, 2013 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This research was supported by the US National Science Foundation through its programs in Long Term Ecological Research (DEB 06-20443). Study Region We examined changes in forest composition within a sample of colonial townships distributed throughout a nine state region of the northeastern U.S. (Figure 1), ranging latitudinally from northern Maine (47u309 N) to southern New Jersey (39u309 N) and longitudinally from western Pennsylvania (67u W) to the Atlantic Ocean (80u309 W). The study area encompasses 4.336105 km2 and spans nine physiographic provinces, primarily in the Appalachian Highlands Division, but also extending into portions of the Atlantic and Interior Plains. The region’s rolling topography is interrupted by several discrete sub-mountain ranges belonging to the greater Appalachian Chain. Several major rivers including the Hudson, Connecticut, Merrimack, Susquehanna, and Penobscot form low-elevation valleys across the study area. Much of the present geology of the region was shaped by the last glaciation (c. 20,000 ybp). The region is influenced by a range of climatic conditions; annual mean temperatures range from 3 to10uC (mean Jan temp = 26uC; mean July temp = 19uC), and average annual precipitation ranges from 79 to 255 cm. The study area includes four USFS designated ecoregions (Figure 1), defined based on a broad set of ecologically-relevant attributes [35], which we used to stratify our samples. Witness tree have been used extensively throughout the northeast to reconstruct historical forest composition, without making comparisons to modern forest conditions [21–27]. Far fewer studies have quantified compositional change and these have all focused on relatively small regions. Their localized perspective limits our understanding of the regional variability and the relative importance of land use history and biophysical setting for determining the degree and nature of forest change. A review of these studies shows some consistent changes. For instance, the loss of American chestnut (Castanea dentata) due to an introduced fungal blight (Cryphonectaria parasitica) is evident throughout the region [28]. But looking across these studies shows that changes in composition have not been uniform throughout the Northeast. Of course, there are a number of reasons to expect different patterns in different places related to variation in: land use, edaphic factors, disturbance regimes or position relative to climatic drivers and floristic boundaries. For example, Bu¨rgi et al. [29] compared witness trees to modern inventory data in two counties in Massachusetts and two in Pennsylvania and found an almost two-fold difference in the degree of overall compositional change between study areas. Study Region Similarly, some studies suggest that the modern forest is compositionally more homogeneous than the forests they replaced [4,30] while others have found the opposite [31]. Trends in the abundance of individual taxa also vary widely. The abundance of oak, for example, has increased in some areas [32] and decreased in others [33]. Variation in land use is often assumed to be a primary determinant of change and, indeed, agricultural clearing, harvesting bark for tanning, and logging have been correlated with compositional change [29,31,34]. F h l l l di k h h f h Introduction Or, does an inherent resilience and species fidelity to environmental conditions drive regional ecosystems back to their pre-disturbance condition? September 2013 | Volume 8 | Issue 9 | e72540 1 PLOS ONE | www.plosone.org Four Centuries of Change in U.S. Forests Because the processes of regional disturbance and recovery occur over such large spatial and long temporal scales, these questions are rarely addressed empirically. producing a landscape that is compositionally more or less diverse to what existed prior to colonization? (4) Is the nature of compositional change consistent across the region? (5) What are the patterns of change among individual taxa? By answering these questions we begin to quantify the resilience of regional ecosystems subjected to large-scale shifts in land use. y p y Here we use a unique dataset, singular in its geographic scope, to quantify the Northeast U.S. region’s response to long-term, broad-scale shifts in land use. We compare modern forest data to historical vegetation data gleaned from archival, colonial land- survey notes collected over a nine-state area. Colonial surveyors described the details of ‘‘witness trees,’’ which served as semi- permanent monuments of survey corners. Ecologists have been using these inadvertent forest inventories to reconstruct pre- colonial forest composition for almost a century (e.g., [17]). The records contain a wealth of information about North America’s historical ecosystems [18]. But because the records were not designed to record vegetation, they do have several limitations and biases, (see [19,20] for reviews). In the Northeast, the witness trees demarcated the corners of lots ranging in size from 0.5 to 65 hectares [2,21]. Unlike the more systematic approach used by the General Land Office, concurrent with westward expansion from Ohio to California, witness tree data in the Northeast were collected using a variety of methods, typically do not include tree size, and commonly identify trees only to genera as opposed to species [19]. Despite these comparative shortcomings, ecologists have made great use of town proprietor records and the references to witness trees therein. Pre-colonial data We used the relative abundance of witness tree taxa (i.e., proportion of each taxon) identified in Table 1 within proprietary towns, as our metric of pre-colonial forest composition (c.f. [21]). Proprietary towns (hereafter ‘‘towns’’) were granted by the colonies and states to absentee individuals to encourage colonization and ‘‘improvement’’ of the land throughout the period spanning from just after English colonization (1620) to after the creation of the Erie Canal (1825). Towns were usually 6-miles square (<100 km2) and regularly shaped. Within each town, individual lots were established and surveyed using witness trees (WT) as markers. The original sources of the WT data typically include proprietors’ records, field books, manuscripts, maps and published records of town land surveys before colonization [21]. Town lotting surveys are the authoritative source, but when unavailable or inadequate other sources containing contemporary tree data were used. The witness trees are thus a relatively objective sample of forest composition prior to colonization. The land surveyors used English colloquial names to describe the trees and while they were skilled naturalists they often did not discern individual species within a genus. To reduce taxonomic uncertainty and ensure consistency across surveys we classified all trees into widely represented genera, following Cogbill et al. [21]. While this introduces some species ambiguity into the groupings, it is unavoidable, since surveyors of pre-colonial witness trees often did not distinguish species within genera. We assembled available town land survey records within the region, totaling 1280 towns and 325,000 trees. WT data are publicly available from individual town halls and archives throughout the region. Approximately 55 percent of the WT towns had been utilized in previous published studies, each of which focused on a smaller region. From these local-scale studies we know that the forests have changed, but we are unable to put these changes in the context of the massive shifts in land-use that reshaped the regional ecosystem during the past 400 years. By assembling a comprehensive geo- database of witness-tree records in the Northeast, including more than 150,000 tree records that have not been published before, we addressed several significant questions: (1) How does the magni- tude of compositional change vary across the region? (2) Do changes in composition reflect the region’s land-use history mosaic and environmental gradients? Pre-colonial data (3) Is the regional forest recovery September 2013 | Volume 8 | Issue 9 | e72540 September 2013 | Volume 8 | Issue 9 | e72540 2 PLOS ONE | www.plosone.org Four Centuries of Change in U.S. Forests Figure 1. The nine-state study region in the northeastern United States. Colors correspond to U.S. Forest Service designated ecoregions. The inner polygons correspond to the 1280 colonial town where pre-colonial forest data were collected. Of these, 701 contained an adequate sample of witness trees and modern forest data to permit comparative analyses. Town with insufficient data are grayed-out in the map. Inset: The location of the study area within the conterminous United States. doi:10.1371/journal.pone.0072540.g001 Figure 1. The nine-state study region in the northeastern United States. Colors correspond to U.S. Forest Service designated ecoregions. The inner polygons correspond to the 1280 colonial town where pre-colonial forest data were collected. Of these, 701 contained an adequate sample of witness trees and modern forest data to permit comparative analyses. Town with insufficient data are grayed-out in the map. Inset: The location of the study area within the conterminous United States. doi:10.1371/journal.pone.0072540.g001 Modern Data change in our findings.) We excluded any towns with ,2 qualifying FIA plots. We then binned the tree species into the same 20 taxa used for the WTs (Table 1) and calculated the relative abundance in each plot. The modern tree data come from the USDA Forest Service Forest Inventory and Analysis (FIA) program. We used the FIA census, spanning 2003–2008. FIA plots were sampled at an intensity of one plot per 2400-ha throughout the study region. Each plot consists of four, 7.3-m fixed-radius subplots (totaling 168-m2), on which all trees .1.3-m in height are identified to species and the dbh recorded. . FIA protocols and data are publically available online (http://apps.fs.fed.us/fiadb- downloads/datamart.html). However, we obtained coordinates for the inventory plots, which allowed us to pair FIA plots with the WT town in which they reside, from the US Forest Service pursuant to a Memorandum of Understanding #09MU11242305123 between the U.S. Forest Service and Harvard University. We excluded FIA plots that were not classed as ‘‘Forest’’ within the FIA Condition table or contained ,10 trees .12.5-cm dbh. In the remaining plots we excluded all trees ,12.5-cm dbh to reduce the potential for bias against smaller trees within the pre-colonial data [29,36]. (Based on the findings of Wang et al [37], we explored a 20-cm dbh threshold for inclusion. This resulted in a large reduction in number of towns meeting the sample intensity criteria outlined below, with little qualitative September 2013 | Volume 8 | Issue 9 | e72540 Assessing the sample intensity The density of WTs and FIA plots within towns varied widely. We used an approach somewhat akin to rarefaction analysis (e.g. [38]) to estimate the minimum density of FIA plots and WTs at which tree compositional diversity had been adequately sampled – i.e. to determine whether or not each town had sufficient tree data to include in our analyses. Our approach relied on the fact that tree diversity increases asymptotically with the addition of each new WT or FIA plot. By using bootstrap sampling and fitting Michalis-Menton (M-M) functions, we estimated the density of WTs and FIA plots at which the full complement of diversity was represented. This density was used as a minimum threshold for determining whether or not to include a town in the analyses. More specifically, our procedure for determining adequate FIA plot density was as follows: (1) We stratified the study region by ecoregions (Figure 1). (2) Within each ecoregion, we selected the most plot-dense towns, taking only those towns with $5 FIA plots 3 September 2013 | Volume 8 | Issue 9 | e72540 PLOS ONE | www.plosone.org September 2013 | Volume 8 | Issue 9 | e72540 3 Four Centuries of Change in U.S. Forests Figure 2. An illustrative example of one bootstrap sample of FIA plots or witness trees used to estimate the minimum sampling intensity necessary to capture the forest composi- tional diversity of a town. Each dot represents the Sørensen similarity between a bootstrap sample of FIA plots or bins of witness trees, and the town’s composition with all plots included, for n in one through the total number of plots/bins in the town. The curve is a Michaelis-Menten function fit to the similarity values. Smax is the curve’s asymptote and Dmin is the plot or tree density at 0.9Smax. Dmin was averaged over 100 sets of bootstrap samples from each of the most plot-dense towns in an ecoregion to determine the ecoregion’s minimum sampling density. doi:10.1371/journal.pone.0072540.g002 Table 1. Taxa groupings with stem counts and occurrences within 701 colonial towns that met the minimum threshold to be included in the analysis. Assessing the sample intensity Taxon Pre-Colonial Modern Towns Stems Towns Stems Ashes 547 3916 500 3996 Basswood 390 1655 180 538 Beech 615 31909 474 7525 Birches 655 9678 657 11467 Blackgum 118 460 94 475 Cedar 132 976 116 2278 Cherries 220 546 462 4565 Chestnut 332 7616 11 11 Cypress 23 82 4 34 Elms 331 1433 152 555 Fir 216 2047 222 6178 Hemlock 605 15817 467 8184 Hickories 309 7493 208 1094 Hornbeam 384 1683 245 1017 Magnolias 63 162 46 108 Maple 692 17017 699 33167 Oak 489 49014 467 11662 Pines 516 12176 442 7398 Poplars 258 1109 345 2401 Spruces 355 9288 296 5076 Sycamore 84 162 4 10 Tamarack 63 181 39 269 Tulip 113 335 73 368 Walnuts 156 421 27 69 TOTAL 701 176715 701 109784 doi:10.1371/journal.pone.0072540.t001 Table 1. Taxa groupings with stem counts and occurrences within 701 colonial towns that met the minimum threshold to be included in the analysis. Figure 2. An illustrative example of one bootstrap sample of FIA plots or witness trees used to estimate the minimum sampling intensity necessary to capture the forest composi- tional diversity of a town. Each dot represents the Sørensen similarity between a bootstrap sample of FIA plots or bins of witness trees, and the town’s composition with all plots included, for n in one through the total number of plots/bins in the town. The curve is a Michaelis-Menten function fit to the similarity values. Smax is the curve’s asymptote and Dmin is the plot or tree density at 0.9Smax. Dmin was averaged over 100 sets of bootstrap samples from each of the most plot-dense towns in an ecoregion to determine the ecoregion’s minimum sampling density. d i /j l doi:10.1371/journal.pone.0072540.g002 Dmin, was taken to be the ecoregion-wide threshold plot density necessary to capture a town’s compositional diversity. Dmin, was taken to be the ecoregion-wide threshold plot density necessary to capture a town’s compositional diversity. We followed a similar procedure for determining the adequate number of WTs necessary to capture the compositional diversity within a town, except that we iteratively sampled bins of 20 trees, as opposed to FIA plots, before fitting the M-M function. We also used only those towns with at least 100 WTs and that were within the upper tercile of tree density (WTs per km2). and that were within the upper tercile of plot density (plots/km2). Assessing the sample intensity (3) For each town within this subset, we iteratively took 100 sets of bootstrapped samples, with each set consisting of a sample of one FIA plot, a sample of two FIA plots (sampled with replacement), and so on up to the total number of FIA plots in that town. (4) For each bootstrap sample within a set we calculated the mean Sørenson distance (see below) between the sample’s relative composition and the relative composition with all plots included. As the number of plots sampled increases, Sørenson similarity tends to initially increase sharply before slowly leveling off as composition stabilizes (Figure 2). (5) Accordingly, for each set of bootstrap samples we fit an M-M curve and recorded the asymptote, Smax Smax is an estimate of the similarity between two random samples of the town’s forest when plot density is impossibly large and is typically slightly less than one, or complete similarity, due to variation introduced by bootstrap sampling. (6) We calculated a threshold plot density, Dmin, as the minimum plot density required to reach a given proportion of Smax. Since reaching Smax would require infinite plot density, we decided that the plot density required to reach 90 percent of Smax would be adequate to approximate a town’s true forest composition. (7) We averaged Dmin over all 100 sample sets from each town, and then over all towns within each ecoregion. This ecoregion grand mean, Results Of 1280 towns in the study area with witness tree data, 904 contained two or more forested FIA plots (Figure 1). Of these, 761 towns contained sufficient WT density to meet the sampling density threshold. Most towns with insufficient WT density were clustered in northern Maine. Within the 904 towns with sufficient WT data, 756 towns had sufficient FIA plot density. In all, 701 towns met the sampling density threshold for both the WT and FIA data. We used only these 701 towns in all subsequent analyses. The average density of WTs in the final sample was 1.77 trees per km2 (s = 2.52) and 252 trees/town (s = 357). The average density of FIA plots in the final sample was 0.27 plots per km2 (s = 0.10) and 4.26 plots per town (s = 2.44). The average density of FIA trees was 0.93 trees per km2 (s = 0.41) or 156 trees per town (s = 86). Historical and modern tree data and town shapefiles are available at the Harvard Forest data archive (http://harvardforest.fas. harvard.edu/data/archive.html) as data set HF-210. We ordinated the pre-colonial and modern towns in forest community space to visually examine changes in community composition. We constructed a non-metric multidimensional scaling (NMDS) ordination (50 random starts; scaling; centering; PC rotation; half-change scaling [41]) using the ‘‘MetaMDS’’ function in the Vegan v2.0-5 library in the R statistical language [42]. Wisconsin double standardization was applied and the Sørensen’s measure was used. The appropriate number of dimensions (axes) was determined by plotting final stress values against the number of dimensions on a scree plot. While most taxa persist, the modern forest is compositionally distinct from the pre-colonial condition (Figure 3). Across the region, beech experienced the largest decline in relative abundance from the pre-colonial to modern era (Figure 4), dropping from an average of 22 percent to seven percent (Table 3). Major changes in beech were clustered in VT, western MA, and northern PA. Beech abundance was relatively stable in the Adirondack Mountains of NY. Oaks also underwent substantial declines in abundance, from 18 percent in the pre-colonial data to 11 percent in the modern data. Oak declines were most pronounced in central MA and southwestern PA. Hemlock declined from 11 to seven at a regional scale. Chestnut trees were extirpated from the modern forests, dropping from three to zero percent region-wide (Figure S1). Results The loss was most pronounced in the Appalachian Forest ecoregion where its pre- colonial abundance was ten percent. Due mostly to changes in the north, the abundance of spruce declined overall from 7.6 to 4.0 percent while fir increased from 2.0 to 4.5 percent. Maples experienced the highest absolute change in relative abundance, but unlike the previously mentioned taxa, average maple abundance increased throughout the region, from an average of 11 to 31 percent. Cherries also increased in relative abundance; climbing from ,0.4 to 4.4 percent (Figure S1). We used several approaches to examine potential evidence of compositional homogenization and changes in the strength of associations between community structure and the environment. First, following Rooney et al. [43], we calculated the average dissimilarity (i.e., Sørensen’s) of each town to all other towns within the study area and within each ecoregion within at each time period. We then compared the mean town-to-town dissimilarity between the pre-colonial and modern data using a permutation test. A reduction in town-to-town dissimilarity over time is evidence that the forests have converged in terms of compositional diversity. To gauge changes in the spatial structure of forest community composition between the time periods, we constructed Mantel correlograms [44] using the ‘‘mantel.correlog’’ function within the Vegan v 2.0-5Library of the R statistical language [42]. In brief, the Mantel’s test of matrix correlation, rM, is calculated at multiple distance classes and plotted across the full range of distances. In those distance classes where rM is positive and significantly different than zero, the multivariate similarity among towns is higher than expected by chance. Conversely, when rM is negative and significantly different than zero, then the towns are more dissimilar than expected by chance. Whether the correlation is significantly different than zero is determine through a permutation test with a Bonferoni corrected a of 0.05. To understand if the strength of associations between climatic and edaphic variables has changed over time, we used Mantel correlation tests between the community distance matrices from both time periods and temperature (GDD), average precipitation, average elevation, and percent sand (as described in Table 2). Note that rM are not equivalent to the more familiar Pearson’s r and should not be directly compared [45]. The degree of overall compositional change from the pre- colonial to modern forests varied widely from town to town (Figure 5). Comparing pre-colonial to modern forest composition We first compared the relative abundance ofeach taxon acrossthe study region and within each ecoregion. We used paired Monte Carlo tests (10,000 randomizations of group membership) to compute p-values describing the probability of encountering differences in relative abundance that were at least as large as those observed from chance alone, given the distribution of data [39]. We calculated the Sørensen’s distance measure for several analyses of compositional change, both in time and in space. Sørensen is defined as: S~ P Dxij{xikD  . P DxijzxikD   Where xij is the abundance of taxon i in town j and xik is the abundance of taxon i in town k. Values of S range from 0, indicating identical composition, to 1 for no overlap in compo- sition. Empirical analyses have shown Sørensen’s distance to be a September 2013 | Volume 8 | Issue 9 | e72540 PLOS ONE | www.plosone.org 4 Four Centuries of Change in U.S. Forests robust measure of compositional dissimilarity in ecological data because it retains sensitivity in more heterogeneous data sets and gives less weight to outliers [40]. subsets that are increasingly homogeneous with regard to the response [46]. We used an implementation of RTA, called conditional inference trees using the ‘‘ctree’’ function in the PARTYv 1.0–7 library [47] within the R statistical language [42]. Conditional inference trees establish partitions based on the lowest statistically significant P-value that is obtainable across all levels of all predictor variables, as determined from a Monte Carlo randomization test. This minimizes bias and prevents over-fitting and the need for pruning [48]. For each town we calculated the Sørensen’s distance from its pre-colonial composition to its modern composition to estimate the degree of change over time. We mapped the change in community composition and compared differences between the ecoregions using a permutation test to compute p-values describing the probability of encountering differences in dissimilarity that were at least as large as those observed from chance alone [39]. To understand whether the degree of community change over time (i.e., Sørensen’s) was spatially structured, we constructed a Moran’s I spatial correlogram. We experimented with several distance classes within the correlogram; based on the distribution of town-to-town distances and the average nearest neighbor distance (<7.4 km based on town centroids), we settled on 15 km uniform distance classes. Comparing pre-colonial to modern forest composition The statistical significance of spatial autocorrelation within each distance class was calculated using a permutation test with a = 0.05. September 2013 | Volume 8 | Issue 9 | e72540 Results Sørenson’s values ranged from 0.13 to 0.94 (x = 0.54, s = 0.131). The Central Appalachian ecoregion had the highest average compositional change (x = 0.558, s = 0.12), followed by the Eastern Broadleaf Forest (x = 0.551, s = 0.15), then the Laurentian Mixed Forest (x = 0.517, s = 0.13) and finally the Adirondack-New England Mixed Forest (x = 0.494, s = 0.12); however, all pair-wise comparisons between ecoregions were statistically insignificant. The Moran’s spatial correlogram showed evidence of clustering in the degree of compositional change at short to moderate geographical distance (,125 km) – i.e., towns that incurred a high degree of compositional change tended to be surrounded by towns that also incurred a high degree of change, Finally, to evaluate the relationships between compositional change and the suite of predictor variables identified in Table 2 further, we used regression tree analysis (RTA) with the Sørenson’s distance between time periods as the response variable. RTA is a non-parametric technique that recursively partitions a dataset into September 2013 | Volume 8 | Issue 9 | e72540 September 2013 | Volume 8 | Issue 9 | e72540 PLOS ONE | www.plosone.org 5 Four Centuries of Change in U.S. Forests Table 2. Land use and bio-physical predictor variables. Variable Explanation/Source Temperature Mean annual growing degree days using a 0uC base [73]. Precipitation Mean annual precipitation in millimeters within each town [73]. Elevation Average elevation above sea level in meters within each town calculated using a 30 meter resolution digital elevation model. Ruggedness Standard deviation of elevation in meters within the town calculated using a 30 meter resolution digital elevation model. Peak agricultural land cover Maximum proportion of land in agriculture between 1850 and 1997 according to county-level census and agricultural survey data [74]. Peak agriculture year Year during which the maximum proportion of a counties land was in agriculture according to county-level census and agricultural survey data from 1850 to 1997 [74]. Rate of agricultural decline Proportion of land taken out of agriculture each year, according to a regression of the proportion of agricultural land (PAL) beginning in the year of peak agriculture and ending when PAL went below 10% or came within 20% of its minimum (i.e., when decline flattens out). Based on county-level census and agricultural survey data [74]. Results Canopy cover Density of tree canopy (as a percentage of the area of the town) based on the Multi-Resolution Land Characteristics consortium’s 2001 National Landcover Database [75]. Soil sand Percentage of sand in the mineral portion of the surface layer of the soil calculated as a weighted area of the town using the U.S. General Soil Map STATSGO2 [76]. Soil clay Percentage of clay in the mineral portion of the surface layer of the soil calculated as a weighted area of the town using the U.S. General Soil Map STATSGO2 [76]. Latitude At centroid of the town. Longitude At centroid of the town. doi:10.1371/journal.pone.0072540.t002 Explanation/Source Mean annual growing degree days using a 0uC base [73]. Percentage of clay in the mineral portion of the surface layer of the soil calculated as a weighted area of the town using the U.S. General Soil Map STATSGO2 [76]. doi:10.1371/journal.pone.0072540.t002 and vice versa (Figure 5). At distances .125 km there was no evidence of spatial patterning (i.e., autocorrelation). have Axis 2 scores .0. The overlay of taxa centroids onto the ordination further demonstrated the differences in composition along Axis 2. For example, cherry had the highest Axis 2 values and chestnut had the lowest (Figure 6a). Axis 1of the ordination captured compositional gradients related to climate Average temperature (i.e., growing degree days) and latitude had the highest correlation with Axis 1 (Figure 6b). Climatic influence on forest composition was In the two-dimensional NDMS ordination (Stress = 9.56) compositional differences between the colonial and modern periods are apparent as directional shifts along Axis 2 (Figure 6). Ninety thee percent (653/701) of the town’s pre-colonial data have Axis 2 scores ,0. Conversely, 86 percent (604/701) of the town’s modern data Figure 3. Relative composition of pre-colonial era Witness Trees and modern inventory trees in 701 colonial townships in the northeastern USA. doi:10.1371/journal.pone.0072540.g003 Figure 3. Relative composition of pre-colonial era Witness Trees and modern inventory trees in 701 colonial townships in the northeastern USA. doi:10.1371/journal.pone.0072540.g003 September 2013 | Volume 8 | Issue 9 | e72540 September 2013 | Volume 8 | Issue 9 | e72540 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 6 Four Centuries of Change in U.S. Forests Four Centuries of Change in U.S. Results There was no significant correlation between composition and annual precipitation in either time period. At the full regional scale, average town-to-town dissimilarity was higher in the pre-colonial forests (x = 0.58, s = 0.24) than in the modern forests (x = 0.55, s = 0.16; Figure 7). In contrast, in each of the ecoregions average town-to-town dissimilarity was higher in the modern forests than in the pre-colonial. Differences were highest in the Laurentian Mixed Forest ecoregion (xWT = 0.51 v. xMOD = 0.56), followed by Adirondack-New England Mixed Forest ecoregion (xWT = 0.43 v. xMOD = 0.48), then the Eastern Broadleaf Forest ecoregion (xWT = 0.50 v. xMOD = 0.52) and finally the Central Appalachian Ecoregion (xWT = 0.46 v. xMOD = 0.49). All comparisons between average pre-colonial and average modern data were significantly different (P,0.001), though, this reflects the high degrees of freedom associated with Table 3. Pre-colonial and modern relative abundance of taxa by ecoregion and throughout the entire study area. Results LAUREN (n = 222) ADIRON (n = 242) BROAD (n = 177) APPAL (n = 60) ALL (n = 701) Pre Mod D Pre Mod D Pre Mod D Pre Mod D Pre Mod D ASHES 2.0% 4.6% 2.7%{{{ 1.9% 3.7% 1.8%{{{ 2.9% 4.8% 1.9%{{{ 1.1% 1.3% 0.2% 2.1% 4.0% 1.9%{{{ BASSWD 1.3% 0.7% 20.6%{{{ 1.0% 0.3% 20.7%{{{ 1.1% 0.5% 20.6%{{{ 0.6% 1.0% 0.4% 1.1% 0.5% 20.5%{{{ BEECH 26.6% 6.6% 220.0%{{{ 30.3% 9.9% 220.4%{{{ 9.7% 2.3% 27.4%{{{ 7.2% 5.0% 22.3%{ 22.0% 6.5% 215.4%{{{ BIRCHES 7.7% 8.3% 0.5% 10.1% 14.2% 4.0%{{{ 3.0% 7.9% 4.9%{{{ 2.5% 7.7% 5.1%{{{ 6.9% 10.2% 3.2%{{{ BLKGUM 0.1% 0.2% 0.1% 0.0% 0.0% 0.0% 0.3% 0.5% 0.2% 1.0% 3.4% 2.4%{{{ 0.2% 0.5% 0.3%{{{ CEDARS 2.3% 3.6% 1.3%{ 1.0% 0.7% 20.3% 0.0% 0.5% 0.5%{{{ 0.0% 0.0% 0.0% 1.1% 1.5% 0.4%{ CHERRIES 0.4% 5.3% 4.9%{{{ 0.2% 2.3% 2.1%{{{ 0.4% 4.6% 4.1%{{{ 0.4% 8.5% 8.1%{{{ 0.4% 4.4% 4.0%{{{ CHESTNUT 2.6% 0.0% 22.6%{{{ 0.8% 0.0% 20.8%{{{ 5.5% 0.0% 25.4%{{{ 9.5% 0.0% 29.5%{{{ 3.3% 0.0% 23.3%{{{ CYPRES 0% 0.0% 0% 0.0% 0.0% 0.0% 0.1% 0.1% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% ELMS 0.8% 0.4% 20.4% 0.8% 0.4% 20.4%{{{ 1.1% 1.4% 0.3% 0.2% 0.2% 0.0% 0.8% 0.6% 20.2% FIRS 2.3% 6.3% 4.0%{{{ 3.7% 6.9% 3.2%{{{ 0.1% 0.4% 0.4%{ 0.0% 0.0% 0.0% 2.0% 4.5% 2.5%{{{ HEMLCK 14.8% 8.0% 26.7%{{{ 12.8% 8.3% 24.5% 5.4% 7.6% 2.2%{ 5.4% 2.3% 23.1%{ 10.9% 7.5% 23.4%{{{ HICKORIES 0.9% 0.8% 20.1% 0.3% 0.3% 0.0% 6.2% 3.1% 23.0%{{{ 5.8% 2.0% 23.7%{{{ 2.4% 1.3% 21.1%{{{ HORNBM 1.3% 1.3% 0.0% 1.0% 1.3% 0.3% 1.3% 0.7% 20.5%{ 0.6% 0.3% 20.3% 1.1% 1.1% 0.0% MAGNOL 0.2% 0.1% 20.1%{ 0.0% 0.0% 0.0% 0.2% 0.1% 0.0% 0.4% 0.8% 0.3% 0.1% 0.1% 0.0% MAPLES 12.6% 30.9% 18.2%{{{ 12.2% 32.4% 20.1%{{{ 9.2% 29.2% 20.0%{{{ 8.6% 28.5% 19.9%{{{ 11.3% 30.8% 19.5%{{{ OAKS 9.0% 8.4% 20.6% 3.8% 4.3% 0.5% 40.5% 18.4% 222.1%{{{ 37.2% 27.6% 29.6%{{ 17.5% 11.1% 26.5% PINES 6.5% 5.3% 21.2% 3.1% 6.3% 3.2%{{{ 9.8% 11.3% 1.5% 14.2% 3.4% 210.8%{{{ 6.8% 7.0% 0.2% POPLARS 0.6% 2.9% 2.3%{{{ 0.5% 1.6% 1.1%{{{ 0.9% 2.4% 1.6%{{{ 0.2% 0.7% 0.5% 0.6% 2.1% 1.5%{{{ SPRUCES 6.5% 4.6% 21.9%{{{ 15.4% 6.6% 28.8%{{{ 0.7% 0.8% 0.2% 1.0% 1.0% 20.1% 7.6% 4.0% 23.6%{{{ SYCMOR 0.2% 0.0% 20.2% 0.0% 0.0% 0.0% 0.1% 0.0% 20.1%{{{ 0.1% 0.0% 20.1% 0.1% 0.0% 20.1%{{{ TAMRAC 0.1% 0.3% 0.2% 0.2% 0.2% 20.1% 0.0% 0.1% 0.1% 0.0% 0.0% 0.0% 0.1% 0.2% 0.0% TULIP 0.1% 0.1% 0.0% 0.0% 0.0% 0.0% 0.4% 1.0% 0.6%{{ 0.0% 0.0% 0.0% 0.2% 0.4% 0.2%{{{ WALNUTS 0.3% 0.0% 20.2%{{{ 0.2% 0.0% 20.2%{{{ 0.2% 0.2% 0.0% 0.4% 0.1% 20.4%{{{ 0.2% 0.1% 20.2%{ P-values were derived from a paired Monte Carlo test that describes the probability of encountering differences in relative abundance that were at least as large as those observed from chance alone, given the distribution of data. Results Fores PLOS ONE | www.plosone.org 7 September 2013 | Volume 8 | Issue 9 | e7254 September 2013 | Volume 8 | Issue 9 | e72540 September 2013 | Volume 8 | Issue 9 | e72540 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org Four Centuries of Change in U.S. Forests Four Centuries of Change in U.S. Forests Figure 4. Town-scale relative abundance of forest taxa within 701 colonial townships. Pre-colonial (left); Modern taxa (center); change in abundance between the eras (right), A. Beech; B. Oak; C. Maple; D. Hemlock. Other taxa can be found in online supplementary materials. doi:10.1371/journal.pone.0072540.g004 Figure 4. Town-scale relative abundance of forest taxa within 701 colonial townships. Pre-colonial (left); Modern taxa (center); change in abundance between the eras (right), A. Beech; B. Oak; C. Maple; D. Hemlock. Other taxa can be found in online supplementary materials. doi:10.1371/journal.pone.0072540.g004 also evident in the placement of taxa centroids along Axis 1. For example, the centroid for oak – a taxa more common in the southern part of the study area – scored quite high on Axis 1, while spruce and fir – both northern taxa – scored quite low. Chestnut was placed high on Axis 1, corresponding to its greater abundance in the southern townships in our study area, while also scoring low Axis 2 due to its absence from modern forests. all pair-wise town-to-town dissimilarities and does not necessarily imply that the differences were biologically significant. The Mantel correlogram showed that pre-colonial forest composition had significant positive spatial correlation from 1 to 400 km and significant negative spatial correlation from 400 to 1500 km (with the exception of sites roughly 700 km apart)–i.e., towns ,400 km apart were compositionally more similar than would be expected by chance and towns that were .400 km apart were more dissimilar than would be expected by chance (Figure 8). The modern forest composition followed a roughly similar pattern, except that the strength of compositional clustering and dispersion were not as strong. Mantel correlations between community composition and environmental data varied widely (Table 4). Annual temperature (i.e., growing degree days) had the highest correlations with forest composition in both time periods, but the correlation with the pre-colonial composition was much higher (0.60) than with the modern composition (0.34). The rM values for elevation and percent sand were much weaker (,0.2) in both eras. Results { p,0.05; {{ p,0.01; {{{p,0.001. doi:10.1371/journal.pone.0072540.t003 al and modern relative abundance of taxa by ecoregion and throughout the entire study area. Table 3. Pre-colonial and modern relative abundance of taxa by ecoregion and throughout the entire September 2013 | Volume 8 | Issue 9 | e72540 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 8 Four Centuries of Change in U.S. Forests Figure 5. Town level compositional change (Sørensen’s distance) between the pre-colonial and the modern forest taxa. Higher values indicate greater compositional change over time. Inset: Spatial autocorrelation in compositional change (Sørensen’s distance) over time as shown in a Moran’s I spatial correlogram with 20 km distance classes. Solid points indicate that spatial autocorrelation in that distance class is significantly different than zero (P,0.01). doi:10.1371/journal.pone.0072540.g005 Four Centuries of Change in U.S. Forests Figure 5. Town level compositional change (Sørensen’s distance) between the pre-colonial and the modern forest taxa. Higher values indicate greater compositional change over time. Inset: Spatial autocorrelation in compositional change (Sørensen’s distance) over time as shown in a Moran’s I spatial correlogram with 20 km distance classes. Solid points indicate that spatial autocorrelation in that distance class is significantly different than zero (P,0.01). doi:10.1371/journal.pone.0072540.g005 The RTA identified five significant partitions using four different predictor variables (Figure 9). The first partition was based on whether the maximum percent of a town historically in agriculture was greater or less than 56 percent. The group with greater extent of agriculture experienced, on average, significantly higher levels of compositional change. The model further partitioned the high-agriculture group based on whether modern forest cover was greater or less than 62 percent; the less forested of these groups had the highest level of compositional change across all terminal nodes in the tree. The towns with lower levels of agriculture was next partitioned based on latitude, with towns north of 44.9u experiencing the lowest level of compositional change across all nodes in the tree. The towns with a lower level of agriculture but south of 44.9u were again partitioned based on whether the town longitude was east or west of 275.3. The more westerly group was split again based on the maximum percent of a town historically in agriculture. Towns where the maximum proportion of land in agriculture was greater than 49 percent had higher compositional change. and completely transformed. It is unchanged insomuch as all the major arboreal taxa remain. Results With few exceptions, the same taxa that made up the forest in the pre-colonial period comprise the forest today, despite ample opportunities for species invasion and loss. In this sense, the regional ecosystem has been quite resilient and the recovery of the eastern forests has been quite real in extent and composition. Yet, at the same time, the forest has been radically transformed. The relative abundance and distribution of most taxa have shifted dramatically; the relationship between forest composition and the environment has been weakened; and variable patterns of land use have imposed a mosaic of impacts whose legacies are evident centuries later. In the discussion that follows we address the nature of these changes in the context of our research questions, starting with the broad-scale compositional changes and working toward specific trends from individual taxa. The scale, pattern, and correlates of compositional change Forest composition changed throughout the region – from Maine to Pennsylvania – and no single ecoregion incurred a significantly higher or lower magnitude of change. The degree and ubiquity of compositional change is clear in the ordination (Figure 6), which showed remarkably little overlap in the distribution of modern and pre-colonial towns. It is important to Four Centuries of Change in U.S. Forests Figure 6. Non-metric multidimensional scaling ordination of pre-colonial and modern forest composition. (A) Points represent each town in each time period. Taxa names are position at the centroid of their distributions within the ordination. (b) Environmental param- eters (Table 3) were overlaid onto the NMDS ordination diagram as fitted vectors. R-Square describe the correlation between ordination axes and environmental vectors only vectors with significant Pearson correlation (P,0.05.) were plotted. doi:10 1371/journal pone 0072540 g006 note, however, that this ‘ubiquitous change’ manifested quite variably at sub-regional scales and that the actual changes – i.e., the rise and fall of individual taxa – occurred non-uniformly. Specific compositional shifts were linked to the specific flora and land-use history of individual towns or small clusters of towns. Indeed, we saw strong local-scale (,125-km) clustering in the magnitude of compositional change but almost no evidence of regional-scale gradients in that change (Figure 2). Why do we see clusters of two to five towns with similar levels of change? The regression tree analysis suggested that local-scale clustering of compositional change may be explained by a hierarchy of land use history and climatic factors (Figure 9). The modern forests arose following land uses of varying types and intensities that had direct impacts on composition–such as forest harvesting , land clearance and agriculture – and indirect impact, through interactions among land use, climate and the biophysical setting. These different effects are apparent in the form of the regression tree. The first partition – and therefore the variable with the greatest explanatory power – is based on the maximum level of agricultural clearing between 1850 and 1997. Towns with less than 56 percent of their area cleared for agriculture experienced significantly less compositional change. The emergence of agricultural land clearing as the most important variable provides strong evidence of the effects of land use on long-term compositional change. The enduring impact of land use on composition has also been documented in some smaller scale witness tree studies [29,37,49] and some detailed field-based studies that document the strong legacy of agriculture versus managed woodlot on modern vegetation [50,51]. Among the towns with higher agricultural clearing, the level of modern forest cover is the next best predictor of change. Towns that have largely reforested (.62%) exhibit less compositional change than those with high levels agriculture of other developed land covers. Discussion Our analyses document a remarkable paradox about the eastern forest after 400 years of land use: it is at once largely unchanged September 2013 | Volume 8 | Issue 9 | e72540 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 9 Four Centuries of Change in U.S. Forests Four Centuries of Change in U.S. Forests Among the towns with lower agriculture cover, the next split in the regression tree is based on latitude with the northerly sites – those in northern Maine – having the lowest level of change across the entire region. Towns with lower historical agriculture but south of Maine were next partitioned based on latitude; towns west of Scranton, PA (75.3u) are less changed, perhaps because these towns were colonized later and, therefore, have a shorter history of land use than towns to the east. Figure 6. Non-metric multidimensional scaling ordination of pre-colonial and modern forest composition. (A) Points represent each town in each time period. Taxa names are position at the centroid of their distributions within the ordination. (b) Environmental param- eters (Table 3) were overlaid onto the NMDS ordination diagram as fitted vectors. R-Square describe the correlation between ordination axes and environmental vectors only vectors with significant Pearson correlation (P,0.05.) were plotted. doi:10.1371/journal.pone.0072540.g006 Figure 7. Changes in landscape b diversity between the pre-colonial and the modern forest taxa as shown though the distribution of town-to-town compositional dissimilarities (Sørensen’s distance). Higher values correspond to higher b diversity. The box represents the inner quartile range of inter-town compositional dissimilarity. The horizontal line indicates the median. The whiskers extend to 1.5 times the inner quartile range. doi:10.1371/journal.pone.0072540.g007 Figure 7. Changes in landscape b diversity between the pre-colonial and the modern forest taxa as shown though the distribution of town-to-town compositional dissimilarities (Sørensen’s distance). Higher values correspond to higher b diversity. The box represents the inner quartile range of inter-town compositional dissimilarity. The horizontal line indicates the median. The whiskers extend to 1.5 times the inner quartile range. doi:10.1371/journal.pone.0072540.g007 September 2013 | Volume 8 | Issue 9 | e72540 PLOS ONE | www.plosone.org 10 Four Centuries of Change in U.S. Forests Figure 8. Mantel correlogram showing the spatial correlation for both the pre-colonial (squares) and modern (triangles) forest composition. Filled symbols indicate that the correlation is significantly different than zero using a Bonferroni adjusted a of 0.05. Significant positive correlations indicate towns that are separated by the geographic distance indicated on the x-axis are compositionally more similar than would be expected by chance, while significant negative correlations indicate that towns are more dissimilar than would be expected by chance. doi:10.1371/journal.pone.0072540.g008 Figure 8. Mantel correlogram showing the spatial correlation for both the pre-colonial (squares) and modern (triangles) forest composition. Four Centuries of Change in U.S. Forests Filled symbols indicate that the correlation is significantly different than zero using a Bonferroni adjusted a of 0.05. Significant positive correlations indicate towns that are separated by the geographic distance indicated on the x-axis are compositionally more similar than would be expected by chance, while significant negative correlations indicate that towns are more dissimilar than would be expected by chance. doi:10.1371/journal.pone.0072540.g008 An overall homogenization and de-coupling of composition and environment We confirmed this empirically and showed that, indeed, pre-colonial forests were compositionally more similar at short distances (i.e., positive spatial autocorrelation in the Mantel correlogram (Figure 5)) and more dissimilar at longer distance (i.e., negative spatial autocorrelation). Smaller scale studies have arrived at opposing conclusions with regard to homogenization of the modern forest. For example, in western NY, the replacement of a late successional species (beech) with several early successional species resulted in increased compositional heterogeneity [27]. In contrast, Foster et al [4] observed a pronounced homogenization and the loss of affinity between taxa abundances and the regional climate gradient in central MA. Such divergent conclusions regarding the impact of historical land use underscore the importance of considering the impacts of a regional-scale disturbance regime at the regional scale. From our analysis, it seems clear that the regional ecosystem has not reestablished the mosaic of forest-types and strong climate- driven compositional gradients. Smaller scale studies have arrived at opposing conclusions with regard to homogenization of the modern forest. For example, in western NY, the replacement of a late successional species (beech) with several early successional species resulted in increased compositional heterogeneity [27]. In contrast, Foster et al [4] observed a pronounced homogenization and the loss of affinity between taxa abundances and the regional climate gradient in central MA. Such divergent conclusions regarding the impact of historical land use underscore the importance of considering the impacts of a regional-scale disturbance regime at the regional scale. From our analysis, it seems clear that the regional ecosystem has not reestablished the mosaic of forest-types and strong climate- driven compositional gradients. Table 4. Mantel rM correlations between climatic, topographic, and edaphic variable (Table 2). Pre-Colonial Modern Temperature GDD 0.60{{{ 0.34{{{ Precipitation NS NS Elevation 0.13{{{ 0.14{{{ Sand 0.07{{{ 0.09{{{ P-values are derived from a Monte Carlo test that describes the empirical probability that the correlations are significantly different than zero. { p,0.05; {{ p,0.01; {{{p,0.001; NS = Not Significant. doi:10.1371/journal.pone.0072540.t004 Table 4. Mantel rM correlations between climatic, topographic, and edaphic variable (Table 2). An overall homogenization and de-coupling of composition and environment regional scale, the variance was more than twice as high in the pre- colonial era, where many town-pairs had Sørensen values close or equal to one (indicating little or no overlap in taxa) and many towns had values close to zero (indicating similar taxa in similar abundances). In contrast, the distribution of modern inter-town dissimilarities was comparatively concentrated around the mean. The modern forest composition is less coupled to its local environment than was the pre-colonial forest, which has coincided with a broad-scale compositional homogenization (sensu [52]). Among the suite of environmental variables we examined, temperature had, by far, the strongest association with forest composition in both time periods; however, the strength of that association was dramatically lower in the modern era (rM = 0.60 versus rM = 0.34; Table 4). This has manifested as a compositional ‘‘smoothing’’ across the region. This also affected the inter-town compositional dissimilarity within each time period, which we used as a straightforward comparison of b-diversity (Figure 7). At the full regional scale, mean dissimilarity was marginally higher in the pre-colonial forests – i.e., the community composition between any two towns, on average, is slightly more similar in the modern era than it was in the colonial period. In contrast, at the ecoregional scale, towns were compositionally more similar to each other in the pre-colonial era than in the modern era. Perhaps more telling than the averages, though, were the differences in variation. At the The regional biotic homogenization is visually apparent in mapped community composition (Figure 3). In the pre-colonial maps, the towns in the south are dominated by oak and hickory and have little in common with the northern towns dominated by spruce and fir. In contrast, most modern towns have a significant component of maple regardless of their location. We confirmed this empirically and showed that, indeed, pre-colonial forests were compositionally more similar at short distances (i.e., positive spatial autocorrelation in the Mantel correlogram (Figure 5)) and more dissimilar at longer distance (i.e., negative spatial autocorrelation). The regional biotic homogenization is visually apparent in mapped community composition (Figure 3). In the pre-colonial maps, the towns in the south are dominated by oak and hickory and have little in common with the northern towns dominated by spruce and fir. In contrast, most modern towns have a significant component of maple regardless of their location. taxa P-values are derived from a Monte Carlo test that describes the empirical probability that the correlations are significantly different than zero. { p,0.05; {{ p,0.01; {{{p,0.001; NS = Not Significant. doi:10.1371/journal.pone.0072540.t004 Our data offer snapshot perspectives on the pre-colonial and modern forest composition. From these we can make inferences about the succession and disturbance processes that shaped the September 2013 | Volume 8 | Issue 9 | e72540 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 11 Four Centuries of Change in U.S. Forests Figure 9. Conditional inference tree showing a hierarchy of relationships between the potential predictor variables (Table 3) and the degree of compositional change (Sørensen’s distance) between the pre-colonial and the modern forest taxa (Figure 4). Partitions in the dendrogram represent the lowest statistically significant P-value that is obtainable across all levels of all predictor variables. doi:10.1371/journal.pone.0072540.g009 Figure 9. Conditional inference tree showing a hierarchy of relationships between the potential predictor variables (Table 3) and the degree of compositional change (Sørensen’s distance) between the pre-colonial and the modern forest taxa (Figure 4). Partitions in the dendrogram represent the lowest statistically significant P-value that is obtainable across all levels of all predictor variables. doi:10.1371/journal.pone.0072540.g009 regionally is the disruption of the forest by deforestation, logging and fire. forests. For example, that late successional species such as beech and hemlock were common in the pre-colonial data is a testament to the relative stability of these forests and their disturbance regimes for the millennia preceding colonization. Beech and hemlock are archetypically late successional species; they are shade tolerant, slow growing, long-lived, and slow to re-colonize a site after disturbance. Based on their pre-colonial abundance, it is clear that the disturbance regime was long dominated by small canopy gaps. forests. For example, that late successional species such as beech and hemlock were common in the pre-colonial data is a testament to the relative stability of these forests and their disturbance regimes for the millennia preceding colonization. Beech and hemlock are archetypically late successional species; they are shade tolerant, slow growing, long-lived, and slow to re-colonize a site after disturbance. Based on their pre-colonial abundance, it is clear that the disturbance regime was long dominated by small canopy gaps. The overall reduction in hemlock is significant but less stark and more multifaceted than that for beech. PLOS ONE | www.plosone.org Four Centuries of Change in U.S. Forests percent. It is worth noting, however, that there were a few small pockets where the relative abundance of oak increased (e.g., in eastern PA) and there was little change along much of the northern extent of it pre-colonial range. McEwan et al. [69] recently dissected the myriad potential causes for the decline in oak; they conclude that a ‘‘multiple interacting ecosystem drivers hypoth- esis’’ is essential to understand long-term oak dynamics. Important factors include a climatic shift toward increased moisture availability, a shift away from Native American burning and a shift first toward low human populations and then to European colonization, plus changes in populations of acorn consuming fauna, including white tailed deer (Odocoileus virginianus). absolute change in relative abundance – a nearly 20 percent rise in average regional abundance. Because the FIA data have species- level resolution, we know that the maple species that dominates the modern landscape is overwhelmingly red maple, a pioneer species with moderate to great shade tolerance and light, wind- dispersed seeds that readily invades open fields after farm abandonment as well as occupy forest gaps following disturbance. It sprouts readily following damage and has great edaphic amplitude ranging from saturated wetland soils to drier uplands. The ecological versatility of red maple allows it to occupy a broad range of edaphic and climatic conditions throughout our study area [59–61]. And, indeed, maple increased in 87 percent of the towns we examined, spanning all the environmental gradients within the region, as demonstrated by its central location within the cloud of modern data in the ordination (Figure 6). percent. It is worth noting, however, that there were a few small pockets where the relative abundance of oak increased (e.g., in eastern PA) and there was little change along much of the northern extent of it pre-colonial range. McEwan et al. [69] recently dissected the myriad potential causes for the decline in oak; they conclude that a ‘‘multiple interacting ecosystem drivers hypoth- esis’’ is essential to understand long-term oak dynamics. Important factors include a climatic shift toward increased moisture availability, a shift away from Native American burning and a shift first toward low human populations and then to European colonization, plus changes in populations of acorn consuming fauna, including white tailed deer (Odocoileus virginianus). The regional stability in average pine abundance belies the fact that pine was among the most dynamic taxa we examined (Figure S1). Four Centuries of Change in U.S. Forests At the regional scale, the relative abundance of pine did not change significantly. However, in the Central Appalachian ecoregion, pines declined by 11 percent, a reduction that was offset by modest increases in pine in the other ecoregions. The relative abundance of pines increased by more than ten percent in 100 towns and decreased by more than ten percent in 99 towns. This high amplitude of change in pine abundance likely speaks to two countervailing characteristics of pine in the region: On one hand, pine is a pioneer taxon that establishes on disturbed sites including post-fire landscapes and abandoned agricultural fields, grows fast, and lives for a long time. On the other hand, pine is vulnerable to natural disturbance (particularly wind) and has always been among the most sought after timber species in the region [6,70]. Similarly, cherry is positioned squarely in the modern data cloud within the ordination; indeed, it has the highest position along axis two of all taxa (Figure 6). Cherry was absent in most of the pre-settlement surveys, but was present at low abundance throughout much of the modern forests. Thanks again to the resolution of the FIA data, we know that references to cherry in the modern data are mostly (.75%) black cherry. The prevalence of this relatively short-lived, pioneer taxon reflects the greater extent and intensity of forest disturbance in the modern era. It shows that disturbance, e.g., from harvesting and meteorological processes, is ongoing throughout the region and the macro shift to reforestation 150 years ago does not imply that succession has progressed uninterrupted. In the absence of anthropogenic influences we would expect succession to increase the similarity to the pre-colonial forests. By examining our two snapshots of forest composition coupled with our understanding of past and ongoing disturbance processes, we can infer that this has not yet occurred because: (1) despite more the 150 years of reforestation, these ecosystems are still in the early stages of recovery, and (2) through that 150-year period there has been ongoing harvests and other land uses as well as exogenous disruptions, including pests (e.g., HWA), pathogens (e.g., chestnut blight), climate change, and atmospheric deposition. Limitations of data sources It is important to acknowledge the limitations of the datasets we employed and discuss how we dealt with them: (1) The WT data were collected over a period of more than 150 years. While the surveys were conducted at a similar timeframe relative to colonization, they still potentially represent different levels of Native American and European influence and different survey methods. We minimized our exposure to this by excluding towns with explicit evidence of colonial influence (e.g., those with apple trees) and by screening out the trees sampled using surveys types known to produce a bias (e.g., road surveys, sensu [71]). (2) The witness trees were not collected as a random sample. Instead we assembled all witness tree records known to exist with the nine- state region. While we know of no particular bias in the towns with existing data, we also cannot say emphatically that our collection of towns is a statistically robust sample. (3) The sample of WT and modern trees per town is sparse, which is why we put the data through an exhaustive analysis whereby we only retained towns where the WT and FIA data adequately captured the composi- tional diversity. (4) The FIA protocol measures in clusters whereas the WTs are dispersed throughout the town, which introduce potential for error and bias. We hedged against this by only including towns with at least two FIA plots and then putting the FIA plots through the rarefaction analysis. Having noted these issues, we still believe that historical survey records offer a singular resource for understanding widespread patterns of forest compo- sition at the time of European colonization. This perspective has been borne out through several studies that have shown that these data provide an accurate account of landscape scale vegetation patterns (e.g. [2,21]). In addition, consistency between WT data and contemporary pollen studies [56,72] further confirm the utility of this resource. taxa In the past 10,000 years, several dramatic declines in hemlock are evident in pollen data , always coinciding with periods of drought and aridity [53–55]. Therefore, hemlock would be expected to be under stress during the greater aridity that coincided with the early colonial period and consequently less able to recover from a regime of intense land use and selective harvesting for its tannins throughout the colonial period [2]. Indeed, an initial decline by hemlock in many pollen diagrams just before European settlement is followed by a more rapid decline with intensive land use [56]. In the modern era, hemlock is being decimated by the invasive hemlock wooly adelgid (HWA; Adelges tsugae). We saw the largest reductions of hemlock in the southern portion of our study area, where it frequently declined to zero, which is consistent with the northward spread of HWA from Richmond, Virginia since 1952 [57]. At present, cold winter temperatures appear to be limiting HWA’s extension into northern New England [58]. The abundance of late successional species is significantly lower on the modern landscape, which offers evidence of the legacy of past land use and to changes in the modern disturbance regime. Of all the taxa we examined, beech experienced the most dramatic reduction in relative abundance, declining in 91 percent of the towns in which its pre-colonial abundance was greater than five percent. We found just one pocket of beech stability (or, at least, low change) located in the Adirondack Mountains of New York, which is the region within our study area that has the most old- growth and primary forest and has had the least exposure to human induced disturbances. This suggests that while climate and disease (particularly beech bark disease) may be contributing factors, the primary cause of beech reduction locally and On the other end of the successional spectrum are species such as red maple (Acer rubrum), black cherry (Prunus serotina), and aspens (Populus spp). Within our analysis maple had, by far, the largest PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org September 2013 | Volume 8 | Issue 9 | e72540 12 September 2013 | Volume 8 | Issue 9 | e72540 Four Centuries of Change in U.S. Forests References Canadian Journal of Forest Research 36: 1266–1275. 9. Boose ER, Chamberlin KE, Foster DR (2001) Landscape and regional impacts of hurricanes in New England. Ecological Monographs 71: 27–48. g g g p 10. Drummond MA, Loveland TR (2010) Land-use pressure and a transition to forest-cover loss in the eastern United States. BioScience 60: 286–298. 27. Wang YC (2007) Spatial patterns and vegetation–site relationships of the pre- settlement forests in western New York, USA. Journal of Biogeography 34: 500– 513. forest-cover loss in the eastern United States. BioScience 60: 2 11. Foster DR, Donahue B, Kittredge DB, Fallon-Lambert K, Hunter M, et al. (2010) Wildland and woodlands: A forest vision for New England. 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Foster DR (1992) Land-use history (1730–1990 ) and vegetation in central New England, USA dynamics. Journal of Ecology 80: 753–771. 30. Schulte LA, Mladenoff DJ, Crow TR, Merrick LC, Cleland DT (2007) Homogenization of northern U.S. Great Lakes forests due to land use. Landscape Ecology 22: 1089–1103. England, USA dynamics. Journal of Ecology 80: 753–771. 14. Turner BL II, Klepeis P, Schneider LC (2002) Three millennia in the southern Yucatan Peninsula: Implications for occupancy, use, and carrying capacity. In: Fedick S, Allen M, Gomez-Pompa A, Jimenez-Osornio J, editors.The lowland Maya area: Three millennia at the human-wildland interface. New York: Haworth Press. 361–387. p gy 31. Wang Y-C, Larsen CPS, Kronenfeld BJ (2009) Effects of clearance and fragmentation on forest compositional change and recovery after 200 years in western New York. Plant Ecology 208: 245–258. 32. References 19. Whitney G, DeCant J (2001) Government land office surveys and other early surveys. The Historical Ecology Handbook. Washington, DC: Island Press. 147– 172. 1. Cronon W (1983) Changes in the land: Indians, colonists and the ecology of New England. New York: Hill and Wang. 1. Cronon W (1983) Changes in the land: Indians, colonists and the ecology of New England. New York: Hill and Wang. 2. Whitney GG (1994) From coastal wilderness to fruited plain: A history of environmental change in temperate North America from 1500 to the Present. New York: Cambridge University Press. 20. Liu F, Mladenoff DJ, Keuler NS, Moore LS (2011) Broadscale variability in tree data of the historical Public Land Survey and its consequences for ecological studies. Ecological Monographs 81: 259–275. 3. Foster DR, Aber JD (2004) Forest in time: Environmental consequences of 1000 years of change in New England. New Haven: Yale University Press. 21. Cogbill C, Burk J, Motzkin G (2002) The forests of presettlement New England, USA: spatial and compositional patterns based on town proprietor surveys. Journal of Biogeography 29: 1279–1304. 4. Foster DR, Motzkin G, Slater B (1998) Land-use history as long-term broad- scale disturbance: regional forest dynamics in central New England. Ecosystems 1: 96–119. g g p y 22. Lutz HJ (1930) Original forest composition in northwestern Pennsylvania as indicated by early land survey notes. Journal of Forestry 28: 1098–1103(6). 5. Chilton ES (2001) The archaeology and ethnohistory of the contact period in the northeastern United States. Reviews in Anthropology 29: 337–360. 23. McIntosh RP (1962) The forest cover of the Catskill Mountain region, New York, as indicated by land survey records. American Midland Naturalist 68: 409–423. 6. Irland LC (1999) The Northeast’s changing forests. Cambridge, MA: Harvard University Press. 7. Donahue B (2007) Another look from Sanderson’s farm: a perspective on New England environmental history and conservation. Environmental History 12: 9– 34. 24. Lorimer CG (1977) The presettlement forest and natural disturbance cycle of northeastern Maine. Ecology 58: 139–148. 25. Seischab F (1990) Presettlement forests of the Phelps and Gorham purchase in western New York. Bulletin of the Torrey Botanical Club 117: 27–38. 8. Foster DR, Aber JD, Melillo JM, Bowden RD, Bazzaz FA (1997) Forest response to disturbance and anthropogenic stress. BioScience 47: 437–445. 26. Black BA, Ruffner CM, Abrams MD (2006) Native American influences on the forest composition of the Allegheny Plateau, northwest Pennsylvania. Acknowledgments We thank D. Orwig for a helpful review of an earlier version of this manuscript and Evelyn Strombom for editing and help with figures. FIA field plot locations were provided by Brett Butler and Elizabeth LaPoint (both with the US Forest Service) pursuant to a Memorandum of Understanding 09MU11242305123between the US Forest Service and Harvard University. Figure S1 Maps of relative abundance and change for all taxa. Figure S1 Maps of relative abundance and change for all taxa. (ZIP) Among individual taxa, we found that late successional trees such as beech, hemlock, and spruce, were once predominant across the region but are now much less so, save for in some small refugia within isolated mountain regions with little history of land use. Oaks also declined throughout most, but not all, of its range. The 400-year history of land use benefited maple the most and it is now a dominant taxon throughout most of the region. Short-lived, early seral species are also much more common in the modern landscape, which indicates that land clearing disturbance is ongoing and widespread and the abandonment of agriculture 150 years ago did not mark a return to the natural disturbance regime of small gap openings. Dynamics of other significant taxa Chestnut suffered a near complete extirpation as a tree-sized individual from the region due to the introduced fugal blight, Endothia parasitica in the early 1900s. Much has been written about the loss of chestnut and its replacement by oak and maple [28,62– 64]. Conventional wisdom holds that chestnut was among the dominant overstory species throughout eastern forests [65–67]. However, the average relative abundance of chestnut in the pre- colonial data was only 3.3-percent. Its highest abundance was in the Appalachian ecoregion, and even there it was less than ten percent and just one-quarter that of oak. Indeed, and in contrast to many other southern and northern taxa, no town in our analysis exceeded 25-percent pre-colonial chestnut abundance. These data suggest that the historical dominance of chestnut is currently often overstated, at least in the Northeast. Nevertheless, even with our comparatively modest estimate of pre-colonial abundance, the loss of chestnut initiated a significant change in forest composition. Like chestnuts, oaks are mid- to late-successional, hard-mast producing canopy tree. There has been growing concern among researchers and managers that oaks are losing their dominance on the landscape [68,69]. And, indeed, oaks declined in 75 percent of the towns in which its abundance was greater than five percent. Across the region, the relative abundance of oaks declined by almost seven percent. Declines were largest in the Eastern Broadleaf ecoregion where relative abundance fell from 40 to 18 September 2013 | Volume 8 | Issue 9 | e72540 13 PLOS ONE | www.plosone.org Four Centuries of Change in U.S. Forests Conclusion one hand, the modern expense of forest is diminished in so many of the components and processes that once characterized the regional ecosystem; on the other, given the extent and magnitude of land use it is remarkable that native species predominate and the forests looks in many ways as it has for millennia. By comparing the modern forest condition to regional database of WT, we have learned much about the changes regional forest composition. While most native taxa remain, the community composition has shifted dramatically. While significant composi- tional changes were ubiquitous throughout the region, the specific attributes of change varied at local scales. One important pattern throughout was the reduction of late successional species in favor of early successional species. Additionally, the modern forest is more homogeneous and less coupled to local climatic controls. Author Contributions Conceived and designed the experiments: JRT DNC CC DRF. Performed the experiments: JRT DNC CC DRF. Analyzed the data: JRT DNC. Contributed reagents/materials/analysis tools: CC. Wrote the paper: JRT DNC CC DRF. Conceived and designed the experiments: JRT DNC CC DRF. Performed the experiments: JRT DNC CC DRF. Analyzed the data: JRT DNC. Contributed reagents/materials/analysis tools: CC. Wrote the paper: JRT DNC CC DRF. 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Faison E, Foster D, Oswald W, Hansen B, Doughty E (2006) Early holocene openlands in southern New England. Ecology 87: 2537–2547. 76. Soil Survey Staff, Natural Resources Conservation Service, United States Department of Agriculture (2013) U.S. General Soil Map (STATSGO2). Available: http://soildatamart.nrcs.usda.gov. Accessed 2013 Aug 13. 56. Fuller JL, Foster DR, McLachlan JS, Drake N (1998) Impact of human activity on regional forest composition and dynamics in central New England. Ecosystems 1: 76–95. 15 September 2013 | Volume 8 | Issue 9 | e72540 PLOS ONE | www.plosone.org 15
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Thyroid Abscess: Challenges in Diagnosis and Management
Journal of investigative medicine high impact case reports
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Case Report Case Report https://doi.org/10.1177/2324709618778709 Journal of Investigative Medicine High Impact Case Reports Volume 6: 1–3 © 2018 American Federation for Medical Research DOI: 10.1177/2324709618778709 journals.sagepub.com/home/hic Keywords y acute suppurative thyroiditis, thyroid abscess, methicillin-resistant Staphylococcus aureus Abstract Thyroid abscess is an uncommon infectious pathology. The thyroid is highly resistant to infection due to high iodine content, capsular encasement, and rich vascularity. Acute suppurative thyroiditis represents <1% of thyroid diseases that could potentially become a life-threatening endocrine emergency. A 48-year-old woman with AIDS presented with 3 days of fever, tender neck swelling, and methicillin-resistant Staphylococcus aureus bacteremia. Apart from leukocytosis, initial laboratory values including thyroid function tests were normal. The initial plain computed tomography scan of the neck and ultrasound scan of the neck were inconclusive as well. By day 4, she worsened, and on repeat computed tomography scan of the neck with contrast, multiloculated abscesses in the thyroid and retro pharynx were seen, which needed emergent drainage. Acute suppurative thyroiditis, a rare disease, occurs in patients with either preexisting disorders of the thyroid or in the immunocompromised. The most common pathogen is Staphylococcus aureus. In our case, we highlight the fact that initial imaging may be negative in the early stages of acute suppurative thyroiditis and lead to an erroneous diagnosis of subacute thyroiditis. There are less than 5 cases of methicillin-resistant Staphylococcus aureus suppurative thyroiditis reported. Thyroid Abscess: Challenges in Diagnosis and Management Niharika Yedla, MD1, Daniela Pirela, MD2, Alex Manzano, MD1, Claudio Tuda, MD1, and Saberio Lo Presti, MD1 Introduction The thyroid gland is essentially protected from being seeded by infections due to its anatomy. It is surrounded by a capsule, and it has excellent blood supply and lymphatic drainage. The iodine content of the gland also prevents infections. Even among those with impaired immune sys- tems, acute suppurative thyroiditis (AST) is a rare disease with incidence being less than 1% of all thyroid diseases.1 It is most commonly caused by gram-positive aerobes, Staphylococcus aureus being the most prevalent, but there are also reports of gram-negative organisms and fungi.1 Extensive PubMed search brought to light less than 5 reported cases of methicillin-resistant Staphylococcus aureus (MRSA) thyroid abscesses.2-5 AST is a life-threatening endocrine emergency. It presents with fever, neck pain, and swelling. Thyroid hormones may be normal or elevated, but they do not aid in diagnosis or management.6 Ultrasound of the thyroid is the suggested imaging modality of choice. Computed tomography (CT) scan and magnetic resonance imaging are generally not needed.7 Due to its rarity, only case reports and case series are available to guide manage- ment. Traditional treatment includes surgical drainage and systemic antibiotics. We present a 48-year-old woman with significant past medi- cal history of uncontrolled diabetes type 2, former intrave- nous drug user, human immunodeficiency virus, noncompliant with antiretroviral therapy, untreated hepatitis C virus, and epilepsy who presented with a severe sore throat. She reported these symptoms for the last 3 days, which were gradual in onset, mild in intensity, accompanied by nausea and difficulty swallowing, and that progressed within hours into difficulty breathing. She denied any fevers or chills, cough, runny nose or earaches, any palpitations, anxiety, heat intolerance, or weight loss. On physical examination, she was noted to be afebrile, tachycardia into 120s, dyspneic, and hypotensive. She had a tender anterior neck swelling without palpable lymph nodes. 1Mount Sinai Medical Center, Miami Beach, FL, USA 2University of Miami, Miami, FL, USA Received March 13, 2018. Revised April 5, 2018. Accepted April 21, 2018. Corresponding Author: Niharika Yedla, Internal Medicine, Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, FL 33140-2849, USA. Email: niharika.yedla@msmc.com Corresponding Author: Niharika Yedla, Internal Medicine, Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, FL 33140-2849, USA. 1Mount Sinai Medical Center, Miami Beach, FL, USA 2University of Miami, Miami, FL, USA Received March 13, 2018. Revised April 5, 2018. Accepted April 21, 2018. 1Mount Sinai Medical Center, Miami Beach, FL, USA 2University of Miami, Miami, FL, USA Received March 13, 2018. Revised April 5, 2018. Accepted April 21, 2018. Corresponding Author: Niharika Yedla, Internal Medicine, Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, FL 33140-2849, USA. Email: niharika.yedla@msmc.com Creative Commons CC BY: This article is distributed under the terms of the Creative Commons Attribution 4.0 License (http://www.creativecommons.org/licenses/by/4.0/) which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). 1Mount Sinai Medical Center, Miami Beach, FL, USA 2University of Miami, Miami, FL, USA Received March 13, 2018. Revised April 5, 2018. Accepted April 21, 2018. Corresponding Author: Niharika Yedla, Internal Medicine, Mount Sinai Medical Center, Introduction Email: niharika.yedla@msmc.com Creative Commons CC BY: This article is distributed under the terms of the Creative Commons Attribution 4.0 License (http://www.creativecommons.org/licenses/by/4.0/) which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). Journal of Investigative Medicine High Impact Case Reports 2 Figure 1 and 2. Computed tomography of the neck with contrast with extensive multilocular ring-enhancing lesions around the thyroid cartilage, extending superiorly into the retropharyngeal and prevertebral space at the C7-T1 level and extending posteriorly to the para-pharyngeal region. Figure 1 and 2. Computed tomography of the neck with contrast with extensive multilocular ring-enhancing lesions around the thyroid cartilage, extending superiorly into the retropharyngeal and prevertebral space at the C7-T1 level and extending posteriorly to the para-pharyngeal region. No exudates were noted in the oropharynx. She had bilateral wheezing without stridor. The rest of the systemic examina- tion was unremarkable. airway edema and observed in the intensive care unit. The cultures from the abscesses also grew MRSA. In the setting of persistent bacteremia and suspicion for endocarditis, a transesophageal echocardiography was done with no evi- dence of vegetations. She was successfully extubated and her repeat thyroid hormone studies postdrainage were in euthy- roid ranges. However, by day 7, she again developed wors- ening neck pain, fevers, and lactic acidosis. Repeat CT scan of the neck showed re-accumulation of abscesses. She was taken to the operating room again for surgical exploration and drainage. She improved postprocedure. She was given a total 14 days intravenous vancomycin. She was successfully discharged from the hospital. Laboratory tests revealed leukocytosis with bands; plate- lets were reduced to 30 000/mL. Chemistry revealed sodium of 135 mmol/L, potassium of 2.9 mmol/L, blood urea nitro- gen of 32 mg/dL, and creatinine of 2.67 mg/dL. The thyroid function test revealed thyroid-stimulating hormone to be within the normal range at 3.740 µIU/mL with elevated free thyroxine (T4) and free tri-iodothyronine (T3) at 2.33 µg/dL and 5.16 ng/dL, respectively. Antithyroperoxidase antibodies and thyroglobulin antibodies were negative. Her CD4 count was 7 cells/mm3, with 873 000 viral copies/mL. Urine analysis revealed significant pyuria and bacteriuria. Due to the acute kidney injury, a noncontrast CT scan of the neck was done, which showed fat stranding in the anterior cervical region and around the thyroid gland with marked narrowing of the subglottic airway. Introduction An ultrasound of the thy- roid gland failed to reveal any obvious fluid collections to suggest an abscess or features of AST-like increased vascu- larity. Based on these findings, we considered subacute thy- roiditis as the probable differential. Conclusion In conclusion, we present a very rare presentation of MRSA suppurative thyroiditis with bacteremia in an immunosup- pressed individual with AIDS. Initial imaging studies may not always be conclusive in the early phase of AST prior to abscess formation. Hence, we emphasize the need to pursue the diagnosis of AST when clinically prompted. Initial imag- ing studies may not always be conclusive. She was admitted to the intensive care unit and placed on broad-spectrum antibiotics, piperacillin-tazobactam with vancomycin. Highly active antiretroviral therapy along with bactrim and azithromycin prophylaxis, acyclovir, and fluco- nazole for opportunistic infections were started. References 1. Paes JE, Burman KD, Cohen J, et al. Acute bacterial suppura- tive thyroiditis: a clinical review and expert opinion. Thyroid. 2010;20:247-255. 2. Lethert K, Bowerman J, Pont A, Earle A, Garcia-Kennedy R. Methicillin-resistant Staphylococcus aureus suppurative thy- roiditis with thyrotoxicosis. Am J Med. 2006;119:e1-e2. 2. Lethert K, Bowerman J, Pont A, Earle A, Garcia-Kennedy R. Methicillin-resistant Staphylococcus aureus suppurative thy- roiditis with thyrotoxicosis. Am J Med. 2006;119:e1-e2. 3. Elorza JL, Echenique-Elizonda M. Acute suppurative thyroid- itis. J Am Coll Surg. 2002;195:729-730. 3. Elorza JL, Echenique-Elizonda M. Acute suppurative thyroid- itis. J Am Coll Surg. 2002;195:729-730. 4. Brook I. Role of methicillin-resistant Staphylococcus aureus in head and neck infections. J Laryngol Otol. 2009;123:1301- 1307. 4. Brook I. Role of methicillin-resistant Staphylococcus aureus in head and neck infections. J Laryngol Otol. 2009;123:1301- 1307. As per our literature search, less than 5 previous cases of MRSA thyroiditis have been reported.2-5 Lethert et  al2 reported a case, with unknown source, diagnosed with ultra- sound and needle aspiration found to have thyrotoxicosis who was successfully treated with vancomycin. Elorza et al3 described a case where a man presented 3 months after total cystectomy for invasive bladder cancer with anterior neck pain. He was diagnosed with a CT scan and treated with sur- gical drainage. Cabizuca et  al5 reported a case of thyroid abscess seeded from infective endocarditis. 5. Cabizuca CA, Bulzico DA, de Almeida MH, Conceição FL, Vaisman M. Acute thyroiditis due to septic emboli derived from infective endocarditis. Postgrad Med J. 2008;84:445-446. 6. Yu EH, Ko WC, Chuang YC, Wu TJ. Suppurative Acinetobacter baumanii thyroiditis with bacteremic pneumonia: case report and review. Clin Infect Dis. 1998;27:1286-1290. 7. Naik KS, Bury RF. Imaging the thyroid. Clin Radiol. 1998;53:630-639. 8. Mohi GK, Datta P, Chander J, Das A. Citrobacter freundii as a cause of acute suppurative thyroiditis in an immunocompetent adult female. Indian J Pathol Microbiol. 2017;60:282-284. In our case, MRSA grew in wound cultures as well as blood and urine cultures. The proposed causal web here is based on immunocompromised state and history of intrave- nous drug use. We suspect skin colonization with MRSA, which entered her blood stream and caused bacteremia. The same bacteria spilled into the urine and seeded the thyroid with the immunosuppressed state favoring the infection. 9. Pearce EN, Farwell AP, Braverman LE. Thyroiditis. N Engl J Med. 2003;348:2646-2655. 10. McAninch EA, Xu C, Lagari VS, Kim BW. Discussion Acute infection of the thyroid is a less seen diagnosis, mostly so as the gland is inherently protected. The encapsu- lation, rich blood and lymphatic supply, and iodine content inside offer the gland protection from being seeded. However, it is a differential to be kept in mind in a patient with a tender neck.8 Her blood cultures came back positive with MRSA; hence, only vancomycin was continued. A transthoracic echocar- diography and carotid Doppler ultrasound were inconclusive. Urine cultures also grew MRSA. By day 4 of hospitalization, the patient had worsening neck pain and stridor. She was taken for a stat contrast CT-neck, which revealed multiple abscesses in the thyroid as well as in the retropharyngeal space (Figures 1 and 2). She was taken to the operating room for transoral and transcervical drainage of the thyroid abscesses. She was intubated postprocedure for possible In children, the presence of a pyriform sinus may predis- pose to this infection, whereas in adults a compromised immune state, especially acquired immunodeficiency syn- drome (AIDS), can do the same.1 History of previous thyroid disease is another risk factor.8,9 Yedla et al 3 Major pathogens are Staphylococcus and Streptococcus species about 35% to 40% of the time. Gram-negative organ- isms cause about 25% cases, whereas anaerobes around 9% to 12%. The rest being fungal etiologies.1,5 Mycobacterial and fungal cases tend to be more common in immunocom- promised patients and are chronic in nature, while bacterial causes are more acute.10,11 Funding The author(s) received no financial support for the research, author- ship, and/or publication of this article. Yu et al6 performed a review of 191 cases from 1980 to April 1997 and compared it with a review of 224 cases (1900-1980). They found that as the numbers of immuno- compromised patients has been increasing, cases of suppurative thyroiditis are increasing. Interestingly, they found bacterial thyroiditis to present in a euthyroid state (83.1%), while mycobacterial were more hyperthyroid (50%) and fungal more hypothyroid (62.5%).6 In our case immunocompro- mised state seems to be the risk factor. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Informed Consent Written informed consent was obtained from the patient for their anonymized information to be published in this article. Early diagnosis is important as the disease can progress rapidly and may prove fatal. Ultrasound is the preferred imaging technique for diagnosis of thyroid diseases.12 It also offers the advantage of needle aspiration. Unless inconclu- sive, one need not obtain a CT scan or magnetic resonance imaging.7 If an iodine scan is done, abscess areas may appear as cold.9 Antimicrobials and surgical drainage of the abscess is the treatment of choice. In cases with pyriform sinus fistu- las, chemocauterization techniques are being utilized, due to being less invasive.13 Ethics Approval Our institution does not require ethical approval for reporting indi- vidual cases or case series. References Coccidiomycosis thyroiditis in an immunocompromised host post-transplant: case report and literature review. J Clin Endocrinol Metab. 2014;99:1537-1542. Due to the initial negative imaging studies and thyroid abscesses being a rare entity, reaching a diagnosis was a challenge. Since this is a life-threatening disease, early rec- ognition and high clinical suspicion is essential. Another rare phenomenon to highlight from our case was the re-accumu- lation of the abscess after initial drainage. We feel that it is important to recognize AST and associated early abscess for- mation in the differential diagnosis of anterior neck pain in immunocompromised patients. 11. Chaudhary V, Bano S. Thyroid ultrasound. Indian J Endocrinol Metab. 2013;17:219-227. doi:10.4103/2230-8210.109667. 12. Das DK, Pant CS, Chachra KL, Gupta AK. Fine needle aspira- tion cytology diagnosis of tuberculous thyroiditis. A report of eight cases. Acta Cytol. 1992;36:517-522. 13. Miyauchi A, Inoue H, Tomoda C, Amino N. Evaluation of chemocauterization treatment for obliteration of pyriform sinus fistula as a route of infection causing acute suppurative thy- roiditis. Thyroid. 2009;19:789-793.
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Habitat and humans predict the distribution of juvenile and adult snapper (Sparidae: Chrysophrys auratus) along Australia's most populated coastline
Estuarine, coastal and shelf science
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Author Author Rees, MJ, Knott, NA, Hing, ML, Hammond, M, Joel Williams, Neilson, J, Swadling, DS, Jordan, A MJ, Knott, NA, Hing, ML, Hammond, M, Joel Williams, Neilson, J, Swadling, DS, Jordan, A Downloaded from University of Tasmania Open Access Repository Please do not remove this coversheet as it contains citation and copyright information. Copyright information This version of work is made accessible in the repository with the permission of the copyright holder/s under the following, Title Title Habitat and humans predict the distribution of juvenile and adult snapper (Sparidae: Chrysophrys auratus) along Australia’s most populated coastline Licence. Rights statement: Crown Copyright © 2021 Published by Elsevier Ltd. This is an open access article under the Creative Commons Attribution 4.0 International (CC BY 4.0) license (http://creativecommons.org/licenses/by/4.0/). If you believe that this work infringes copyright, please email details to: oa.repository@utas.edu.au Bibliographic citation Rees, MJ; Knott, NA; Hing, ML; Hammond, M; Williams, Joel; Neilson, J; et al. (2021). Habitat and humans predict the distribution of juvenile and adult snapper (Sparidae: Chrysophrys auratus) along Australia’s most populated coastline. University Of Tasmania. Journal contribution. https://figshare.utas.edu.au/articles/journal_contribution/Habitat_and_humans_predict_the_ https://figshare.utas.edu.au/articles/journal_contribution/Habitat_and_humans_predict_the_distribution_of_juven Is published in: 10.1016/j.ecss.2021.107397 Habitat and humans predict the distribution of juvenile and adult snapper (Sparidae: Chrysophrys auratus) along Australia’s most populated coastline M.J. Rees a,*, N.A. Knott a, M.L. Hing a, M. Hammond a, J. Williams b, J. Neilson c, D. S. Swadling a, A. Jordan b M.J. Rees a,*, N.A. Knott a, M.L. Hing a, M. Hammond a, J. Williams b, J. Neilson c, S. Swadling a, A. Jordan b a Fisheries Research, NSW Department of Primary Industries, PO Box 89, Huskisson, NSW, 2540, Australia b Fisheries Research, NSW Department of Primary Industries, Locked Bag 800, Nelson Bay, NSW, 2315, Australia c Marine Planning, NSW Department of Primary Industries, 84 Crown St, Wollongong, NSW, 2520, Australia A R T I C L E I N F O Ecosystem-based fisheries managers are increasingly seeking quantitative and spatially-explicit information on species distributions to assist with the management of fisheries and aquatic habitats. In this study, we used boosted regression trees (BRT) to build species distribution models for a highly valued coastal teleost – pink snapper (Sparidae: Chrysophrys auratus) across rocky reefs adjacent to Australia’s most urbanised coastline. BRT models for juvenile (<25 cm total length) and adult (>32 cm total length) snapper were created using a suite of environmental and habitat predictors. A surrogate for multiple anthropogenic stressors, measured as surrounding human population density, was also included in the models. The BRT model for juvenile snapper performed well (cross-validated AUC = 0.78) and identified habitat features as the most important drivers of their distribution across the region. Juvenile snapper were commonly associated with small patch reefs of low relief adjacent to large estuarine water bodies. In contrast, the performance of the BRT model for adult snapper was weak (cross- validated AUC = 0.68) but identified human population density over habitat features as the strongest predictor of adult snapper distributions. Lower occurrences of adult snapper were associated with reef habitats adjacent to large metropolitan centres, suggesting anthropogenic stressors, such as water pollution, noise and fishing may be negatively impacting adult snapper in the region. Our results highlight essential habitats for snapper populations, notably the importance of large estuaries in the coastal seascape, which are nurseries for juvenile snapper. Knowledge of the demographic habitat associations and spatial distribution of snapper across this highly urbanised coastline will support ongoing management and monitoring of snapper populations and their key habitats. Keywords: Baited remote underwater video (BRUV) Ecosystem-based fisheries management Essential fish habitat Fisheries management Species distributions modelling SDMs to produce maps of predicted species distributions also provides a visual means to engage key stakeholders, such as recreational and commercial fishers, in the management of a fishery. Contents lists available at ScienceDirect Contents lists available at ScienceDirect * Corresponding author. E-mail address: matthew.rees@dpi.nsw.gov.au (M.J. Rees). University of Tasmania Open Access Repository Library and Cultural Collections University of Tasmania Private Bag 3 Hobart, TAS 7005 Australia E oa.repository@utas.edu.au CRICOS Provider Code 00586B | ABN 30 764 374 782 utas.edu.au Estuarine, Coastal and Shelf Science 257 (2021) 107397 Available online 11 May 2021 0272-7714/Crown Copyright © 2021 Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.ecss.2021.107397 Received 22 February 2021; Received in revised form 1 April 2021; Accepted 3 May 2021 0272-7714/Crown Copyright © 2021 Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction Detailed information on the spatial distribution of fishes and their essential habitats, such as nursery and spawning grounds is critical for effective ecosystem-based fisheries management (Valavanis et al., 2008). Species distribution models (SDMs) have become a powerful tool to describe species-habitat relationships and identify critical habitats for species’ life-history processes in marine ecosystems (Elith and Leath­ wick, 2009; Guisan and Thuiller, 2005; Leathwick et al., 2006). The ability of SDMs to reliably predict species distributions across unsam­ pled locations may offer an opportunity to cost-effectively inform management over broad spatial scales (1–100’s km) (Pittman et al., 2007). As a result, SDMs are becoming increasingly sought after by ecosystem-based fisheries managers (Moore et al., 2016). The utility of ii Across temperate coastlines many studies have demonstrated strong environmental and habitat associations for demersal fishes. For example, previous research has shown that variation in depth (Bacheler et al., 2019; Malcolm et al., 2011; Williams et al., 2019); benthic sub­ strata (Curley et al., 2002; Fulton et al., 2016; García-Charton and P´erez-Ruzafa, 2001; Guidetti, 2000; Young and Carr, 2015), habitat area (Rees et al., 2014), structural complexity (Pygas et al., 2020; Rees et al., 2018b; Young et al., 2010) and connectivity to estuaries and vegetated nursery habitats (Galaiduk et al., 2017; Rees et al., 2018a; Swadling et al., 2019) can have pronounced effects on the spatial distribution of temperate reef fishes. Despite demonstrating strong spatial patterns, Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) 0272-7714/Crown Copyright © 2021 Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Estuarine, Coastal and Shelf Science 257 (2021) 107397 M.J. Rees et al. much of this previous research has described temperate fish-habitat relationships without predicting species distributions beyond surveyed areas (Chatfield et al., 2010). In part, this is likely due to limited availability of broad-scale habitat data required to extrapolate spatial predictions. However, with greater access to remotely-sensed habitat information and tools to implement appropriate survey designs (Foster et al., 2020; Linklater et al., 2019; Lucieer et al., 2019) the opportunities to build SDMs for demersal temperate fishes are growing (Galaiduk et al., 2017; Monk et al., 2010; Young and Carr, 2015). presence or absence of macroalgae and sponges. 2.1. Study region The study was completed in the Greater Sydney region, which is situated in central NSW, Australia and covers approximately 350 km of coastline and extends offshore to 3 nautical miles from Stockton to Shellharbour (Fig. 1). The region comprises the highest coastal popu­ lation density in Australia, including NSW’s three largest cities - Sydney, Wollongong and Newcastle, which as of 2016, had populations of 4.8, 0.2 and 0.15 million people, respectively (Australian Bureau of Statis­ tics, 2017). Between these three major metropolitan areas are terrestrial National Parks and coastal towns resulting in high variation of human population densities throughout the region. Seabed type mapping has been done for approximately 62% of marine waters between 0 and 60 m in the region using a range of methods including multibeam sonar (https://portal.aodn.org.au), LiDAR (https://elevation.fsdf.org.au), side scan sonar and aerial photography (Gordon and Hoffman, 1989; Jordan et al., 2010; Kinsela et al., 2020; Linklater et al., 2019). Seabed mapping of the region has revealed extensive rocky reef habitat between depths of 20 and 40 m – the target habitat for this study. y g p In this study, we develop SDMs for pink snapper (Sparidae: Chrys­ ophrys auratus; hereafter referred to as snapper) on coastal reefs (15–45 m water depth) in the Greater Sydney region, New South Wales (NSW), Australia. This region extends from Stockton to Shellharbour and en­ compasses the large coastal cities of Sydney, Wollongong and Newcastle. As a coastal species, snapper have a high social value and are extremely important for the recreational and commercial fishing sector (West et al., 2015). Snapper are managed and assessed as a single east-coast biological stock along eastern Australia (Wortmann et al., 2018) and there is mounting evidence suggesting snapper stocks should be assessed and managed at local or regional scales (1–100’s km scale) (Stewart et al., 2020). For example, previous research has indicated that the majority of snapper in eastern Australia are primarily resident, dis­ playing spatially restricted movement patterns for substantial periods of time (Harasti et al., 2015a; Stewart et al., 2020; Sumpton et al., 2003) and are likely to recruit locally based on modelled larval projections (Roughan et al., 2011). Further, juvenile snapper use estuaries and shallow-water habitats as nurseries before migrating to deeper shelf waters (Curley et al., 2013; Parsons et al., 2014). 2.1. Study region Otolith chemistry has demonstrated that most offshore commercial catch of snapper in the Greater Sydney region originates from local estuaries (Gillanders, 2002). Consequently, spatially-explicit information on the distribution of snapper and their essential habitats over regional scales is likely to benefit the contemporary and future management of snapper pop­ ulations. This is particularly pertinent in the Greater Sydney region, which covers Australia’s most urbanised coastline with the greatest range and intensity of anthropogenic stressors of any regional coastline in Australia (Birch, 2000). 2.2. Sampling design To survey snapper populations representatively across the Greater Sydney region, spatially balanced sampling designs were created using the ‘MBHdesign’ package in R (Foster et al., 2020). Survey sites were restricted to rocky reef habitat between depths of 20 and 40 m to align with data from the surrounding NSW coastline (Knott et al., 2021). Given estuaries are important nursery grounds for snapper and often a direct sink for catchment derived contamination and its discharge into open coastal environments, we added unequal inclusion probabilities to the spatially balanced designs to ensure rocky reef sites across a gradient of distances to estuaries were selected. Further sampling design details are in Appendix A. 1. Introduction The effect of multiple anthropogenic stressors on snapper distributions were also examined using surrounding human population density as a surrogate measure (likely correlated to catchment development, water pollution, sedi­ mentation, fishing and maritime activities). Model outputs were extrapolated across mapped rocky reefs in the Greater Sydney region to produce predictive maps of juvenile and adult occurrence. Development of SDMs and predicted distribution maps provides a supplementary tool to support ecosystem-based fisheries management of the snapper fishery and importantly, provides a template for their application to other fisheries. Globally, temperate regions are becoming increasingly affected by urbanisation and to some degree industrialisation (Diaz and Rosenberg, 2008; Lotze et al., 2006). Highly populated coastlines are likely to have a greater range and intensity of anthropogenic stressors (e.g. catchment development, water pollution, sedimentation, fishing and maritime ac­ tivities) that place fish populations and their habitats under increased pressure (Todd et al., 2019). Not surprisingly, previous studies have identified substantial negative impacts of increasing human population density and human accessibility in marine environments on reef fish assemblages (Cinner et al., 2018; MacNeil et al., 2020; Stuart-Smith et al., 2008). Therefore, in highly urbanised and industrialised systems, SDMs for demersal fishes may benefit from including direct or indirect measures of anthropogenic stressors as these factors could be strong drivers of species distributions. Accounting for anthropogenic stressors in SDMs may also provide an opportunity to better understand and quantify potential effects of human disturbances on demersal fishes across relatively large spatial scales. 2.3. Underwater video sampling (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 1 1 Fig. 1. Position of the 308 successful stereo baited remote underwater video station deployments (grey circles) in the Greater Sydney region on rocky reef habitat between depths of 15 and 45 m. Overlaid grid represents population per km2 with darker regions containing a greater population density than lighter regions. Blue areas represent open estuarine waters. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 2.3. Underwater video sampling Juvenile and adult snapper occurrences were surveyed using baited remote underwater stereo-video systems (stereo-BRUVs; Langlois et al., 2020) baited with ~500 g of fresh chopped pilchards (Sardinops sagax). Sampling was done in the Austral winter and spring of 2019 between the months of June and October. At each site, one stereo-BRUVs was deployed on the seafloor for a minimum of 35 min to achieve a 30 min sample. Previous research has shown that a 30 min sample is adequate to representatively survey temperate reef fish assemblages on the east coast of Australia (Harasti et al., 2015b). Each stereo-BRUVs was con­ structed as per Malcolm et al. (2007), which included a galvanized metal frame containing a video camera (SONY ×3000 or digital Canon HG21) pointed at a bait bag mounted horizontally at the end of a 1.5 m long bait arm. Cameras were housed within high-pressure polyvinyl chloride pipe with flat acrylic endports yielding a field of view of 110◦. Prior to, and during the sampling campaign, stereo-BRUVs were calibrated using the software CAL (SeaGIS, seagis.com.au) following the 3D calibration The aim of this study was to build species distribution models for juvenile and adult snapper on open coast rocky reefs within the Greater Sydney region. Two demographically-explicit models were developed for juveniles and adults as habitat associations are likely to differ be­ tween the life stages (Compton et al., 2012; Parsons et al., 2014). Snapper <25 cm and >32 cm total length were defined as juveniles and adults, respectively. These size classes were based on previous research finding ~60% of individuals at 32 cm and ~15% of individuals at 25 cm were mature in the central and southern coastal regions of NSW (Stewart et al., 2010). The probability of occurrence for the two life stages was modelled with various environmental and habitat predictors including neighbouring estuary area, reef area, depth, seafloor relief and the 2 M.J. Rees et al. Estuarine, Coastal and Shelf Science 257 (2021) 107397 1 procedure outlined by Boutros et al. (2015) to accurately measure 2.4. Video analysis 1 Fig. 1. Position of the 308 successful stereo baited remote underwater video station deployments (grey circles) in the Greater Sydney region on rocky reef habitat between depths of 15 and 45 m. Overlaid grid represents population per km2 with darker regions containing a greater population density than lighter regions. Blue areas represent open estuarine waters. 2.7. Model formulation and evaluation To assess the relative importance of habitat, environmental and anthropogenic variables on the spatial distribution of juvenile and adult snapper we used boosted regression trees (BRT). BRTs were chosen over other approaches as they are a flexible modelling approach that can model non-linear relationships, incorporate numerous data types and provide robust spatial predictions (Elith et al., 2008). BRT models were performed on presence/absence data for juvenile and adult snapper with a Bernoulli error distribution and a logit link function using the “gbm” package version 2.1.5 (Greenwell et al., 2018) in R using code adapted from Elith et al. (2008). We modelled the presence/absence of snapper rather than the MaxN or relative biomass due to the high proportion of zeros in the juvenile and adult response measures. Due to our sample size, models were developed using 5-fold cross-validation where the learning-rate, tree complexity and bag-fraction settings were optimised to build models containing between 1000 and 2500 trees and best all-round performance. The “gbm.simplify” function was used to reduce the number of variables by an iterative backwards stepwise removal of the least influential variables using 5-fold cross-validation until the change in predictive deviance was minimised. Partial plots of predictors in each of the final models were plotted using the “ggplot2” package (Wickham, 2016) and “ggBRT” package (Jouffray et al., 2019), where bootstrapping was used to calculate 95% confidence intervals. Model performance for each juvenile and adult snapper was evaluated on the portion of the training data withheld for cross-validation during model building. The cross-validated Area Under the receiver operating Curve (AUC) was used to assess model performance, which quantifies the ability of the model to discriminate between presences and absences in the dataset (Franklin, 2010). AUC values range from 0 to 1, where >0.9 indicate excellent model performance, 0.7–0.9 moderate to high per­ formance, 0.5–0.7 low performance and <0.5 indicates performance was no better than random. Percent deviance explained by each model was also used as a measure of overall model goodness-of-fit, which was calculated as (1 – (cross-validated deviance/mean total deviance)). Spatial autocorrelation in model residuals was assessed using spline correlograms (BjØrnstad and Falck, 2001), which indicated that there was limited evidence of spatial autocorrelation for both models To quantify the surrounding seascape characteristics of each survey site, a range of broad-scale environmental and anthropogenic predictors were calculated. 2.4. Video analysis procedure outlined by Boutros et al. (2015) to accurately measure snapper lengths, estimate water clarity and standardise the area sur­ veyed (Langlois et al., 2020). As stereo-BRUVs had a camera separation of 800 mm and a convergence angle of 8◦, length measurements of ju­ venile and adult snapper observed during the study were likely to have a high accuracy (±5% error) (Gardner et al., 2021). Of the 350 stereo-BRUVs deployed, 308 had unobstructed field of views in both cameras enabling length measurements. Videos of 30 min duration were annotated using EventMeasure software (SeaGIS, seagis.com.au) and a standardised field of view of 5 m distance from the centre of the cameras. For each replicate stereo-BRUVs deployment, we recorded snapper MaxN, which was the maximum number of snapper individuals in any frame at any point in time (Parker and DeMartini, 1995). At time of MaxN the individual fork lengths of each snapper were measured. Data were extracted from EventMeasure software and checked following Langlois (2017). Fork length 3 Estuarine, Coastal and Shelf Science 257 (2021) 107397 M.J. Rees et al. measurements were converted to total lengths following formula out­ lined in Ferrell and Sumpton (1997) and recoded to presences or ab­ sences of juvenile (<25 cm total length) and adult snapper (>32 cm total length) for each deployment based on Stewart et al. (2010). such as sea surface temperature, were included as model predictors as the study was completed within a bioregion where there is relatively limited broad-scale oceanographic variability. For example, during the study the mean difference in sea surface temperature between the northern and southern survey sites was 0.44 ◦C calculated from the satellite derived IMOS - SRS - SST - L3S – Single Sensor - 6 day - day and night time measurements (www.aodn.org.au). 2.6. Preliminary data exploration Prior to modelling, data exploration was performed to examine po­ tential outliers, homogeneity and collinearity of explanatory variables. The habitat types; macroalgae and sponges were converted to presence or absence due to their highly skewed distribution in percentage cover. Further, the consolidated and unconsolidated habitat types were removed due to their strong collinearity and skewed distributions. Reef area calculated at 50 m, 100 m, and 200 m radii scales were heavily right skewed with a large proportion of sites having a 100% reef cover in the surrounding seascape. As a result, only the 500 m radius scale of reef area was included for further analysis. Square root transformations of the human population density predictors, estuary area predictors and reef area (500 m) were performed to improve homogeneity. For each of the explanatory variables calculated at multiple spatial scales (human population density and estuary area), only one was included for further analysis, which was determined by visual inspection of plotted re­ lationships. The final subset of continuous explanatory variables had Pearson’s correlation coefficients of less than 0.2. 2.5. Explanatory variables A number of habitat and environmental variables were included in the analysis which were either recorded in situ, from stereo-BRUVs footage or in a Geographic Information System using remotely-sensed environmental data (Table A.1). Fine-scale habitat features and verti­ cal relief of each site were analysed in the program TransectMeasure (SeaGIS, seagis.com.au) following the method described in McLean et al. (2016). A 5 × 4 grid was overlaid on a high definition still frame for each stereo-BRUVs deployment splitting the image into 20 cells. The domi­ nant habitat type and vertical relief was then scored for each cell following the CATAMI classification scheme (Althaus et al., 2015). Habitat types included consolidated (rocky reef), unconsolidated (san­ d/rubble), macroalgae, sponges and ascidians. For each cell over benthos an estimate of vertical relief was scored between 0 and 5 following Wilson et al. (2007). Data were exported from Trans­ ectMeasure software and checked and manipulated using R scripts available from Langlois (2017). Cells of open water were removed before calculating a mean estimate for habitat type percentage cover and vertical relief for each deployment. Depth was recorded in situ using the research vessel sounder at each survey site. Maximum visibility was also quantified for each deployment by making a 3D point measurement of the furthest identifiable object (for example, substrate, alga and fish) in EventMeasure at the maximum range of visibility (Goetze et al., 2019). Deployments in areas of greater water clarity and with unobstructed field of views resulted in larger maximum visibility measurements. Maximum visibility was included as a potential model covariate as variation in visibility among deployments could possibly influence the detectability of snapper. 2.7. Model formulation and evaluation Using a spatial layer of rocky reef habitat throughout the Greater Sydney region derived from previously collected singlebeam and multibeam survey data (Gordon and Hoffman, 1989; Jordan et al., 2010), surrounding reef area was calculated for each deployment using 50 m, 100 m, 200 m, and 500 m radii buffers in ArcGIS (ESRI, 2011). Following a similar procedure, surrounding area of open estuaries in the Greater Sydney region was calculated for each deployment using buffers with a radius of 10 km and 20 km. Surrounding human population density for each deployment was calculated from Australian Bureau of Statistics Australian Population Grid 2016 (Australian Bureau of Sta­ tistics, 2017) within circular buffers of 10 km and 20 km radius using zonal statistics function in R. Human population density was included in the species distribution models as a surrogate for multiple anthropo­ genic stressors, where greater population density is likely correlated to catchment development, water pollution, sedimentation and maritime activities. Human population density is also an appropriate surrogate for fishing effort and accessibility as distance to nearest boat ramp was correlated with human population density. No oceanographic variables, Table 1 Table 1 Summary of the optimal parameters and predictive performance measures for the juvenile and adult snapper BRT models, tc = tree complexity, nt = number of trees, lr = learning rate, AUC = Area Under the receiver operating Curve and TSS = True Skill Statistic. Model tc nt lr Cross- validation Test with independent data AUC R2 AUC TSS Juvenile 2 1700 0.003 0.78 15.3 0.81 0.39 Adult 2 1200 0.001 0.67 5.69 0.50 – Summary of the optimal parameters and predictive performance measures for the juvenile and adult snapper BRT models, tc = tree complexity, nt = number of trees, lr = learning rate, AUC = Area Under the receiver operating Curve and TSS = True Skill Statistic. 4 Estuarine, Coastal and Shelf Science 257 (2021) 107397 M.J. Rees et al. M.J. Rees et al. above. Estimates of reef area was derived from a rocky reef shapefile digitised from NSW Multibeam Surveys (AODN https://portal.aodn.org. au) and NSW marine LiDAR (Geosciences Australia https://elevation.fsd f.org.au/) data (Jordan et al., 2010). Deployments in the independent data set ranged from 14 km to 100 km south of the most southern survey site in the Greater Sydney region. To evaluate the Greater Sydney region models with independent data, confusion matrices were run to calculate two model accuracy indices, AUC and true skill statistic (TSS). To calculate the TSS, the predicted probability of occurrence values (0–1) were converted into categories of suitable and unsuitable localities using M.J. Rees et al. 3. Results Juvenile snapper (<25 cm total length) and adult snapper (>32 cm total length) were recorded on 72 and 71 of the 308 stereo-BRUVS de­ ployments across the Greater Sydney region, respectively. The BRT model for juvenile snapper performed moderately well (cross-validated AUC 0.78) explaining 15.3% of the variation in their occurrence in the region (Table 1). Habitat and seascape variables were the most impor­ tant predictors of juvenile snapper occurrence with a combined relative influence of 79% within the BRT model (Fig. 2). Juvenile snapper preferred small patch reefs with low relief adjacent to large estuarine water bodies (Fig. 2). A higher probability of juvenile occurrence was also observed with increasing population density within 20 km until approximately 1,000,000 people where occurrence sharply declined. Model validation against an independent data set revealed that the optimal juvenile snapper model performed well (AUC = 0.80, TSS = 0.31), indicating that habitat associations and spatial predictions are reliable for management purposes. Furthermore, a high count of oc­ currences in the independent data set were observed at sites of greater predicted suitability (Fig. A.4). , ; , ; g , ) The overwhelming influence of habitat and seascape variables in explaining the occurrence of juvenile snapper is not surprising consid­ ering the species’ life history. Snapper often recruit into estuaries, bays and shallow-water inlets where they use these systems as nursery hab­ itats before migrating to the open coast (Curley et al., 2013; Gillanders, 2002; Parsons et al., 2014). Expectedly, the area of surrounding estuary within a 20-km seascape was one of the most important drivers of ju­ venile snapper distributions, with higher occurrences observed on open coast reefs adjacent to large open estuaries. This finding supports those by Swadling et al., (in review) who recorded a higher abundance and smaller size class of snapper on coastal reefs within 10 km of estuary entrances in the neighbouring Batemans Marine Bioregion. Juvenile snapper were also strongly associated with seascapes dominated by small patch reefs of relatively low structural complexity. Laboratory experiments have demonstrated that juvenile snapper choose sheltered habitat when exposed to a predatory threat, yet field research indicates they are most abundant over soft sediments adjacent to rocky reef (Ross et al., 2007). (Fig. A.2). Model performance was also evaluated using an independent stereo- BRUVs dataset collected in the adjoining Batemans Marine Bioregion to the south of the Greater Sydney region between Kiama (34.6738◦S, 150.8444◦E) and Ulladulla (35.3572◦S, 150.4613◦E) in 2015 and 2016 (Fig. A.5). These data were collected by NSW Department of Primary Industries Fisheries Research as part of the state-wide monitoring of rocky reef fishes (Knott et al., 2021) and were accessed from the Global Archive repository (https://globalarchive.org/). Fine-scale habitat and seascape variables were calculated for each deployment as described 5 1 endency plots with 95% confidence intervals for the most influential variables in the juvenile and adult snapper mod r on the probability of occurrence of juvenile and adult snapper while keeping all other variables at their mean. Th within the BRT model is shown in brackets next to the variable name. Grey rugs along the top of each plot indica 1 1 Fig. 2. Partial dependency plots with 95% confidence intervals for the most influential variables in the juvenile and adult snapper models. The plots show the effect of a given predictor on the probability of occurrence of juvenile and adult snapper while keeping all other variables at their mean. The relative importance of each predictor variable within the BRT model is shown in brackets next to the variable name. Grey rugs along the top of each plot indicate observed data points. Estuarine, Coastal and Shelf Science 257 (2021) 107397 M.J. Rees et al. a threshold of 0.5. All predicted probabilities of occurrence for adult snapper were <0.5 and therefore, TSS was only calculated for juvenile snapper. The TSS ranges from −1 to 1, where 0 denotes the threshold between models with some predictive skill (model skill increases to­ wards 1) and models that are no better than random (model skill de­ clines towards −1) (Allouche et al., 2006). To further validate the juvenile and adult models, we plotted the distribution of observed oc­ currences in the independent data set across the range of predicted probabilities of occurrence derived from the Greater Sydney region models. A high count of observed occurrences at sites of greater suit­ ability would indicate that the models are a valuable decision-support tool. it continued to remain low with increasing population density (Fig. 2). (Fig. A.2). Validation of the optimal adult snapper model against an independent data set was poor (AUC = 0.51) although a positive trend between predicted suitability and adult snapper occurrences across the inde­ pendent data set was observed (Fig. A.4). Predicted species distributions of juvenile and adult snapper from the optimal BRT models showed a high degree of spatial variation across the Greater Sydney region (Fig. 3). Hotspot locations for juvenile snapper were strongly associated with seascapes characterised by patchy reef systems in close proximity to large estuarine water bodies. Important locations included reefs in the northern section of the Greater Sydney region and adjacent to the Hawkesbury River and Port Hacking. Higher occurrences of adult snapper were predicted along remoter coastlines of the region with low population densities, which were generally adjacent to terrestrial National Parks. Spatial variation in the error surrounding predictions were generally positively related to predicted probability of occurrences (Fig. A.3). 4. Discussion Identifying important habitats and understanding how they shape the spatial distribution of targeted fishes is valuable for effective ecosystem-based fisheries management (Moore et al., 2016; Valavanis et al., 2008). In this study, a BRT modelling approach was used to assess habitat preferences as well as the influence of anthropogenic pressures on the distribution of snapper on open coast rocky reefs across Aus­ tralia’s most densely populated coastline. The occurrence of juvenile snapper (<25 cm total length) in the region was driven largely by habitat and seascape features, with juveniles preferring small patch reefs of low structural complexity in close proximity to large estuarine water bodies. In contrast, habitat and seascape variables were poor predictors of adult snapper occurrence (>32 cm total length). Rather, adult snap­ per were observed more frequently in the remoter areas of the region where surrounding human population density was relatively low. These findings confirm other research highlighting the importance of model­ ling habitat associations for snapper at different life stages (Compton et al., 2012; Galaiduk et al., 2017; Swadling et al., in review). l 2.8. Spatial predictions Maps of the predicted probability of occurrence of juvenile and adult snapper throughout the Greater Sydney region were generated following the general procedures of Leathwick et al. (2006) and Hill et al. (2014). Firstly, a fish net layer was created in ArcGIS (ESRI, 2011), where points were separated by 500 m and clipped to the rocky reef shapefile used for creating the spatially balanced design. For each point, explanatory variables included in the optimal BRT models were calculated following procedures outlined above. Fine-scale variables from stereo-BRUVs im­ agery that could not be estimated for each fish net point (for example, mean relief) were kept at their mean value. The optimal BRT models were fitted to a bootstrap sample of the fish net points, which was then used to make predictions across the spatial extent of rocky reef in the region. This procedure was repeated 1000 times to generate 1000 spatial layers of predicted probabilities of juvenile and adult snapper occur­ rences. The mean probability of occurrence and its standard deviation was generated for each point and converted to a raster layer. Maps of the mean predicted probabilities of occurrence and associated error were generated for juvenile and adult snapper. 3. Results Therefore, a likely explanation for our results is that ju­ venile snapper prefer reef-sand boundaries as these edges provide food resources (Langlois et al., 2005) and the presence of reef may act as shelter to assist predator avoidance. In contrast to juveniles, only weak habitat and seascape associations were observed for adult snapper across the region. This finding aligns with that of Swadling et al., (in review), who found habitat variables, such as distance to estuary, es­ tuary area and depth to be poor predictors of adult snapper with their distribution being relatively even on open coast reefs between depths of 20–40 m in southern NSW. The BRT model for adult snapper had relatively poor performance (cross-validated AUC 0.68) explaining only 5.7% of the variation in their occurrence (Table 1). In contrast to juvenile snapper, population density was a much more important predictor of adult snapper than habitat and seascape variables, accounting for over half (52.9%) of the total varia­ tion explained by the BRT model (Fig. 2). The seascape and habitat variables, surrounding estuary area and mean relief had a relative in­ fluence of 26.8% and 20.3% respectively, however, these relationships were relatively weak. Higher probabilities of adult snapper occurrence were associated with population densities less than 150,000 people within 20 km. Probability of occurrence dropped substantially between population densities of 150,000 to 300,000 people within 20 km where Human population density was the strongest predictor of adult snapper in the region, where substantially lower occurrences were 6 Estuarine, Coastal and Shelf Science 257 (2021) 107397 M.J. Rees et al. Fig. 3. Maps of the predicted probability of occurrence of A) juvenile and B) adult snapper across rocky reefs between depths of 20 and 40 m in the Greater Sydney region, NSW, Australia. 3. Maps of the predicted probability of occurrence of A) juvenile and B) adu NSW A t li lity of occurrence of A) juvenile and B) adult snapper across rocky reefs between depths of 20 and 40 m in the Greater Sydney Fig. 3. Maps of the predicted probability of occurrence of A) juvenile and B) adult snapper across rocky reefs between depths of 20 and 40 m in the Greater Sydney region, NSW, Australia. 3. Results A number of previous studies have also demonstrated negative impacts of greater surrounding human popula­ tion density and human accessibility to reef fish assemblages (Cinner et al., 2018; MacNeil et al., 2020; Mora et al., 2011; Stuart-Smith et al., 2008). Similarly, in Western Australia, Navarro et al. (2020) observed lower recreational catches of dhufish, a targeted demersal fish in waters surrounding the Perth metropolitan area relative to regional coastal areas in the State. Many anthropogenic stressors associated with high population densities in the Greater Sydney region could be driving the negative association with adult snapper. For example, the highly urbanised and industrialised coastlines of the region’s three major cities experience elevated levels of pollution, maritime industries and catch­ ment development (Birch, 2000) possibly leading to reductions in adult snapper occurrence. However, as juvenile snapper did not display a clear negative relationship with increasing human population density and are protected from harvesting by a minimum legal size limit (NSW Fisheries Management Act, 1994), our results suggest fishing may be the responsible driver of adult snapper distributions in the region. However, given the relatively poor performance of the adult snapper model, further research is required to better assess whether fishing is the pre­ dominant anthropogenic stressor influencing adult snapper distributions in the region. Future studies could test fishing related hypotheses by including spatially-explicit measures of recreational and commercial fishing effort in species distribution models (Griffin et al., 2020; Navarro et al., 2020) or by using manipulative experimental approaches. Identifying habitat associations and important locations for juvenile and adult snapper has a number of implications for their management in the region and temperate Australian coastlines. Firstly, our results indicate that large open estuaries are important nursery habitats for snapper in the Greater Sydney region, as higher occurrences of juvenile snapper were recorded on reefs adjacent to these systems. Similarly, using otoliths Gillanders (2002) found that most snapper (89%) caught in the region’s commercial fishery originated from local estuaries. Together, these findings suggest that local estuaries play a major role in supplying individuals to the region’s open coast environment. 3. Results juvenile snapper included reefs between Newcastle (32.93◦S, 151.78◦ E) and Lake Macquarie (33.03◦S, 151.56◦E), Terrigal (33.45◦S, 151.44◦E) and the Hawkesbury River (33.53◦S, 151.24◦E) and reefs adjacent to Port Hacking (34.07◦S, 151.12◦E) in southern Sydney. As human population density was the primary driver of the spatial distri­ bution of adult snapper, higher frequencies of occurrence were pre­ dicted along the region’s more remote coastlines. These locations were generally adjacent to terrestrial National Parks, including the northern Illawarra and southern Sydney coastlines surrounding the Royal Na­ tional Park (34.14◦S, 151.12◦E), the Bouddi National Park (33.52◦S, 151.39◦E) and the National Parks and conservation areas surrounding Norah Head (33.28◦S, 151.57◦E) in the north of the region. observed on reefs adjacent to population centres with more than 150,000 people within 20 km. A number of previous studies have also demonstrated negative impacts of greater surrounding human popula­ tion density and human accessibility to reef fish assemblages (Cinner et al., 2018; MacNeil et al., 2020; Mora et al., 2011; Stuart-Smith et al., 2008). Similarly, in Western Australia, Navarro et al. (2020) observed lower recreational catches of dhufish, a targeted demersal fish in waters surrounding the Perth metropolitan area relative to regional coastal areas in the State. Many anthropogenic stressors associated with high population densities in the Greater Sydney region could be driving the negative association with adult snapper. For example, the highly urbanised and industrialised coastlines of the region’s three major cities experience elevated levels of pollution, maritime industries and catch­ ment development (Birch, 2000) possibly leading to reductions in adult snapper occurrence. However, as juvenile snapper did not display a clear negative relationship with increasing human population density and are protected from harvesting by a minimum legal size limit (NSW Fisheries Management Act, 1994), our results suggest fishing may be the responsible driver of adult snapper distributions in the region. However, given the relatively poor performance of the adult snapper model, further research is required to better assess whether fishing is the pre­ dominant anthropogenic stressor influencing adult snapper distributions in the region. Future studies could test fishing related hypotheses by including spatially-explicit measures of recreational and commercial fishing effort in species distribution models (Griffin et al., 2020; Navarro et al., 2020) or by using manipulative experimental approaches. observed on reefs adjacent to population centres with more than 150,000 people within 20 km. Author statement M.J. Rees: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Project administration; Roles/Writing - original draft; Writing - review & editing. N.A. Knott: Conceptualization; Data curation; Investigation; Methodology; Project administration; Re­ sources; Supervision; Roles/Writing - original draft; Writing - review & editing. M.L. Hing: Data Curation; Investigation; Methodology; Re­ sources; Writing - review & editing. M. Hammond: Data Curation; Investigation; Methodology; Resources; Writing - review & editing. J. Williams: Data Curation; Investigation; Resources; Writing - review & editing. J. Neilson: Data Curation; Investigation; Resources; Writing - review & editing. D.S. Swadling: Data Curation; Investigation; Writing - review & editing. A. Jordan: Conceptualization; Funding acquisition; Project administration; Resources; Supervision; Writing - review & editing. Declaration of competing interest Globally, coastal ecosystems are becoming increasingly urbanised, leading to a greater range and intensity of human pressures on important fisheries species and nearshore habitats (Todd et al., 2019). Conse­ quently, information on the spatial distributions, critical habitats, and impacts of multiple human stressors on target species is valuable to assist ecosystem-based fisheries management in urbanised environ­ ments. In this study we demonstrated the use of BRTs in assessing the habitat associations and effect of multiple human stressors on the dis­ tribution of a species of significant socio-economic value along Aus­ tralia’s most densely populated coastline. We showed a strong relationship between juvenile snapper, estuaries, and adjacent low relief nearshore habitat, emphasising the importance of preserving these habitats for the ongoing recruitment of this species. In contrast, adult snapper did not share strong habitat associations. Rather their distri­ bution was negatively related to surrounding human population density, suggesting impacts in their occurrence due to multiple human threats. Our study provides a clear example on the application of demographically-explicit species distribution models to assess human pressures and habitat associations for an important fisheries species. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 3. Results Further­ more, as snapper can display restricted movement patterns in open coast environments (Harasti et al., 2015a; Stewart et al., 2020; Sumpton et al., 2003) with modelling suggesting limited larval dispersal capabilities (Roughan et al., 2011), there is a growing consensus that management across regional scales may be required to sustain local fisheries (Stewart et al., 2020). Consequently, it is important that the condition of estu­ aries, including their vegetated habitats (seagrass and saltmarsh) and water quality are maintained or enhanced to ensure recruitment of ju­ veniles to the open coast snapper population. Management initiatives Spatial predictions of the distribution of juvenile and adult snapper showed a number of hotspots in their occurrence throughout the Greater Sydney region. Generally, three important locations were identified for juvenile snapper, which were seascapes characterised by patchy reef systems in close proximity to large estuarine water bodies. Hotspots for 7 Estuarine, Coastal and Shelf Science 257 (2021) 107397 M.J. Rees et al. aimed at improving the health of estuaries and their resilience to increasing and ongoing anthropogenic pressures are likely to benefit the region’s snapper population. This information can be used to support effective ecosystem-based management of important fisheries species. The predictive performance of the optimal models evaluated using an independent data set varied greatly between juvenile and adult snapper. The optimal juvenile snapper model had a relatively high de­ gree of transferability, predicting the occurrences of juvenile snapper in the neighbouring northern section of Batemans Marine Bioregion rela­ tively well. In contrast, the optimal model for adult snapper had poor transferability. However, for both juvenile and adult snapper, a positive trend was observed between predicted suitability and observed occur­ rences in the independent data set suggesting model outputs are useful for management purposes. It is important to acknowledge that our Greater Sydney region models have a temporal limitation as samples were collected in Austral winter and early spring. We encourage future repeat baited remote underwater video sampling in the region to further assess model performance and quantify temporal variation in snapper distributions, which has been shown to vary among seasons and years (Egli and Babcock, 2004; Malcolm et al., 2015). Acknowledgements We acknowledge the traditional owners and custodians of the Awa­ bakal, Eora, Kuring-gai, Tharawal and Yuin nations where this research took place. This research was funded by the NSW Government under the Marine Estate Management Strategy. The ten-year Strategy was devel­ oped by the NSW Marine Estate Management Authority to coordinate the management of the marine estate. We thank NSW Fisheries techni­ cians and compliance officers; Kye Adams, Cameron Baber, Luke Bidg­ ood, Joel Cox, Jason Delamont, Eddie Douglas, Amie Hibberd, Daniel Minter, Alex Musumeci and Tristan New for assistance with field work, Curtis Champion for modelling advice, Kylie Brown for the fish graphics, and Hamish Malcolm, Rodney James and the two anonymous reviewers for reviewing the manuscript. APPENDIX A To survey snapper populations representatively across the Greater Sydney region, spatially balanced designs were created following Foster et al. (2020). Survey sites were restricted to rocky reef habitat between depths of 20 and 40 m to align with data from the surrounding NSW coastline (Knott et al., 2021). Given estuaries are important nursery grounds for snapper and often a direct sink for catchment derived contamination and its discharge into open coastal environments, we added unequal inclusion probabilities to the spatially balanced designs to ensure rocky reef sites across a gradient of distances to estuaries were selected. This was achieved by creating a distance from major estuary layer using the Euclidian Distance tool in ArcGIS (ESRI, 2011), for each of the major estuaries in the region (permanently open estuaries: Hunter River, Lake Macquarie, Tuggerah Lake, Hawkesbury River, Port Jackson, Botany Bay, Port Hacking, Port Kembla and Lake Illawarra). The distance from major estuary layer was clipped to a shapefile of rocky reef habitat between 20 and 40 m in depth, which represented the potential survey area for the region (Fig. A.1). As rocky reef between 20 and 40 m within the Greater Sydney region generally exists as a long narrow belt running parallel to the coastline the region was separated into 5 sub­ regions based on natural breaks in reef habitat; i) Port Kembla to Bellambi, ii) Coal Coast, iii) Royal National Park to Long Reef, iv) Long Reef to Avoca and, v) Avoca to Newcastle. For each subregion, a spatially balanced design with biased inclusion probabilities was created in R statistical computing language (R Core Team, 2020) using the ‘MBHdesign’ package (Foster et al., 2020) with the number of ‘distance bins’ set to 4. The density of sites among subregions was kept consistent at approximately 1 site per square kilometre of rocky reef. The spatial layout of the sites was then reviewed to ensure sites were separated by a minimum of 250 m. Occasionally, sites were within 250 m of one another and in these instances one site was re-positioned to achieve the minimum distance of separation. In total 350 sites were allocated across the Greater Sydney region. M.J. Rees et al. Estuarine, Coastal and Shelf Science 257 (2021) 107397 Fig. A.1. Proximity of deep rocky reef habitat (20–40 m) to estuaries within the Greater Sydney region. APPENDIX A Colour gradient represents distance from the entrance of the nearest permanently open major estuary. The raster layer was included in the spatially balanced design to create unequal inclusion probabilities to favour site selections close to open estuaries. ef habitat (20–40 m) to estuaries within the Greater Sydney region. Colour gradient represents distance from the entrance of the Th l i l d d i h i ll b l d d i l i l i b bili i f i Fig. A.1. Proximity of deep rocky reef habitat (20–40 m) to estuaries within the Greater Sydney region. Colour gradient represents distance from the entrance of the nearest permanently open major estuary. The raster layer was included in the spatially balanced design to create unequal inclusion probabilities to favour site selections close to open estuaries. Table A.1 Details of the explanatory variables used to model the distribution of snapper with boosted regression trees. Table A.1 Table A.1 Details of the explanatory variables used to model the distribution of snapper with boosted regression trees. Predictor Description and calculation References Habitat/Seascape Estuary area Area of estuarine water within a 10 km and 20 km buffer of each survey site (ArcGIS - ESRI, 2011). Gillanders (2002); Swadling et al., (in review) Reef area Area of rocky reef within a 50 m, 100m, 200m and 500 m buffer of each survey site (ArcGIS - ESRI, 2011). Ross et al. (2007); Rees et al. (2014); Swadling et al. (2019) Macroalgae presence/ absence Presence or absence of macroalgae on BRUVs footage recorded at each survey site following McLean et al. (2016) in Transect Measure (SeaGIS, seagis.com.au). Fulton et al. (2016); Curley et al. (2002) Sponge presence/ absence Presence or absence of sponges on BRUVs footage recorded at each survey site following McLean et al. (2016) in Transect Measure (SeaGIS, seagis.com.au). Choat and Ayling (1987); Curley et al. (2002) Depth Bathymetry of survey site recorded in situ using depth sounder. Williams et al. (2019); Rees et al. (2014); Parsons et al. (2016) Relief Mean relief score recorded from BRUVs footage at each survey site following definitions of (Wilson et al., 2007). Quantified in Transect Measure (SeaGIS, seagis.com.au) following procedures of McLean et al. (2016). Rees et al. (2018b); Pygas et al. (2020) Visibility Maximum distance of the furthest object (benthic substrate or fish) in the stereo cameras field of view measured in EventMeasure (SeaGIS, seagis.com.au). Bacheler et al. (2019) Anthropogenic stressors Human population density Sum of the number of people that reside within a 10 km and 20 km radius buffer surrounding each survey. Calculated from the Australian Population Grid 2016 (Australian Bureau of Statistics, 2017) in R using zonal statistics. References M.J. Rees et al. Estuarine, Coastal and Shelf Science 257 (2021) 107397 Fig. A.2. Spline correlograms examining spatial autocorrelation in the residuals of the optimal juvenile and adult snapper boosted regression trees. Shaded areas depict 95% pointwise bootstrap confidence intervals. Fig. A.2. Spline correlograms examining spatial autocorrelation in the residuals of the optimal juvenile and adult snapper boosted regression trees. Shaded areas depict 95% pointwise bootstrap confidence intervals. 10 Estuarine, Coastal and Shelf Science 257 (2021) 107397 M.J. Rees et al. Fig. A.3. Maps of the standard deviation in mean predicted probabilities of A) juvenile and B) adult snapper from optimal boosted regression trees. References Cinner, J.E., Maire, E., Huchery, C., MacNeil, M.A., Graham, N.A., Mora, C., McClanahan, T.R., Barnes, M.L., Kittinger, J.N., Hicks, C.C., 2018. Gravity of human impacts mediates coral reef conservation gains. Proc. Natl. Acad. Sci. Unit. States Am. 115, E6116–E6125. Allouche, O., Tsoar, A., Kadmon, R., 2006. Assessing the accuracy of species distribution models: prevalence, kappa and the true skill statistic (TSS). J. Appl. Ecol. 43, 1223–1232. Althaus, F., Hill, N., Ferrari, R., Edwards, L., Przeslawski, R., Sch¨onberg, C.H., Stuart- Smith, R., Barrett, N., Edgar, G., Colquhoun, J., 2015. A standardised vocabulary for identifying benthic biota and substrata from underwater imagery: the CATAMI classification scheme. PloS One 10, e0141039. https://doi.org/10.1371/journal. pone.0141039. 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Species distribution models: ecological explanation and prediction across space and time. Annu. Rev. Ecol. Evol. Syst. 40, 677–697. Table A.1 The standard deviation of predictions were generated from 1000 bootstrap samples. Fig. A.3. Maps of the standard deviation in mean predicted probabilities of A) juvenile and B) adult snapper from optimal boosted regression trees. The standard deviation of predictions were generated from 1000 bootstrap samples. Fig. A.4. The relationship between observed probabilities of occurrence in the independent data set and predicted probabilities of occurrence from the Greater Sydney region models for A) juvenile and B) adult snapper. A higher number of observed occurrences on independent deployments predicted to have high suitability indicates reasonable reliability of the models for management purposes. Fig. A.3. Maps of the standard deviation in mean predicted probabilities of A) juvenile and B) adult snapper from optimal boosted regression trees. The standard deviation of predictions were generated from 1000 bootstrap samples. Fig. A.4. The relationship between observed probabilities of occurrence in the independent data set and predicted probabilities of occurrence from the Greater Sydney region models for A) juvenile and B) adult snapper. A higher number of observed occurrences on independent deployments predicted to have high suitability indicates reasonable reliability of the models for management purposes. Fig. A.4. The relationship between observed probabilities of occurrence in the independent data set and predicted probabilities of occurrence from the Greater Sydney region models for A) juvenile and B) adult snapper. A higher number of observed occurrences on independent deployments predicted to have high suitability indicates reasonable reliability of the models for management purposes. 11 11 M.J. Rees et al. Estuarine, Coastal and Shelf Science 257 (2021) 107397 Estuarine, Coastal and Shelf Science 257 (2021) 107397 Fig. A.5. Map of the southern Greater Sydney region baited remote underwater stereo-video systems samples (grey circles) and the independent baited remote underwater stereo-video systems deployments (white circles) used for model validation. M.J. Rees et al. Fig. A.5. Map of the southern Greater Sydney region baited remote underwater stereo-video systems samples (grey circles) and the independent baited remote underwater stereo-video systems deployments (white circles) used for model validation. Choat, J., Ayling, A., 1987. The relationship between habitat structure and fish faunas on New Zealand reefs. J. Exp. Mar. Biol. Ecol. 110, 257–284. ESRI, 2011. ArcGIS Desktop: Release 10. Environmental Systems Research Institute, Redlands, CA. References R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. 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Belonging: A Study of the Relationship Between Migrant Women’s Social Interactions and Their Perception of Their Own Integration
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Belonging: A study of the relationship between migrant women’s social interactions and their perception of their own integration. Belonging: A study of the relationship between migrant women’s social interactions and their perception of their own integration. Nafisah Graham-Brown UCL Institute of Education Doctor in Education (EdD.) 1 1 Declaration I, Nafisah Graham-Brown, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Nafisah Graham-Brown Nafisah Graham-Brown Word count 45,982 2 2 Abstract Belonging: A study of the relationship between migrant women’s social interactions and their perception of their own integration. Belonging: A study of the relationship between migrant women’s social interactions and their perception of their own integration. Migrant women settling in the UK face multiple barriers to integration. This qualitative study explored the experiences of six newly arrived migrant women who attended community ESOL classes (English for Speakers of Other Languages), focusing on the women’s social interactions in English, the places in which social interactions took place and their sense of belonging. Belonging is defined as feeling at ease with oneself and one’s surroundings (Miller, 2003). Data was gathered over one and a half years, using narrative interviews and oral diaries, and analysed using thematic and classic content analysis methods. The study concluded that women’s social interactions took place in four main spheres: local community, public services, work and home. Data demonstrated that social interactions in English affected women’s feelings of belonging from four aspects: material, relational, cultural and temporal. Findings indicated that social interactions supported women to build trust in people who were different to them and to learn cultural knowledge, practices and acceptable behaviours not formally taught in their ESOL sessions, which supported participants to gain access and to feel at ease in unfamiliar spheres. This study will help teachers to recognise the contribution of social interactions to their students’ language development and integration; policy-makers to plan for future ESOL and integration programme development and funding; and assist organisations in designing more effective community ESOL programmes. Recommendations include for practitioners to plan for activities to support social interactions outside the classroom, to consider the importance of digital skills in enabling ESOL learners to participate in online social interactions, and a more joined-up approach with public service organisations to support staff in those roles to understand how to best support migrant clients who are learning English. 3 3 Impact Statement As the EdD is a professional doctorate, the rationale for study presented in 2.4 are rooted in the future impact that the study will have. As an ESOL practitioner and leader, I am heavily involved in sector improvement through my role as National Co-Chair of NATECLA and management committee of NATECLA London. As part of my role, I also represent ELATT at various forums. I am passionate about disseminating what I have learnt to improve the quality of provision amongst providers of adult and community learning, and through providers, improving the experiences and outcomes of women who are settling in the UK. This research has supported me to continue sharing best practice with the sector. This impact statement presents impact that the thesis research and findings have already had and possible future impact of the thesis. Impact that this research has already achieved At my organisation, findings about the use of Whatsapp in supporting sustained relationships have informed teaching practice on our programmes. Involvement in an Erasmus+ project (see reflective statement) allowed me to apply my insights about Second Language Socialisation (SLS) and the process of integration to the project. Through my role at NATECLA, I have disseminated analysis about ESOL and Integration in England to inform providers and practitioners in the sector. I have led on a developing a framework to support organisations helping migrants and refugees with language to be aware of the value of informal and non-formal learning using knowledge gained from my early analysis about the value of social interactions. I have been involved in the consultation about the development of the government’s ESOL strategy and the knowledge I have gained from the thesis has informed the advice given to Department for Education and Learning and Work Institute. Future possible impact of the thesis In my organisation, it is anticipated that we will use the findings about increasing opportunities for social interactions for our future funding applications. Also, the research will inform the design of our programme for this particular client group as we are more aware of their experiences. For example, creating courses that give participants confidence to interact with council staff more effectively. From a local area perspective, as per the research recommendations (6.2), we may develop migrant and refugee awareness workshops to improve communication by local authority staff. 4 4 From an academic perspective, two areas of potential impact are the methods I used (oral diaries for authentic and independent data gathering and narrative interviewing for ESOL students) and the theories and concepts: SLS and belonging. The use of these methods and concepts in research with ESOL students can provide a basis for future researchers’ work, especially as these are currently under-researched concepts in the UK. Finally, I will be using my time in the United States in 2019-20 to learn more about English as a Second Language in the US. The discoveries and insights from my thesis will hopefully be valuable to the international TESOL community to further enrich TESOL scholarship. 5 5 Acknowledgements Subhanallah for the Blessings bestowed on me, and Strength I have found within me with Your guidance. Ameen. I am eternally grateful to my supervisor David Mallows, without whom I would not be at this stage of my academic journey. I could not have asked for a more knowledgeable, conscientious and supportive supervisor. Words cannot express my gratitude for my long-suffering husband Sam, who has motivated me to complete this journey through his unwavering belief in me. Cheering me on and sustaining me with his cooking kept me focused on completing this EdD. I would like to thank my parents, Hassan and Markizah, for instilling in me the drive for social justice, and the desire to make social change. Also, thanks to my half-mother Maggie for always being the voice of reason, and to Peter for looking after my well- being over these past seven years. Thanks to all my family who have encouraged me! Thank you to the amazing women for taking part in this research, and for sharing with me their stories. Thank you to ELATT for truly being a ‘learning organisation’. The encouragement to take part in further study has enabled me to feel supported throughout my MA and Doctoral studies. Thank you also to my colleagues at ELATT for the opportunities I have been given over the years, and to my team for their encouragement whilst I have been completing this thesis. I am extremely grateful to the three independent readers of my work Dr Sam Duncan, Dr Geraldine Rowe and Valisa Iskandar. Their supportive and incisive feedback has helped me to shape this thesis. Thank you to Alex Hart for his help. Thank you to my amazing friends for their patience whilst I have been concentrating on the EdD. Their meaningful and caring friendship has kept me going through tough times. To my EdD Cohort and Google Group, the support of the group has been invaluable. Thank you for the camaraderie and friendship, which I hope will endure post-IoE. Last, but not least, I have to thank Bryan, for being an additional support for every EdD student. Our thesis years have been all the more enjoyable because of his workshops! 6 Contents 7 Contents List of figures and tables ............................................................................................. 10 Reflective Statement ................................................................................................... 11 List of abbreviations .................................................................................................... 17 1. Introduction ............................................................................................................. 18 2. Background to the research .................................................................................... 22 2.1 UK strategy for integration and cohesion ........................................................... 22 2.2 UK government reports on integration and cohesion ......................................... 23 2.3 English language for migrants in the UK ............................................................ 25 2.4 Research rationale ............................................................................................ 27 3. Literature Review .................................................................................................... 28 3.1 Ontological and epistemological viewpoint ........................................................ 28 3.2 English: ESOL and Second Language Learning ................................................ 29 3.2.1 Language socialisation ............................................................................... 29 3.2.3 Researching language socialisation ............................................................ 33 3.3 Integration ......................................................................................................... 35 3.3.1 Integration and social cohesion ................................................................... 36 3.3.2 Dimensions of integrations .......................................................................... 38 3.3.3 Research on measurements and indicators of integration ........................... 41 3.3.4 Recent research about participants’ own perceptions of integration ............ 42 3.4 Belonging .......................................................................................................... 44 3.4.1 Belonging as a way of conceptualising ‘being integrated’ ............................ 44 3.4.2 Social fields and socialisation ..................................................................... 45 3.4.3 Definition of social interactions .................................................................... 46 3.4.4 Places of interactions .................................................................................. 46 3.5 Aspects of belonging ......................................................................................... 49 3.5.1 Material Belonging ...................................................................................... 50 3.5.2 Cultural Belonging ...................................................................................... 52 3.5.3 Relational Belonging ................................................................................... 55 3.5.4 Temporal belonging .................................................................................... 59 3.6 The framework for belonging ............................................................................. 61 3.7 Research Aims and Questions: ......................................................................... 62 4. Methodology ........................................................................................................... 63 4.1 Design ............................................................................................................... 63 4.1.1 Research questions .................................................................................... 64 4.2 Methods of data collection ................................................................................. 65 4.2.2 Narrative interviews .................................................................................... 65 4.2.3 Oral diaries ................................................................................................. 69 4.3 Sample .............................................................................................................. 72 4.3.1 Challenges with this participant group ......................................................... 73 4.4 Ethical Considerations ....................................................................................... 75 4.5 Reliability and Validity ....................................................................................... 77 4.5.1 Access ........................................................................................................ 78 4.5.2 Trustworthiness, verisimilitude and authenticity .......................................... 79 4.5.3 Transferability ............................................................................................. 79 4.6 Analysis ............................................................................................................. 81 4.6.1 Analytical approach ..................................................................................... 81 4.6.2 Participants’ experiences prior to migration ................................................. 83 4.6.3 Participants’ experiences as new migrants ................................................. 85 4.6.4 Places of social interactions ........................................................................ 85 4.6.5 Identifying and describing social interactions .............................................. 87 4.6.6 Participants’ perception of belonging .......................................................... 88 5. Findings and Discussion ......................................................................................... 90 5.1 RQ 1: What are participants’ experiences at the start and at the end of the research project ...................................................................................................... 90 5.1.1 Participants’ life stories ............................................................................... 90 5.1.2 Participants’ experiences as newly arrived migrants affecting their belonging ............................................................................................................................ 99 5.1.3 Participants’ early sense of belonging ....................................................... 104 5.2 RQ2: What are the places in which social interactions take place? ................. 105 5.2.1 Local Community sphere .......................................................................... 105 5.2.2 Public services sphere .............................................................................. Contents 106 5.2.3 Work ......................................................................................................... 107 5.2.4 Private ...................................................................................................... 108 5.3 RQ3: What is the nature of the social interactions? ......................................... 110 5.3.1 Local Community Sphere .......................................................................... 110 5.3.2 Public Services Sphere ............................................................................. 116 5.3.3 Work sphere ............................................................................................. 121 5.4 Main summary of findings ................................................................................ 125 5.5 What are participants’ perceptions of their belonging ...................................... 126 5.5.1 Material Belonging- access to places ........................................................ 127 8 5.5.2 Relational belonging ................................................................................. 131 5.5.3 Cultural belonging ..................................................................................... 137 5.5.4 Temporal belonging .................................................................................. 140 5.6 Discussion on significant findings .................................................................... 142 6. Conclusion ............................................................................................................ 144 6.1 Limitations of the study .................................................................................... 145 6.2 Implications for professional practice and contribution to professional knowledge .............................................................................................................................. 146 6.3 Recommendations for further research ........................................................... 148 Bibliography .............................................................................................................. 149 Appendices ............................................................................................................... 168 9 9 Journey to the EdD My initial experience of further education was when I got a ‘second chance’ to complete my A-levels. Whilst working in the retail and catering sector, I realised that I wanted to teach and I decided to re-train as a college teacher. I completed a Post Graduate Certificate in Education (PGCE) Post-Compulsory at the Institute of Education (IoE). On the PGCE, my preconceptions about education and meritocracy were challenged. I learnt about the role of power imbalances and contexts in teaching and learning. I started working for an education charity in East London as an ESOL teacher on projects for newly arrived migrant women in London. Whilst on my MA in Comparative Education, my passion for social justice, which I had been so enthusiastic about in my youth, was re-ignited. I realised that I was interested in the experiences of the women I was supporting in my day-to-day work. As soon as I had finished my MA I set my sights on the EdD. The start of my EdD coincided with a promotion to Head of Curriculum at my organisation. I chose the EdD for the structured first year of the programme. It was a challenging year as my research knowledge and skills were stretched, whilst learning my new role at work. It was also an enriching year and those in my year became a support network that has endured over the past seven years. “We don’t see things as they are; we see things as we are”. Anon* “We don’t see things as they are; we see things as we are”. Anon* This reflective statement is of my personal and professional development as an educator, leader and researcher during my EdD journey. I present motivations for undertaking the EdD, and how the elements on the programme have supported me to develop professionally and academically. Over the past seven years of the EdD, I have experienced continuous growth in my knowledge of teaching and learning, my education sector (ESOL- English for Speakers of Other Languages), and the field of refugee and migrant integration. List of figures and tables Figure 1:Dimensions of integration vs aspects of belonging ........................................ 50 Figure 2: Diagram of framework of belonging ............................................................. 61 Figure 3: Interview 1 times .......................................................................................... 68 Figure 4: Oral Diary Questions .................................................................................... 71 Figure 5: Participants and their countries of origin ...................................................... 72 Figure 6: Table of number of interviews with participants ............................................ 74 Figure 7: Table of the analytical process ..................................................................... 82 Figure 8: Places of social interactions ......................................................................... 86 Figure 9: Participants' perception of their own belonging ............................................ 89 10 10 Foundations of Professionalism Foundations of Professionalism (FoP), allowed me to explore multiple meanings of professionalism with reference to the experiences I had at my organisation. I was able to re-familiarise myself with the concepts and revisit them with the benefit of 5 years’ teaching experience gained since my PGCE. I investigated the role of ESOL tutors 11 supporting migrant and refugee women in my organisation and how this differed from traditional FE tutor roles, using Celia Whitchurch’s ‘Third Space Theory’ (Whitchurch, 2008). There were differences in expectation, which when not well communicated, could cause performance-related issues. I was able to re-examine my organisation recruitment and performance-management systems and introduce a coaching-based approach. Method of Enquiry 1 and 2, and Initial Specialist Course I was aware of the gaps in my knowledge about empirical research, as I had previously carried out secondary research. The modules were crucial in developing my knowledge of methodological frameworks and perspectives. I had to be explicit about my epistemological position. This was also my first experience of ‘insider research’ (Robson, 2011). I was surprised that we were encouraged to acknowledge our personal stances and values towards professional practice rather than to be neutral (Robson, 2011). I became more aware of my democratic and participatory values, which influenced my social constructivist stance (Garrison, 1995), believing in the cooperative construction of knowledge that is rooted in multiple social perspectives built through social interaction (Plowright, 2011). The stance became the basis for my theoretical and methodological framework. An area of interest from professional practice at my organisation was the experiences of newly arrived women, who were settling in the UK and learning English. We had designed programmes to support them to become ‘integrated’ and I wanted to examine the effectiveness of our courses, as well as be able to develop future courses. I was interested in learning about the perceptions of the women themselves regarding the contested concept of integration (Morrice, 2011). My MoE1 assignment was about a proposal for research to explore migrant women’s own views. Unfortunately, I failed to sufficiently articulate, explain, defend and justify my assignment topic, which led to me writing an essay where the research questions were not adequately formed. The process taught me the importance of clarity about my topic. In Methods of Enquiry 2 (MoE 2) I carried out a pilot project to better understand the experiences of out-of-classroom interactions of migrant spouse women using oral diaries, a method which I had not seen previously used on this type of research. I experienced the steepest learning curve and struggled to manage the demands of the research assignment together with work demands. The new understandings I gained were related to how research questions worked in practice and the types of errors that In Methods of Enquiry 2 (MoE 2) I carried out a pilot project to better understand the experiences of out-of-classroom interactions of migrant spouse women using oral diaries, a method which I had not seen previously used on this type of research. Method of Enquiry 1 and 2, and Initial Specialist Course I experienced the steepest learning curve and struggled to manage the demands of the p g p g diaries, a method which I had not seen previously used on this type of research. I experienced the steepest learning curve and struggled to manage the demands of the research assignment together with work demands. The new understandings I gained were related to how research questions worked in practice and the types of errors that 12 could happen in data collection. In designing my proposal for the research questions I became more aware to pay extra attention to the wording of research questions to avoid ambiguity. However there were weaknesses in clarifying the link between theory and research for my readers. Feedback in the MoE 2 assignment was that my literature review, methodological frameworks and methods did not link as well as they could; the lack of clarity in writing has been an area I have worked hard to improve throughout the EdD. My understanding of the complexity of social science research deepened, particularly the acknowledgement of multiple perspectives and different research data interpretation. The feedback received in my EdD interview and MoE 1 was both frustrating and constructive as it caused me to question the reality of my thought processes: I was unknowingly applying a ‘cause and effect’ understanding of research when constructing research questions and methodological frameworks. By MoE 2 and especially after the Initial Specialist Course (ISC), I started thinking of qualitative social science research differently. I chose Rethinking Education: Psychoanalytic Perspectives on Learning and Teaching as my ISC. On this, I experienced a fundamental shift in how I viewed research and education. Through learning about psychoanalytic perspectives, I developed critical understandings of the complex relationships within the learning process, and how these could be interpreted vis-à-vis wider cultural and social power relations. Before starting the EdD I was convinced that my research would measure the effectiveness of projects at my organisation. During the taught element, my view changed to being more experience-focussed as I recognised that an exploration of the experiences of our client group would be more valuable than an evaluation exercise. A greater understanding of the nature of insider and social science research influenced this. My research topic evolved to be about migrant women’s experiences vis-à-vis settling and belonging. Thesis The thesis has by far been the most challenging project I have ever completed. Based on the aim of the research, I selected a narrative inquiry method. Although I was the first in my cohort to have their proposal approved, the logistical issues related to gathering data, specifically the using the interview method with my participants, was one I had not anticipated. Coupled with that was an unprecedented cut (30%) to the adult education budget for all further education providers. This meant that work commitments increased as we tried to maintain our provision with fewer staff. The delays to the data gathering activity pushed my timeline back making completing this project particularly challenging. My work role also became increasingly strategic as I joined the Senior Management Team, and I struggled to maintain balance between my job and the EdD thesis workload. Reading literature from broader fields of study was required to find a theory that could form a framework to explain the relationship between English language and integration. It took several years before I found the ideal concept to link my theories. IFS IFS The Institution Focussed Study (IFS) was a study of the role of out-of-class interactions migrant women’s perceptions of their language development and integration. It was the most exciting piece of research I have carried out, mainly because of the research design, involving participatory research methods developed from Reflect for ESOL (Cardiff, Newman, & Pearce, 2007), and the oral diary method that I had developed in MoE2. I wanted to capture migrant women’s voices with minimal researcher involvement. The process was enjoyable and the findings were fascinating. Findings 13 indicated that the migrant women had limited interactions in English. Participants accessed formal interactions (such as with staff at schools) to be able to access informal interactions (with other parents) (Graham-Brown, 2015). I felt I gained confidence in research skills using the case study method, especially in different methods of analysis. Feedback from the IFS suggested more a more qualitative research project for my doctoral thesis as it was not possible to study the role and nature of social relationships of migrant women in the IFS. I was intrigued by the development of social relationships by migrant women settling in this country and wanted to research this longitudinally. Contribution to professional development and knowledge Reflecting on the development of my academic and research skills, I can truly see a difference in how I perceive the world. I am more reflective, analytical and critical and I continue to develop. I am also constantly assessing reliability and validity in all the assertions I make when reporting results of outcomes and achievements (professionally and academically). Working in charity education sector and education research has required me to use different genres of writing for different audiences: writing for ESOL students, writing for work, fundraising, and academic writing. The Feedback from my EdD supervisor has 14 developed my writing skills to become more precise and succinct, and I have been able to make successful grant applications for my organisation. Another contribution is in the assessment of student experience in my organisation. My acknowledgement of multiple perspectives (of experiences) has affected the way I view student experience. The understanding of research methodology from the EdD, has given me the tools to best capture this data and to analyse it critically. I have more conviction in my own knowledge as an expert in my field. The understanding I developed in the field of ESOL and integration led to my organisation being invited to partner with UCL and others in an Erasmus+ research project. The project, which was about volunteer support in migrant language learning, raised the profile of my organisation and me in the ESOL field. At the same time my role at my organisation becomes more strategic and external-facing. I had been involved in NATECLA, the national body for ESOL previously and was invited to apply for the Co- chair role, which I have now been carrying out since September 2018. From a personal development point of view, the EdD has undoubtedly forced me to become highly organised with time management. Consequently, my delegation skills improved and I have since grown a successful team. I have become a more reflective manager, taking a step back to look at issues from different perspectives. I believe that my research skills have developed me as a teacher and trainer. Ultimately, the aim of this doctoral thesis was to better understand the experiences the of migrant women we support through giving them a voice I feel that I have met this aim. The personal and professional development I have gained as part of the process has been invaluable. Contribution to professional development and knowledge Although it has taken me four years, it has been necessary to fully experience this process of ‘becoming’ an academic, one where I have been able to reflect on my thinking within my practice. A process that I would argue is crucial in a professional doctorate. I am excited to share the findings of my study with colleagues and partners in the sector and look forward to future opportunities for further research and dissemination in this field. Cardiff, P., Newman, K. & Pearce, E. (2007). Reflect for ESOL Resource Pack. London: ActionAid * There is disagreement about the origin of this quote- it has been attributed to Nin (1961), P i k ( ) d h T l d * There is disagreement about the origin of this quote- it has been attributed to Nin (1961), Patrick (1890) and the Talmud. Garrison, J. (1995). Deweyan Pragmatism and the Epistemology of Contemporary Social Constructivism. American Educational Research Journal, 32(4), 716–740. https://doi.org/10.3102/00028312032004716 date accessed: 27.05.19 Morrice, L. (2011). Being a Refugee : Learning and Identity (A Longitudinal Study of Refugees in the UK). Stoke on Trent: Trentham Books. Robson, C. (2011). Real World Research (3rd ed.). West Sussex. References Cardiff, P., Newman, K. & Pearce, E. (2007). Reflect for ESOL Resource Pack. London: ActionAid Cardiff, P., Newman, K. & Pearce, E. (2007). Reflect for ESOL Resource Pack. London: ActionAid 15 16 List of abbreviations APPG All Party Parliamentary Group BERA British Educational Research Association BNIM Biographical Narrative Interview and/or Interpretive Method CCA Classic content analysis CIC Commission for Integration and Cohesion CV Curriculum Vitae DCLG Department for Communities and Local Government DFE Department for Education DWP Department for Work and Pensions ESFA Education and Skills Funding Agency ESOL English for Speakers of Other Languages EU European Union FANI Free Association Narrative Interviewing GLA Greater London Authority GP General Practitioner IFS Institute Focussed Study LS Language Socialisation MHCLG Ministry of Housing, Communities and Local Government NATECLA National Association of Teaching English and Community Languages to Adults ONS Office for National Statistic PIN Particular incident narratives PTA Parent-Teacher Association PTSD Post Traumatic Stress Disorder SCT Socio-Cultural Theory SLA Second Language Acquisition SLS Second Language Socialisation SQUIN Single question aimed at inducing narrative VLE Virtual Learning Environment List of abbreviations 17 17 1 Data tables showed that in 2015 migrant women represented 75% of the total adults given family and dependent visas. 1. Introduction 18 18 arrived migrant women are women who have arrived in the UK within the previous ten years. In this study, the terms migrant women and women refer to newly arrived migrant women. Migrants face difficulties to be able to communicate in English when they are new to the UK (Mallows, 2014). The role of language and communication in enabling migrants to be able to function in their new country and community has been widely researched (Bryers, Winstanley, & Cooke, 2013; Esser, 2006; Morrice, Tip, Collyer, & Brown, 2019). The most important indicator of a person’s ability to integrate is their confidence in using the language of the country they are settling in (Esser, 2006; Phillimore, Ergun, Goodson, & Hennessy, 2007; Simpson, 2012). Besides language issues, migrant women especially, face multiple barriers to settling in the UK, (Eaves, 2015). They often have difficulties understanding their new environment and how systems work (Simpson & Whiteside, 2012). For example, many do not know how the schooling system works in England and how to support their children at school (Court, 2014; Simpson, 2011). They may not be aware of the rights they have in the UK in comparison to the rights in their previous countries (Eaves, 2015). Additionally, women may not be used to the diverse and multicultural mix of people in their new neighbourhoods and be at a disadvantage when it comes to understanding people from other countries (Roberts & Cooke, 2009). This includes knowing what constitutes acceptable behaviour when they meet and communicate with others (Morrice, 2007). This lack of knowledge together with the lack of language skills, often affect migrant women’s level of confidence and how independent they are in everyday life (Eaves, 2015). In this situation, migrant women have to construct new social relationships with people in their wider local community: people who are outside of their family circle and immediate ethnic community (Eaves, 2015). Wider local community comprises neighbours, parents of other children at the school or nursery their children go to, shop owners and service staff, public transport workers and public services staff (such as at council service centres, Jobcentres or libraries) (Graham-Brown, 2015). The low level of confidence, together with other barriers affect how migrant women participate in their wider communities. 1. Introduction In this chapter I introduce my research and briefly discuss the issues that my research aimed to address. I also outline the organisation of this thesis. My research concerns the experiences of migrant women learning English as they attempted to integrate into their local communities, with a focus on the social interactions in English that migrant women participated in and how these affected their perception of their own integration. Their perception of their integration has been explored through using a framework of belonging based on Vanessa May's concept of belonging (2013), discussed further in section 3.4. For over 10 years, I have been delivering projects to support newly arrived migrant and refugee women to improve their English language skills, and to get involved in their local communities. My role is Head of Life Skills and Community Projects at ELATT, a London education charity. ELATT has been supporting migrants for over 30 years, specialising in delivering ESOL (English for Speakers of Other Languages) with community programmes to support our client groups with their integration in London, one group being newly arrived migrant and refugee women. The Institute of Migration defines a migrant as ‘a person who moves away from his or her place of usual residence, whether within a country or across an international border, temporarily or permanently, and for a variety of reasons’ (Institute of Migration, 2019, p.130). The umbrella term can include ‘refugees, displaced persons, economic migrants, and persons moving for other purposes, including family reunification’ (Eaves, 2015, p.16). The term migrant women in this study refers to women from non- EU countries, who have moved to the UK for refugee and family reasons or fear of persecution, and who do not come from countries where English is the majority language. They are reflective of the proportion of migrant women who move to the UK annually for family reasons on family and dependent visas (Home Office, 2017b)1. Although some may consider them voluntary migrants, based on our experience at ELATT, factors such as socio-cultural norms and other inequalities have meant that they often do not have agency over their decision to migrate. Therefore, they face similar challenges as refugee women when they are new to London and the UK. Newly 1 Data tables showed that in 2015 migrant women represented 75% of the total adults given family and dependent visas. 1. Introduction If they are unable to overcome barriers to taking part in social interactions with their wider community groups, and/or live within communities where they are not connected or interacting with the wider community, they are less likely to feel they are integrated (Eaves, 2015). 19 One of the main challenges ELATT’s clients tell us they face is to find opportunities to have meaningful English language interactions with others in their local communities. This is because they do not often have readily available contact with people outside of their family and immediate ethnic community in order to have social interactions in English. In 2013, research I carried out found that most of the reported out-of-class interactions in English by research participants were in shops and when using public transport (Graham-Brown, 2015, discussed in 3.3.4). The language used in these interactions was often limited to language around buying goods and accessing services. We at ELATT perceived these interactions to have limited potential to support development of complex language fluency and contribute to participants’ integration. As a result, we designed projects that included opportunities for participants to take part in more meaningful social interactions, through either volunteering or engagement with external volunteers to our organisation. The value of social interactions in migrants’ language development and integration is an under-researched area (Heckmann, 2005). There is little existing research about migrants’ out-of-class language use (Simpson, 2011), particularly social interactions that migrant women participate in during their daily lives and how these social interactions contribute to their perceptions of integration. Norton (1995) researched social relationships from an identity perspective, but did not specifically focus on wider social relationships. Cooke (2006) referred to the lack of opportunities for migrants to practice English outside of their classroom setting. There is also research carried out in America, Canada and Australia about the difficulties ESL students have in participating in their new communities (Warriner, 2007), social isolation of second language speakers (Miller, 1999) and the importance of social acceptance and peer support in learning a new language (Baquedano-Lopez & Mangual Figueroa, 2011). Many researchers say that migrants make friends in their ESOL/ ESL classes (Cooke, 2006; Domínguez & Maya-Jariego, 2008; Graham-Brown, 2015; Kanno & Varghese, 2010). Research by Dimitriadou (2004) with refugees studying in ESOL classes in London showed that friendships and social relationships developed between migrants in their ESOL classes. 1. Introduction However she also found that there was little evidence that social relationship were being developed outside of the ESOL departments, or whether the classroom relationships continued after the courses had ended (Dimitriadou, 2010). This thesis has built upon my earlier Institution Focused Study (IFS) (Graham-Brown, 2015), in which I compiled a typology of the interactions migrant women had with people in their wider community. The IFS findings were that women accessed formal places, such as ESOL classes and children’s schools, to be able to gain access to 20 informal social interactions. Participating in informal social interactions in turn affected migrant women’s perceptions of their confidence to use English language. I further discuss the IFS research along with other recent research, in section 3.3.4. In this study, I explored the experiences of newly arrived migrant women and their social interactions in English to answer the following research question: informal social interactions. Participating in informal social interactions in turn affected migrant women’s perceptions of their confidence to use English language. I further discuss the IFS research along with other recent research, in section 3.3.4. In this study, I explored the experiences of newly arrived migrant women and their social interactions in English to answer the following research question: In this study, I explored the experiences of newly arrived migrant women and their social interactions in English to answer the following research question: What is the relationship between migrant women’s social interactions in English and their perception of their own integration? The definition of integration I used was: The definition of integration I used was: Integration is the process by migrants who are new to the UK, of becoming part of UK society. This process has multiple responsible parties including host communities and government. Integration is the process by migrants who are new to the UK, of becoming part of UK society. This process has multiple responsible parties including host communities and government. (Graham-Brown, 2015, p.13) In order to examine migrant women’s perceptions of their own integration, I used a framework of belonging. Belonging is defined as feeling at ease with one’s surroundings (Miller, 2003). I developed this framework using existing literature about integration, second language socialisation (SLS) and concepts of belonging (further discussions in sections 3.5-3.6). This thesis is organised in the following way: Chapter 2 is the background to the research including the research rationale. In Chapter 3, I review existing literature and present a framework of belonging. The first part of Chapter 4 is a description of the methodological framework, including research methods, sample, ethical consideration and discussions of reliability and validity. The second part of Chapter 4 presents my analytical approach, and stages and methods of analysis. Findings and Discussions are presented in Chapter 5. Both findings and discussion are organised around my research questions, followed by a summary and discussion of findings. Chapter 6 contains conclusions about the study, limitations and recommendations for further research. 21 21 2 A version of this chapter was published in the journal Language Issues (Graham-Brown, 2018 2. Background to the research2 In this chapter, I discuss the background to my research project, and provide rationale for the research project from academic and professional perspectives. 2.1 UK strategy for integration and cohesion UK integration strategy is derived from and influenced by EU integration policy. The EU’s definition of integration is: “Integration is a dynamic, long-term, and continuous two-way process of mutual accommodation, not a static outcome. It demands the participation not only of immigrants and their descendants, but of every resident. The integration process involves adaptation by immigrants, both men and women, who all have rights and responsibilities in relation to their new country of residence. “It also involves the receiving society, which should create the opportunities for the immigrants' full economic, social, cultural, and political participation. “Accordingly, Member States are encouraged to consider and involve both immigrants and national citizens in integration policy, and to communicate clearly their mutual rights and responsibilities.” “Accordingly, Member States are encouraged to consider and involve both immigrants and national citizens in integration policy, and to communicate clearly their mutual rights and responsibilities.” (Council of The European Union, 2004) The definition has been used by nation states to formulate national integration policies (Council of The European Union, 2004). The definition suggests that integration has multiple responsible parties and therefore it is important that there is clarity about national integration aims. 22 22 22 3 The Department for Communities and Local Government (DCLG) had a name change in 2018 to the Ministry of Housing and Local Government (MHCLG). 4 See http://tedcantle.co.uk/about-community-cohesion/ for a year-by-year account of differing government reports and policy responses for community cohesion. 2.2 UK government reports on integration and cohesion Successive governments have been concerned about integration and cohesion (Casey, 2016; Community Cohesion Review Team, 2001; Crick, Brighouse, Brown, Coussey, & Hira, 2003). In the early 2000s, the disturbances in Northern England led the then government to set up the Commission for Integration and Cohesion (CIC) to review integration and cohesion and produce reports to inform policy-makers (Community Cohesion Review Team, 2001; Department for Communities and Local Government3, 2008). The CIC recommended promoting ‘meaningful interactions’, and stated that socio- economic factors often affected community cohesion, resulting in a situation where people were living parallel lives (Community Cohesion Review Team, 2001). It recommended setting a time limited expectation for migrants to achieve fluency in English (Community Cohesion Review Team, 2001). The language and citizenship requirements for those naturalising as British citizens were introduced in 2003, and later for those settling in the UK (Cooke, 2009). Subsequent reports focussed on recommended clearer rights and responsibilities for migrants, including learning English (Commission for Integration and Cohesion, 2007)4, and the obligations of host communities (House of Commons Communities and Local Government Committee, 2008). The concept of parallel lives was revived in Dame Louise Casey’s 2016 review into social integration (Casey, 2016). Parallel lives is when different community members are perceived to live segregated lives to others in the wider area, and when the term is used it is mainly aimed towards minority ethnic groups, seen as being segregated from White British groups (Simpson & Finney, 2010). Casey’s report highlighted that women from certain groups (majority ethnically from Muslim majority countries such as Pakistan and Bangladesh) were more likely to have parallel lives in segregated and closed communities with no social mixing (2016). Casey identified lack of English as a key factor in the women’s inability to integrate (2016). 23 Therefore, government commission reports concerning integration appear to have changed in the past 10 years. The CIC’s reports recognised the effect of inequality and socio-economic deprivation on cohesion (Community Cohesion Review Team, 2001), especially as different groups perceived unfair distribution of services (Commission for Integration and Cohesion, 2007) and recognised the barriers people faced to access language provision (Commission for Integration and Cohesion, 2007). Therefore, government commission reports concerning integration appear to have changed in the past 10 years. 2.3 English language for migrants in the UK The importance of English for migrants settling in the UK and recommendations for increasing English language provision have featured in the above reports by researchers and review teams. English for Speakers of Other Languages (ESOL) is the main type of English language course for migrants and refugees settling in the UK. Courses are generally taught in general further education (FE) colleges and adult learning providers. From 2013 onwards, the body responsible for adult education in England, the Skills Funding Agency (SFA) (later renamed the Education and Skills Funding Agency (ESFA) under the Department for Education (DFE)), reduced funding for adult skills and this affected the funding allocated for ESOL classes (Foster & Bolton, 2017). It was estimated that the reduction in funding for ESOL between 2009 to 2016 was by £113million (Exley, 2017). The real term annual loss was approximately 60% when comparing figures in 2009 with those in 2016 (Foster & Bolton, 2017). Because of the recommendations from reports mentioned earlier (CIC reports, DCLG reports and Casey Review), alternative funding by MHCLG, The Home Office and EU programmes has been made available for English language projects, which are distinct from FE and adult education ESOL (see Foster & Bolton, 2017). However from my calculations, the alternative funding listed in the briefing report (Foster & Bolton, 2017) and EU sources (Home Office, 2014) is an estimate of £40million annually, whereas the reduction of ESOL funding in FE has been estimated at £132million annually as at 2016 (Foster & Bolton, 2017). Therefore, contrary to the recommendations of the earlier reports, and the announcements made about specific funds for learning English, these figures show there has been an overall reduction in funding for ESOL for migrants settling in the UK. At the same time, there has been a further tightening of the language testing requirements for applications for settlement and citizenship. It appears that the government chose to interpret the findings from the integration and cohesion reports as suggesting a need for indicators of competency levels that migrants must achieve to prove they are integrated (Blackledge, 2009; Cooke, 2009; Home Office, 2013), rather than a need for increased opportunities for migrants to learn English to be able to integrate. Additionally, the ESFA continues to have strict eligibility rules for those applying for funded courses. 2.2 UK government reports on integration and cohesion The CIC’s reports recognised the effect of inequality and socio-economic deprivation on cohesion (Community Cohesion Review Team, 2001), especially as different groups perceived unfair distribution of services (Commission for Integration and Cohesion, 2007) and recognised the barriers people faced to access language provision (Commission for Integration and Cohesion, 2007). Casey’s review seemed to mark a change of tone. There was an increased emphasis on the idea that people were leading parallel lives, sometimes presented as out of choice, and that certain Muslim minority groups were resistant to integration (Casey, 2016). The responsibility was placed on the migrant to ‘adapt’ into their new society (Stone, 2017). Casey’s report findings have been questioned by researchers as the research methodology is not clear (Crossley, 2018). However the findings were widely publicised in national newspapers with a pervasive message perception that migrants do not want to learn English and want to be segregated (see for example national news reports: Asthana & Walker, 2016; Peev, 2017; Stone, 2017). Media reporting of reports such as the Casey review above, and reports by researchers over the past three years (Burnett, 2017; CIVITAS, 2016; Duru, Hanquinet, & Cesur, 2017) suggest that public perception of migrants has been influenced by government policy and media reporting. The UK does not yet have an integration strategy (All Party Parliamentary Group on Social Integration, 2017), which would be a plan to enable the process of integration of migrants into British society, setting out what parties are responsible and how those parties should work together. In 2018 the UK government produced a green paper “Integrated Communities” to inform initial consultation for a national strategy for England (HM Government, 2018). The responses to the consultation, along with an Action Plan were published in February 2019 (HM Government, 2019a, 2019b), and a national strategy is due in late 2019. 24 24 2.3 English language for migrants in the UK For example, people on partner visas were not eligible for SFA skills funding until they had lived in the UK long enough to satisfy residency requirements 25 (Skills Funding Agency, 2016). Many migrant women who are mostly on dependent visas, have been ineligible for fully-funded courses. Therefore, even when satisfying residency criteria, they have not been eligible for a free course (Skills Funding Agency, 2016). Those of us working in ESOL know the migrant women we support are usually highly motivated to study and improve their English skills to become integrated. However, the reduction of available funding and restrictions on eligibility has meant that they might not have been able to access the services to learn the language they need to take part in social interactions. Negative perceptions of migrants may also affect the ability of migrant women to mix with host community members. 26 3. Literature Review In this chapter, I present the main theoretical perspectives, which formed the conceptual framework for this research project. I set out my personal perspective about knowledge and the study of knowledge (ontological and epistemological viewpoint). I present the theory of second language socialisation to understand how English language is learnt by people who move to, and are settling in the UK. I then present and discuss theories of integration and belonging and examine the relationship between English language and belonging. In order to research migrant women’s perceptions of their own integration, I used the concept of belonging as part of the conceptual framework for my research. Finally, I present my research questions. 2.4 Research rationale Academic rationale: contribution to a body of research on how learning English supports integration and can be used to advise policy-makers on models of English language learning for integration. As mentioned earlier, government commissioned reports about integration and cohesion have highlighted the importance of English to support social mixing between people in communities. Reports state that if migrants have a better command of English they will be able to converse with people better (All Party Parliamentary Group on Social Integration, 2017; Casey, 2016). However, neither reports refers to current relevant research about the relationship between interactions in English and being integrated. There is a lack of research of how someone who achieves a level of language competency will become integrated (Court, 2017). This thesis contributes to this growing body of research about the integration experiences of migrant women (discussed in 3.3.4) and in doing so, suggests ways in which learning English can support integration. Along with other research emerging in the field, it will be available to influence policy-makers when they are planning for ESOL funding and English and integration programmes to ensure effective and efficient use of public money. Practice rationale: to support practitioners and delivery providers, such as my organisation, to create better programmes and identify best practice to share with other organisations. ELATT has successfully developed several programmes to respond to the needs of local migrant women. The aims of ELATT to support our students to integrate into their communities continue to be of great importance to our organisation’s vision. As an organisation that values research as a basis for creating our projects, ELATT is using this research to support the development of our future integration and cohesion programmes. It is hoped that other teachers and providers will also use the findings from this study to inform teaching and learning and future programmes in this field. In this chapter, I have presented the background to my research, setting out key definitions to be used, policy contexts within which my research sits, and important dimensions to the research. Drawing together both the fields of ESOL and integration to set up my research background and context, I have highlighted the academic, policy and practice rationale for my research. More in-depth discussion on the concepts discussed in this chapter such as ESOL, social interactions, integration, cohesion are found within Chapter 3, Literature Review. 27 27 3.1 Ontological and epistemological viewpoint A researcher’s ontological position is their assumption of what constitutes reality (Grix, 2002). My ontological position, which forms the foundation of this research, is that there is plurality of realities, as opposed to the positivist scientific position that there is one objective reality (Cohen, Manion, & Morrison, 2005). My positioning is that a reality or truth, can only be known through humans’ interpretations and construction of meanings (Blaikie, 2007). Therefore, the nature of reality is subjective. Where ontology is about the nature of reality, epistemology is about how we gain knowledge about the nature of that reality (Grix, 2002), or ‘how we know what we know’ (Crotty, 1998). My epistemological standpoint is a social constructivist one, which can be both an epistemological position (Larochelle & Bednarz, 1998), and a methodological perspective (Flick, 2004). The constructivist position is that knowledge is constructed rather than inherently known (Crotty, 1998). Social constructivism suggests that the construction of knowledge is through individuals’ social interactions with others and their environment (Crotty, 1998). The migrant women in this study may have had different perceptions of their experience of integration; my intention was not to fully know their ‘truth’, rather to gain an in-depth understanding of their experiences. Where ontology is about the nature of reality, epistemology is about how we gain knowledge about the nature of that reality (Grix, 2002), or ‘how we know what we know’ (Crotty, 1998). My epistemological standpoint is a social constructivist one, which can be both an epistemological position (Larochelle & Bednarz, 1998), and a methodological perspective (Flick 2004) The constructivist position is that knowledge 28 3.2 English: ESOL and Second Language Learning As described in the Introduction, it is widely accepted that lack of language proficiency, especially at a functional level, is one of the greatest barriers to integration (see Esser, 2006; Simpson, 2012). Indeed if a person settling into a new society has no knowledge and ability to use the main language of communication, then it is seen to be entirely limiting to the person’s ability to integrate (Eaves, 2015; Ipsos Mori, 2007; Phillimore et al., 2007). England has a long history of migrant language education. ESOL was formalised in national adult education provision in the 1990s (Rosenberg, 2007). The area of study of how people learn another language as an adult is second language acquisition (SLA). The term second language is taken to mean any language learnt as an adult, additional to the person’s first language (Van Patten & Williams, 2014). Theories of SLA have evolved and developed over many years. One approach to SLA comes under the umbrella of socio-cultural theory. Developed from Vygotsky’s theories, SCT’s central principle is that human cognitive development takes place as a result of social participation (Lantolf & Thorne, 2006). Although there are ‘experts’ and ‘novices’ in learning interactions, SCT posits that knowledge is co-constructed and the nature of the exchange in learning means that is it not only top-down (Swain, Brooks, & Tocalli-Beller, 2002). Negotiation of meaning is within a context and through social interactions using mediation artifacts (in this case language) (Lantolf, 2011). 3.2.1 Language socialisation A theory of SLA that is interested in the entire context of learners’ experience is language socialisation (Watson-Gegeo & Nielson, 2003). Language socialisation proposes that ‘novices are socialized into communities of practice across the life course’ to ‘use language appropriately in culturally significant activities’ through socialisation using the same ‘language, into local values, beliefs, theories, and conceptions of the world’ (Howard, 2008, p.187). Language socialisation is a theory of language learning that initially focused on child language development, (termed L1 socialisation), but in the past 30 years has been seen as a ‘lifespan process’ spanning a variety of settings and spaces (Ochs & Schieffelin, 2011). It has been used in identity and ethnographical research. Recently, language socialisation has been used to study the experiences of adult second (or additional) language learning, termed Second Language Socialisation (SLS) (Baquedano-Lopez & Mangual Figueroa, 2011; Cook, 2011; Duff, 2010; Ortaçtepe, 2013). SLS is the process of a novice being inducted into a new group or community through the use of a ‘new’ language and to use the language (Duff, 2007). Regardless 29 of age, to be able to use a new language involves linguistic and social input, instruction and interaction to be able to participate with the new language in group-determined communicative practices (Duff, 2007). At the same time, this process is experienced using the new or destination language, for example how to use the language for functioning in daily life, through using the destination language. The term 'language socialisation' is derived from Sapir's work on culture, language and identity (Ochs & Schieffelin, 2011). Sapir argued that language was not just functional and was an individuals' vocalisation of a symbolic representation of what they saw as reality (Sapir, 1949). Sapir also identified that speaking a common language was an integral symbol of social solidarity stating that 'Language is a great force of socialisation, probably the greatest that exists’. (Sapir, 1949, p.15). The theory is built upon the social theories of Bourdieu (Baquedano-Lopez & Mangual Figueroa, 2011). Bourdieu, in his theory of practice, hypothesised that society was organised into social fields (Hilgers & Mangez, 2015). These are socially ranked spaces, within which there are interrelations between individuals (players) mainly for the purposes of accumulation or re-distribution of different types of capital (Bourdieu, 1991). Capital is not only monetary and does not only exist in the economic form (money) (Bourdieu, 1983). 5 There is a third form of social capital: linking social capital. This is the capital derived from the relationship between community and public agency (Zetter et al., 2006) 3.2.1 Language socialisation It also exists as cultural capital (knowledge, skills and other cultural attainments such as from qualifications) and social capital (the amount of resources or potential resources that one could get from the participation or membership of a network or group) (Bourdieu, 1983). Therefore language knowledge and skills can be considered cultural capital to be gained by migrants when interacting within fields. Social capital can also be divided into two main types depending on the relationship between players5. Bonding social capital is the social capital accumulated between close groups, such as families, religious groups, and friends from similar class backgrounds (Office for National Statistics, 2001). Bridging social capital is where there are relationships between people from groups that are different from each other such as differences in cultural groups, socio-economic background and educational background (ONS,2001). Research has shown that both types of social capital are important to help people get on in life, but that bridging social capital has wider benefits 30 to communities because of the accumulated group relationships that can benefit many people (Granovetter, 1973). Fields are not two dimensional spaces but constructed through the perceptions and experiences of players in their fields, and have a set of regulatory practices also commonly known as ‘rules of the game’ (Edgerton & Roberts, 2014). For example, in ESOL classes or workplaces there are practices regulating acceptable behaviour. Successful participation in these fields require individuals to have dispositions or habitual ways of being, that are compatible with those fields. This includes the ingrained knowledge and language of how to behave and to act/react in certain ways (Bourdieu, 1991). These dispositions are termed habitus, and formed from participation in social fields from early life, whereby situations and contexts create long-lasting and transposable ways of being and acting (Bourdieu, 1977). Habitus has also been likened to a person’s identity (Bottero, 2010). Schools are often used to illustrate the interactions between these key concepts (Bourdieu, 1977; Davey, 2009; Farrell, 2010). A commonly used example is that of a child attending school (see for example Morrice (2007) and Reay (1997). When a child is from a good socio-economic background and who has previously attended nursery starts school, they are likely to already possess cultural and social capital that could enable them to do well in the school (field). 3.2.1 Language socialisation Their socialisation into becoming a ‘good’ school pupil (habitus) may be easier and it is likely that their behaviours and actions (practice) are all considered desirable in the schooling system. This situation would be different for a child from a lower socio-economic background, who may not have previously had learning experiences and the associated cultural and social capital that might come with those prior experiences. Socialisation is the process of construction of habitus through the social interactions that take place within a field and the process is influenced by the systems and structure of the field (Davey, 2009). Therefore, part of the socialisation process of migrants who are new to the UK is learning English, including how to interact in English in the different fields in which they participate. The participation of the ‘novice’ in new communicative practices is influenced by a whole range of cultural, social and legal structures and systems (Duff, 2007; Moore, 2008; Ochs & Schieffelin, 2011). Examples of places where a migrant could experience socialisation could be the following: public services (such as with service providers or government services, children’s schools), English classes, at work and or places where you look for work. Additionally, one of the main influencing aspects in SLS is usually the novice’s first language socialisation as 31 this is shaped influenced by the range of linguistic, cultural and discursive knowledges that a person already has (Duff, 2007), which could also be interpreted as their habitus. According to Duff (2007), a key tenet of language socialisation is the role of experts or more proficient speakers of the language, acting as interlocutors of language for the novice, and implicitly or explicitly teaching them to ‘think, feel and act in accordance with the values, ideologies and traditions of the group’ (Duff, 2007, p. 311). Social interactions are key in developing language through creating opportunities for both positive and negative feedback from interlocutors during the ‘process of negotiation for meaning’ (Long, 2014, p. 53). Long’s ‘Interaction Hypothesis’ posits that during communication, when learners experience difficulties in understanding negotiation for meaning takes place, which ‘triggers interactional adjustments made by the…interlocutor’ and this ‘facilitates acquisition because it connects input…and output in productive ways.’ (Long 1996, pp. 451–452) He suggests that the adjustments by the interlocutor’s input, will cause explicit focus on language form by the learner, in order for the learner to adjust their response (output) (Long, 2014). 3.2.1 Language socialisation Experts and interlocutors can be native speakers, non-native speakers but are more competent speakers than those who are learning (Gass & Mackey, 2014). They could also be from learners’ own background and be second language speakers who have themselves gone through a process of socialisation (Atkinson, 2011). According to Duff (2007), a key tenet of language socialisation is the role of experts or more proficient speakers of the language, acting as interlocutors of language for the novice, and implicitly or explicitly teaching them to ‘think, feel and act in accordance with the values, ideologies and traditions of the group’ (Duff, 2007, p. 311). Social interactions are key in developing language through creating opportunities for both positive and negative feedback from interlocutors during the ‘process of negotiation for meaning’ (Long, 2014, p. 53). Long’s ‘Interaction Hypothesis’ posits that during communication, when learners experience difficulties in understanding negotiation for meaning takes place, which ‘triggers interactional adjustments made by the…interlocutor’ and this ‘facilitates acquisition because it connects input…and output in productive ways.’ (Long 1996, pp. 451–452) He suggests that the adjustments by the interlocutor’s input, will cause explicit focus on language form by the learner, in order for the learner to adjust their response (output) (Long, 2014). Experts and interlocutors can be native speakers, non-native speakers but are more competent speakers than those who are learning (Gass & Mackey, 2014). They could also be from learners’ own background and be second language speakers who have themselves gone through a process of socialisation (Atkinson, 2011). According to Duff (2007), a key tenet of language socialisation is the role of experts or more proficient speakers of the language, acting as interlocutors of language for the novice, and implicitly or explicitly teaching them to ‘think, feel and act in accordance with the values, ideologies and traditions of the group’ (Duff, 2007, p. 311). Social interactions are key in developing language through creating opportunities for both positive and negative feedback from interlocutors during the ‘process of negotiation for meaning’ (Long, 2014, p. 53). Long’s ‘Interaction Hypothesis’ posits that during communication, when learners experience difficulties in understanding negotiation for meaning takes place, which ‘triggers interactional adjustments made by the…interlocutor’ and this ‘facilitates acquisition because it connects input…and output in productive ways.’ (Long 1996, pp. 3.2.1 Language socialisation 451–452) He suggests that the adjustments by the interlocutor’s input, will cause explicit focus on language form by the learner, in order for the learner to adjust their response (output) (Long, 2014). Experts and interlocutors can be native speakers, non-native speakers but are more competent speakers than those who are learning (Gass & Mackey, 2014). They could also be from learners’ own background and be second language speakers who have themselves gone through a process of socialisation (Atkinson, 2011). One marked difference between SLS and SCT is that notions of ‘asymmetry of power’ are always acknowledged as part of a novice-expert relationship (Poole, 1992). Research by (Court, 2017; Norton, 1995; Roberts & Cooke, 2009) discussed the unequal power between interlocutors or experts, especially interlocutors outside the classroom environment . Norton’s research participants showed how an interlocutor who is perceived to be non-sympathetic can have a negative effect on a learners confidence, and this is especially so with migrants and refugees who already possess feelings of inferiority (Norton, 1995). Hann’s research of family members acting as interlocutors for ESOL learners showed participants who were more comfortable with speaking English with family members as they felt interactions with family members were more legitimate, and they were more likely to engage in interactions with them (Hann, 2017). These research examples shows that the lack of opportunities for migrants to interact with others, or interaction with people who are not sympathetic interlocutors can have negative effects on their socialisation. However, in SLS there is also a strong emphasis that the traditional view of who is expert and novice is fluid and changeable according to environment, situation and new 32 knowledge to be developed (Ochs & Schieffelin, 2011). For example, a new language learner might be a novice in their new setting in the UK, but they may also be an expert as a parent, an area that their teacher or peers may in turn be novices (Duff, 2011). Therefore, if interpreted in this way, SLS is a democratic approach to teaching and learning second languages as the existing knowledge and experiences of learners are acknowledged. The appeal of SLS for me is that it takes a multi-disciplinary approach and has been researched in cross-cultural societies (Baquedano-Lopez & Mangual Figueroa, 2011). 3.2.1 Language socialisation It is not only a study of SLA in English language, but also one that could be applicable universally as the language acquisition process is part of a larger process of socialisation into new places. 3.2.3 Researching language socialisation As discussed earlier, much of SLA focuses on the acquisition of the new language. Pavlenko and Lantolf (2000) call this the ‘acquistion metaphor’ (p.155), and this is based on work by Sfard on learning theories as metaphors. Sfard’s research presents metaphors as powerful agents of conceptual change (Sfard, 1998). There are two main metaphors to describe the learning process: the acquisition metaphor- or a description of knowledge as something to be acquired by the learner; and the participation metaphor- to describe knowledge as something that a learner participates in or that enables participation (Sfard, 2012). Based on Sfard’s research, Pavlenko and Lantolf presented an alternative lens through which to view second language learning, stating that second language learning is the ‘struggle of concrete socially constructed and always situated beings to participate in the symbolically mediated lifeworld of another culture’ (Pavlenko & Lantolf, 2000, p.155). This metaphor is one of being physically immersed in a new culture and trying to participate within it, using language as one form of connection and communication. The two metaphors for second language acquisition: • the acquisition metaphor, which is interested in the language level elements of SLA such as utterances, pronunciation, grammar rules or codes, and • the participation metaphor, which views learning as a process of ‘becoming a member of a certain community’ (Sfard, 1998, in Pavlenko & Lantolf, 2000, p.155) (Pavlenko & Lantolf, 2000) 33 Previous LS/SLS research design has generally involved research in two ways: The first has an analytical focus on ‘speech, writing and gesture’ (Ochs & Schieffelin, 2011, p.10). The second is in ethnographic studies of ‘shifting socio-culturally meaningful practices, events, situations, institutions, relationships’ (Ochs & Schieffelin, 2011, p.11). LS/SLS generally uses longitudinal study to capture changes in communicative practices over time. A key part of LS/SLS is researching the detailed changes to novices’ communication practices within their evolving social and public spheres, especially recognizing the imbalance of power structures. The method of analyzing semiotic resources was problematic for this research project as the research aim was to study the out-of-class use of language in authentic private and public settings that participants were living. Recording of speech was not possible. Therefore I focused on the participation element, using self-reporting of social interactions, to research the relationship between participants’ social interactions and their perception of their own integration. 3.3 Integration In this section I review literature about definitions of integration and social cohesion, effective models of integration, how integration is perceived by people who are settling in the UK, specifically migrant women, and the role of English language in that process. As presented in the introduction, for this study I used the definition of integration from my earlier research (2015): Integration is the process by migrants who are new to the UK, of becoming part of UK society. This process has multiple responsible parties including host communities and government. Integration is the process by migrants who are new to the UK, of becoming part of UK society. This process has multiple responsible parties including host communities and government. This definition recognizes that there are many players in the integration process responsible for different aspects of integration. This definition recognizes that there are many players in the integration process responsible for different aspects of integration. Integration is a complex and contested concept (European Commission, 2003). A large and varied body of literature exists about the concept depending on whether the context is national (Dorling, 2007b; European Commission, 2003; Han, Starkey, & Green, 2010); historical (Rex, 2016); political (Bosswick & Heckmann, 2006a; Department for Communities and Local Government, 2012; HM Government, 2018; Saggar et al., 2012; Webb, Hodge, Holford, Milana, & Waller, 2016); social (Ager & Strang, 2008; Baquedano-Lopez & Mangual Figueroa, 2011; Morrice, 2007b); or individual (from an identity perspective) (Duff, 2008; Miller, 1999, 2004; Morrice, 2017; Ochs, 1993). Integration is a complex and contested concept (European Commission, 2003). A large and varied body of literature exists about the concept depending on whether the context is national (Dorling, 2007b; European Commission, 2003; Han, Starkey, & Green, 2010); historical (Rex, 2016); political (Bosswick & Heckmann, 2006a; Department for Communities and Local Government, 2012; HM Government, 2018; Saggar et al., 2012; Webb, Hodge, Holford, Milana, & Waller, 2016); social (Ager & Strang, 2008; Baquedano-Lopez & Mangual Figueroa, 2011; Morrice, 2007b); or individual (from an identity perspective) (Duff, 2008; Miller, 1999, 2004; Morrice, 2017; Ochs, 1993). Some definitions define social integration as assimilation, whereby a person is expected to alter their habits and culture, and change their identity to become more like the people in the host community (Bowskill, Lyons, & Coyle, 2007). 3.2.3 Researching language socialisation Previous LS/SLS research design has generally involved research in two ways: The first has an analytical focus on ‘speech, writing and gesture’ (Ochs & Schieffelin, 2011, p.10). The second is in ethnographic studies of ‘shifting socio-culturally meaningful practices, events, situations, institutions, relationships’ (Ochs & Schieffelin, 2011, p.11). 34 34 6 See Appendix 1 for the Common Basic Principles for Immigrant Integration Policy in the EU 3.3 Integration This definition has its origins in the models of acculturation of immigrants in the United States, theorised by researchers in the mid-1900s (Heckmann, 2005). In this definition, integration is viewed as a one-way process, in which migrants change their own culture and take on the culture or the new society (Heckmann, 2005). The responsibility is placed solely on the migrant and there is no obligation by the host community to support or be part of the process (Bowskill et al., 2007; Rudiger & Spencer, 2003). The Labour government in the late 1960s began to adopt a multicultural approach to migration with a strong emphasis on equal rights and opportunity (Rex, 2016), for migrants who had come from post-war New Commonwealth regions such as South Asian, Caribbean and African immigrants (Saggar, Somerville, Ford, & Sobolewska, 35 2012). They enacted early race relations laws to promote multiculturalism, which were based on the rights of citizenship (Rex, 2016). In the early 2000s, responding to the riots in Northern England, the then Labour government developed the concept of ‘shared values’ as part of wider social inclusion policies to support social cohesion (Young, 2003). The government adopted the Common Basic Principles for Immigrant Integration Policy in the EU (Berry, 2011) (see 2.1)6. Principles such as ‘participation’ and ‘equity’ promoted a more inclusive form of integration, whereby each group worked together towards shared values, as opposed to earlier assimilationist ideas (Berry, 2011). Integration was seen as a way to acknowledge and celebrate cultural diversity across EU countries (Berry, 2011). EU and UK policy objectives on integration have largely been in response to the increasing diversity of ethnic and cultural groups in post-modern societies across Europe, and challenges that pluralistic societies present (Zetter, Sigona, Flynn, & Pasha, 2006). One challenge is how pluralistic societies can be organised to reduce differences in opportunities and attainment between people and groups (Heckmann, 2005). Another is how to support groups within communities to ‘get along’ with each other- also termed social cohesion (Saggar et al., 2012). General understandings of social cohesion refer to the common values and purpose of people in society enabling people from different backgrounds to feel a sense of belonging and solidarity with each other (Hope Cheong, Edwards, Goulbourne, & Solomos, 2007). 3.3.1 Integration and social cohesion The terms integration and social cohesion are often used together and interchangeably (Saggar et al., 2012) but there is a difference. Integration is about ensuring migrant groups are included into the existing structures of society and able to participate equally (see for example Ager & Strang, 2008a; Heckmann, 2005; Jansen, Chioncel, & Dekkers, 2006; Saggar et al., 2012). Social cohesion is about how people ‘get along’ with other people in their communities (Cantle, 2007; Hope Cheong et al., 2007; Jansen et al., 2006). As described in the previous chapter (2.3) the UK government initiatives for social cohesion are through promotion of English language and social mixing. The CIC defined a cohesive community as one where there is common vision, similar opportunities, respect for diversity and a sense of belonging (Cantle, 2007). It was seen 36 that ethnic diversity and cultural differences between groups could result in low trust (Hope Cheong, Edwards, Goulbourne, & Solomos, 2007) and so social mixing would promote shared values and morals (Jansen et al., 2006). However, some researchers suggest that cohesion is not only affected by cultural differences as when inequalities increases, ‘everyone becomes more different from everyone else’, leading to less cohesive societies (Dorling, 2007). There appear to be two broad approaches to social cohesion (Phillips, 2003). The first is based on creating shared vision and purpose (Jenson, 2010), through building social capital based on Putnam’s theories of social capital; specifically developing bridging, bonding and linking social capital (Forrest & Kearns, 2001) through social mixing. Social mixing is seen to develop social networks and foster a shared identity (Forrest & Kearns, 2001). This approach is now considered mainstream and generally accepted (Hope Cheong et al., 2007). The second approach is by reducing inequalities of opportunity, poverty and minimizing exclusion (Phillips, 2003). This approach is less favoured as it requires greater state intervention (Phillips, 2003). In the UK, this approach could be seen to be opposite to the recent governments’ policies on state involvement (Institute of Community Cohesion, 2010). The approach of promoting social mixing to build social capital and enable social cohesion can be seen as one where responsibility can be placed on people in communities, specifically migrants in those communities (see for example Casey, 2016; HM Government, 2019b). 3.3.1 Integration and social cohesion Where there are divisions seen in groups living together, there is a risk that migrant groups will be seen to not be doing enough to enable social cohesion (All Party Parliamentary Group on Social Integration, 2017). In many cases ‘diversity’ is presented as being problematic in discourses about social cohesion (Jansen et al., 2006), resulting in negative perceptions of migrants as discussed earlier in the Background chapter (2.2 and 2.3). It was important to highlight the distinction between integration and cohesion as the two terms have been used interchangeably by other researchers and in government documents. Cohesion is also sometimes suggested as an indicator of successful integration (Heckmann, 2005) and has been linked to a sense of belonging (Laurence & Heath, 2008). It is likely that living in a local area where everyone ‘gets along’ with each other will contribute to a person’s perception of their integration. However, based on the earlier discussion, social cohesion is usually seen from a group, community or societal perspective, rather than an individual perspective, which is the focus of this study. 37 Integration and cohesion are related, because if communities are not cohesive, it is likely to affect a migrant’s ability to take part in social mixing and feel belonging in their local community. 7 often nicknamed a two-way street (Bryers et al., 2013) 8 Bosswick& Heckmann (2006a) suggested that acculturation and socialisation mean the same. According to Berry (2014) socialisation only applies to primary socialisation, and secondary socialisation is always acculturation. 7 often nicknamed a two-way street (Bryers et al., 2013) 3.3.2 Dimensions of integrations From the early 2000s the Council of Europe commissioned reports into effective models of integration (Rudiger & Spencer, 2003). The Common Basic Principles presented in 2.1 are an example of policies based on commissioned research reports (Niessen & Huddleston, 2009). Models of successful integration proposed integration as a two-way process7 whereby legal and political rights are conferred to new migrants (European Commission, 2003), there is meaningful interaction between migrants and host communities, and migrants have access to jobs and other opportunities (Saggar et al., 2012). Berry and Heckman describe the process of integration as one where there is mutual change on both the part of the migrant and the host community as a by- product of the process of socialisation or acculturation8 (Berry, 2011; Heckmann, 2005). At the individual level, integration is dependent on the motivations and goals of a person, and can ‘mean different things to different people at different times’ (Bryers, Winstanley, & Cooke, 2013, p.6). Ager and Strang, in research commissioned by the Home Office, suggested that an individual could be considered integrated when they: ● achieve public outcomes within employment, housing, education, health etc., which are equivalent to those achieved within the wider host communities; ● achieve public outcomes within employment, housing, education, health etc., which are equivalent to those achieved within the wider host communities; ● are socially connected with members of a (national, ethnic, cultural, religious or other) community with which they identify, with members of other communities and with relevant services and functions of the state; and ● have sufficient linguistic competence and cultural knowledge, and a sufficient sense of security and stability, to confidently engage in that society in a manner consistent with shared notions of nationhood and citizenship.’ (Ager & Strang, 2004, p.5) 8 Bosswick& Heckmann (2006a) suggested that acculturation and socialisation mean the same. According to Berry (2014) socialisation only applies to primary socialisation, and secondary socialisation is always acculturation. 38 This suggests that the ability to integrate is highly dependent on migrants being able to access structures and systems and participate equally to others. This could include being awarded rights to access structures such as housing and education, language support so that migrants can participate in employment, and ensuring equal opportunities to access and participate through anti-discrimination laws. 3.3.2 Dimensions of integrations For Lockwood, there were two parts to the integration process: system integration, related to institutions and systems, and social integration, which is enacted by individual actors (Lockwood, 1976). System integration is considered foundational to the ability to fully integrate, as it includes legal aspects such as rights and laws to allow a person to settle in the UK, institutional policy governing organisations, and organization of markets including mechanisms for payment/working between these (Esser, 2001). In contrast, social integration is ‘the inclusion (or exclusion) of actors into a social system…and following on from this the equal or unequal distribution of characteristics among…categories of actors’ (Esser, 2006, p.8). Within social integration there are four dimensions of integration, developed from Esser’s (2000) work: placement, acculturation, interactions and identification (Esser, 2000). These were later further developed and adapted by Bosswick & Heckmann (2006) as below: • Structural integration- rights to access core institutions, • Cultural integration- the mutual development of a new shared culture, • Interactive integration- inclusion into the host community’s social networks, • Identificational integration- when a migrant person identifies that they belong in the new society. However, Esser’s original version of structural integration, or which he called ‘placement’, referred to an individual’s ability to gain a place within core institutions (e.g. housing, health systems and labour market) (Esser, 2000) rather than the rights to access them (as per Bosswick and Heckmann 2006). The idea of ‘placement’ as a way of understanding structural integration creates a distinction between the two. System integration is awarding of rights, legal process and government policy, which are pre- conditions for integration (Bosswick & Heckmann, 2006). Whereas social integration is the ability to exercise those rights (Esser, 2000). An example of the distinction is the government stating that only people on certain visas are allowed to study at a college (system integration). Social integration would be the ability for ‘eligible’ people to access the courses at the college. 39 Esser also proposed that capital (e.g. social and cultural) affects the ability to access the dimension of structural integration as part of social integration (Esser, 2006, p.8). Participation and continual participation in the dimension (through activities such as work, attending education and visiting health services) is affected by the social, cultural and economic capital that a person has. 3.3.2 Dimensions of integrations For my study, I selected Esser’s conceptualisation of integration (Esser, 2001, p.16) for two reasons: 1) I could see that from the perspectives of women in my earlier research, Esser’s framework better explained their personal construction of integration. 2) As I was focusing on social integration from an individual perspective, it was more helpful use Esser’s ‘placement integration’ as a way of understanding the dimension of structural integration (Esser, 2001, p9-10). Therefore, in this study the four dimensions of integration are understood as the following: Therefore, in this study the four dimensions of integration are understood as the following: • Structural integration- being able to gain a place within core institutions. For newly arrived women to the UK, this involves getting support to be able to access services that they have the right to access such as housing, and health through their council and local healthcare providers. Either through English classes that are contextualized, through citizenship lessons about citizens’ rights, or peer sharing/support. • Cultural integration or acculturation- involves migrants gaining knowledge, behaviours and competencies to participate in the host society (Heckmann, 2005). However the process is mutual and the host community also changes (Bosswick & Heckmann, 2006). Learning the host country’s language, English in this case, usually sits within cultural integration. Other examples are supporting new migrants to learn about the places of cultural and historical interest, such as museums and historical buildings and to share cultural knowledge such as ‘British values’. It can also involve creating opportunities for settled members of the community to come together with new migrants to foster community relations through shared understandings. • Interactive integration- concerns migrants’ inclusion into the host community’s social networks. For new migrants to the UK this continues to be a barrier to integration (Dimitriadou, 2004; Eaves, 2015; Norris, 2004; Ortaçtepe, 2013). Recent reports cited in 2.2 propose social mixing. Examples at a local level are events and activities, and participating in volunteering. Recent reports cited in 2.2 propose social mixing. Examples at a local level are events and activities, and participating in volunteering. 40 • Identificational integration is when a migrant person identifies that they belong in the new society (Nimmerfeldt, 2009). 3.3.2 Dimensions of integrations This dimension is seen as the final stage, when a person feels a sense of belonging to a new community, the stage where a migrant’s has formed a new identity with a part to play in their life in the UK (Bosswick & Heckmann, 2006). Cultural knowledge could be both a precursor to, and a result of structural integration, and vice versa (Esser, 2006). At the same time, in these dimensions (cultural and structural), interactive integration could be taking place (Esser, 2006). A combination of these dimensions are necessary to develop identificational integration (Bosswick & Heckmann, 2006), which is often described as the feeling of ‘belonging’. 3.3.3 Research on measurements and indicators of integration There have been several research reports about measuring integration (see for example Ager & Strang, 2004; Amit & Bar-Lev, 2014; Entzinger & Biezeveld, 2005; European Commission, 2003; Jenson, 2010; Saggar et al., 2012). Saggar et al (2012) used empirical research of social impact of integration; Amit and Bar-Lev (2014) used life satisfaction as a measurement; Eaves (2013) involved migrants themselves to select the goals they set themselves and to measure those in longitudinal research; Entzinger and Biezeveld (2005) and Ager and Strang (2004) proposed indicators of integration based on migrants’ participation and outcomes in certain domains. Recently the Home Office have adopted Ager and Strang’s framework of indicators of integration (Ndofor-Tah et al., 2019). The framework was produced as part of a research report for the Home Office and used the four dimensions of integration in section 3.3.2 as the starting point (Ager & Strang, 2004, p.9). Ager and Strang’s framework has 10 domains of integration grouped under four main headings: foundation, facilitators, social connections, and markers and means. Education, work, health and housing are domains that are key areas for participation for people in communities. They are considered markers as they give an indication of success in achieving participation, but are also considered means as participation in these domains can also support integration (Ager & Strang, 2004). The framework included a list of indicators that could be used to gather data for evaluation and planning of refugee services, for example % in paid work under the employment domain (Ager & Strang, 2004). In the suggested indicators, Ager and Strang listed potential sources such as Home Office Citizenship Survey, Health Authority reports, Labour Force Survey, Department for Work and Pensions (DWP) records (Ager & Strang, 2004). The framework provides a set of common indicators for 41 funders and organisations working with refugee services. I had considered outcomes- based measurements early on in my research journey but recognized that they were not compatible with my research strategy. However, the domains that were identified by Ager and Strang as key areas of participation were useful to my study. The domains identified in the research: Education, Housing, Work, Health were used to anticipate places that migrant women participated in social interactions within. 3.3.4 Recent research about participants’ own perceptions of integration As the focus of my study is migrants’ perceptions of their own integration, I looked at previous research involving the perspectives of migrants themselves. There were four recent studies looking at migrants’ perceptions of what integration meant to them (Bryers et al., 2013; Court, 2017; Eaves, 2015; Graham-Brown, 2015). Based on these studies, I suggest that ‘being integrated’ is complex, non-linear and may have multiple pathways within the overall process. Participants of these studies reported that integration was influenced by their ability to fully access services, make relationships with people outside of their immediate family, ethnic and community circles, and gain independence through employment (Bryers et al., 2013; Eaves, 2015; Graham-Brown, 2015). The dimensions mentioned earlier- structural integration, cultural integration and interactional integration- could be used to explain the self-reported areas. Additionally, participants for two of the studies highlighted the role of UK immigration policy and the power that the UK government had over allowing people permission to integrate through awarding residency status and rights to political participation (Eaves, 2015; Graham-Brown, 2015). Participants in Bryers et al’s research identified barriers such as racism encountered in the communities they were settling in, which they viewed as influenced by wider UK immigration policy (Bryers et al., 2013). I saw these perspectives as belonging to Esser’s system integration (Esser, 2000) (in earlier section 3.3.2), which is a dimension outside of the control of migrants themselves. Time was also identified as an important part of the integration process, with some migrants taking longer to feel integrated than others (Eaves, 2015). Therefore, if the process of integration is viewed on a continuum, then it is reasonable to expect people to be ‘integrated’ in different areas of their lives and at different levels simply because of differing personal situations and length of time in those contexts. A person can perceive themselves to be well integrated in one area of their life, such as at their workplace, and not in another area, such as in their local neighbourhood, where they might spend less time (Graham-Brown, 2015). 42 Bryers et al’s (2013) also re-conceptualised and further extended the existing integration metaphor. Previously, integration has been likened to a two-way street by researchers and policy makers, with migrants and the host community having a role to play (Strang & Ager, 2010). Through participatory activities in their study, Bryers et al observed integration as a ‘spaghetti-junction’ of opportunities and responsibilities. 3.4 Belonging So far, I have discussed theories of integration, establishing that structural, cultural and interactional integration are related to identificational integration. Next, I examine belonging as an indicator of identificational integration, and propose a conceptual framework to link social integration, SLS and belonging. 3.3.4 Recent research about participants’ own perceptions of integration ‘I.e. a complicated, dense set of intersections, crossroads and junctions going in lots of different directions.’ (Bryers et al., 2013, p.33). Participants felt that integration was not static but a ‘series of transitory moments of belonging’ (Bryers et al., 2013, p.26). Participants of the Eaves study also used the term belonging, expressing that belonging was to feel that they were comfortable and at ease, and to be accepted and welcomed whilst retaining their own cultural identities (Eaves, 2015). Similarly, in early findings on my IFS research, participants had expressed that to be integrated was to feel comfortable, safe and ‘welcomed in their new home’ (Graham-Brown, 2015, p.57). 43 3.4.1 Belonging as a way of conceptualising ‘being integrated’ Identificational integration is related to a new migrant’s feelings of belonging (Bosswick & Heckmann, 2006). As discussed earlier in this section, the term belonging has often been used to describe when someone feels integrated. Belonging has been identified as an indicator of integration (Ager & Strang, 2004) and as a way of demonstrating the feeling of being integrated (Heckmann, 2005). Belonging is defined as a feeling of being at ease with oneself and one’s surrounding (Miller, 2009). In my review of literature on integration and cohesion, I found many references to belonging, but without any accompanying definition (see for example Ager & Strang, 2008a; Department for Communities and Local Government, 2012; Forrest & Kearns, 2001; Saggar et al., 2012). There seemed to be an assumption of a shared understanding of what belonging is vis-à-vis integration and cohesion. Some writers suggest belonging is related to acquisition of rights through being a citizen of a nation-state (Strang & Ager, 2010), and contingent on whether migrants feel included or excluded (Morrice, 2011). I was unable to find sufficient explanation of the relationship between integration and belonging. Outside of integration, there appear to be two main research fields where the concept of ‘belonging’ has been researched. The first is within Lave and Wenger’s community- of-practice (Wenger, 2010). This type of ‘belonging’ refers to professional groupings (Ionescu, 2016; Whitchurch, 2009). I explored using this understanding of belonging as it has been applied to groups studying in adult education and ESOL previously (Duff, 2007; Ibarz & Webb, 2007; Norton & Toohey, 2001). The premise is that by studying together in a classroom setting, participants feel that they belong to a community of practice and this supports the development of their skills and confidence through non- formal and informal learning (Duff, 2007). However, when assessing suitability for my study, I felt that this understanding would not be suitable to support the exploration of belonging in relation to all the dimensions of integration, as it focused on classroom- based interactions and relationships. Another type of belonging is related to identity studies (Conde, 2011; Krzyzanowski & Wodak, 2008; Said, 2018; Stets & Burke, 2000). This type of belonging has been 44 researched with reference to nationhood and transnationalism (Baquedano-Lopez & Mangual Figueroa, 2011; Erdal & Oeppen, 2013) and in relation to politics of belonging (Morrice, 2017; Sandu, 2013; Sicakkan & Lithman, 2005; Youkhana, 2015; Yuval- Davis, 2006). 9 See May, 2013 and Sicakkan& Lithman, 2005 for several other reasons for using belonging instead of identity. 3.4.1 Belonging as a way of conceptualising ‘being integrated’ Again, I considered these concepts but I felt that neither concept was suitable to adequately explain the ‘feeling’ of belonging in relation to participants’ social interactions in the community where those interactions took place. Therefore I selected place belonging, which is an understanding of belonging as an emotional attachment (Antonsich, 2010). This type of belonging exists across academic disciplines, from geographers, sociologists, to cultural theorists and educators (see Antonsich, 2010, for a review of literature on belonging across disciplines). This concept of belonging describes belonging as a sense of ease with ‘one’s self and one’s social, cultural, relational and material contexts’ (May, 2013, p.3), and often unnoticed until there is change (May, 2013). It is also related to feeling comfortable, familiar and having confidence in the place (Yarker, 2019). Belonging is often described as a human need (Baumeister & Leary, 1995) and this particular concept is built upon the basis of relational self: that our identities and sense of self is formed based on our relationships with others (May, 2013). As it is intrinsically linked to individuals’ identity, but rooted in context and place (May, 2013), I favoured using this explanation of belonging as a way of explaining migrant women’s identity, as belonging allows identity to be explained as a transitory process of being and becoming (Yuval-Davis, 2006)9. Nevertheless, some findings in 5.3.2 suggest that ‘the politics of belonging’ could be used as a lens for a future study into experiences of women on their pathways to integration. In this research, I will be using the term belonging to mean belonging as an emotional attachment to place also known as place belonging (Antonsich, 2010). The following is an explanation of the significance of the concepts of social spaces, places and integration. 3.4.3 Definition of social interactions Social interactions are interactions that take place in the social space, rather than interactions with content that is social in the informal and friendship sense. This is based on the understanding of social interactions of Sandstrom & Dunn's (2014) study, Ochs' (1991) discussions of language practices for novice learners, and Sharma (1997)’s theory of social process. Sharma suggests that ‘social interaction is communicative interaction, and that interaction is the simplest form of social process’ (Sharma, 1997, p.162). Therefore all interactions between participants and other people will be considered social interactions for this research project, regardless if the interactions are purely transactional or include more meaningful transfer of knowledge. 3.4.2 Social fields and socialisation The process of socialisation is not static and can be seen as a continuous process of socialisation through and into different Habitus is helpful in conceptualizing an individual’s relationship between self and society. The theory has been criticized for social reproduction of inequalities (see Bottero, 2010; Edgerton & Roberts, 2014; Nash, 1999). In order to overcome this, May suggested using belonging as a way of further extending habitus (May, 2013). Belonging is a relational concept with a focus on social interactions to enable access to, and changing of, social fields and habitus. The process of socialisation is not static and can be seen as a continuous process of socialisation through and into different fields (Davey, 2009), including unfamiliar fields in the case of migration to a new place. Therefore the process of socialisation could be seen as the integration process in order to become socialized into a community, that is to feel identificational integration or a sense of belonging. 3.4.2 Social fields and socialisation Belonging suggests that we will feel at ease in a familiar social field (Bourdieu, 2000) and we will understand how to behave and ‘play the game’ to retain or accumulate capital. Habitus is largely unseen and comes across as natural or second nature (May, 45 2013). However, we will not feel comfortable if we are not familiar with a place (Yarker, 2019) or do not know how systems work (May, 2011). Integration at the local level is based on the successful inclusion of migrants into existing neighbourhoods and communities (Heckmann, 2005), which may be new and unfamiliar. Migration from one set of familiar social fields to a new place with unfamiliar social fields will affect one’s feeling of belonging (Marshall & Foster, 2002). In order to feel at ease again, a person will have to become socialized into the fields of the new society. Part of this socializing is learning to use the new language, through the use of the new language in the field (SLS, section 3.2.1). Habitus is helpful in conceptualizing an individual’s relationship between self and society. The theory has been criticized for social reproduction of inequalities (see Bottero, 2010; Edgerton & Roberts, 2014; Nash, 1999). In order to overcome this, May suggested using belonging as a way of further extending habitus (May, 2013). Belonging is a relational concept with a focus on social interactions to enable access to, and changing of, social fields and habitus. The process of socialisation is not static and can be seen as a continuous process of socialisation through and into different fields (Davey, 2009), including unfamiliar fields in the case of migration to a new place. Therefore the process of socialisation could be seen as the integration process in order to become socialized into a community, that is to feel identificational integration or a sense of belonging. Habitus is helpful in conceptualizing an individual’s relationship between self and society. The theory has been criticized for social reproduction of inequalities (see Bottero, 2010; Edgerton & Roberts, 2014; Nash, 1999). In order to overcome this, May suggested using belonging as a way of further extending habitus (May, 2013). Belonging is a relational concept with a focus on social interactions to enable access to, and changing of, social fields and habitus. 3.4.4 Places of interactions Places that migrant women’s social interactions take place in are a representation of the social fields that they are integrating into. Therefore, it is important to clarify what is meant by a ‘field’ and to provide terminology for the research. 46 As described earlier in section 3.2.1, a field is not a two-dimensional space, rather a constructed representation of a social space that a person encounters, lives-in or interacts with. The study of spaces has usually been of interest to geographers (Massey, 1994). Recently, a greater understanding of how material objects can influence social spaces, social relationships and metaphorical aspects of space has meant that more consideration has been given to the physical aspects of place in studies of identity and belonging (Antonsich, 2010). The terms ‘out of place’ and ‘know your place’ are both indicative of how both the physical and social space can make someone feel. Whereas space might describe the physical area or location that structures, enables or constrains activities and interaction, place describes the social meanings that those interactions give to a space (Dourish, 2006). Therefore for this research I use the term ‘place’ to describe the social fields that participants interact with and in. Place does not mean a specific physical building or institution, for example the ESOL classroom in ELATT’s centre, but represents the socially constructed place of activity and interaction. The ESOL class, which may be within or outside of the physical room location, and consists of the students and teachers who form that class, along with behaviours, conventions and understandings that create boundaries for that particular place. Place can be both imagined and embodied simultaneously (Bennett, 2014). In my analysis and findings chapter, I have described each place that the research participants interact in based on their perceptions to support the reader to have an understanding of the places. Spheres The term sphere represents a loose general grouping of places, which have similar characteristics and activities. Habermas first used the concept of spheres when describing ‘public sphere’, which is an imaginary community: groups of people coming together to discuss issues of common concern (Fraser, 1962). Similar to places, spheres are not physical divisions but theoretical concepts (Martin, 2010) on a more macro-level (Calhoun, 2010). The term sphere has been more commonly used to describe the division between the public and private sphere (Biesta, 2012; Martin, 2010; Rudiger & Spencer, 2003) but sphere has also been used to describe other theoretical concepts: e.g. social sphere, political sphere, cultural sphere, academic sphere and economic sphere (Reay, 1997; Weedon, 2011; Whitchurch, 2012). The concept of spheres have been linked to Bourdieu’s concept of fields through the interpretation of the public sphere as a site of political struggle for power (Conde, 2011). Calhoun, (2010) suggests that fields can be nested within spheres, fields being 47 more specific, and spheres more general. In this study I have grouped the specific places of social interactions within more general spheres. The basis for these groupings is discussed in 4.6.4 (Analysis) and 6.2 (Findings). more specific, and spheres more general. In this study I have grouped the specific places of social interactions within more general spheres. The basis for these groupings is discussed in 4.6.4 (Analysis) and 6.2 (Findings). more specific, and spheres more general. In this study I have grouped the specific places of social interactions within more general spheres. The basis for these groupings is discussed in 4.6.4 (Analysis) and 6.2 (Findings). 48 3.5 Aspects of belonging Antonsich (2010) identified five factors that contribute to belonging. These are autobiographical, relational, cultural, economic and legal (Antonisch, 2010, p.647). May identified three key sources of belonging: cultural belonging, relational belonging and material belonging (which comprises place and objects) (May, 2013). In a later paper she described the temporal aspects of belonging (May & Muir, 2015). Other writers look at belonging from a variety of legal, political, social, cultural, physical, spiritual, ethical and emotional dimensions (Buonfino & Thomson, 2007; Sicakkan & Lithman, 2005; Sumsion & Wong, 2011). For this study, I used May’s categorisation of sources of belonging as they complement the dimensions of integration (see Figure 1 below). Additionally, the importance of time and life histories has featured in the prior research discussed in 3.3.4. I used the term ‘aspects’ (Antonsich, 2010) rather than ‘sources’ (May, 2013) to describe the different dimensions of belonging. All aspects are equally important and interlinked. The aspects are not linear and a person could experience one aspect of belonging and not another (May, 2013). Additionally, they could experience belonging differently across relationships and places (Hedetoft, 2002). However, in order for a person to feel that they fully belong to a place all aspects would have to be present (May, 2013). Previously, my challenge was to operationalize the dimensions of integration, whereby I could gather meaningful data of participants’ experiences. Using May’s aspects of belonging I was able to map the dimensions of integration together with the aspects of belonging to identify experiences of belonging in these four aspects. If identificational integration is when an individual feels they fully belong (3.3.2), the aspects of belonging can be conceptualised as pre-conditions to achieving identificational integration. 49 Figure 1 below are the dimensions of integration mapped to aspects of belonging: Identificational integration: when a migrant person identifies that they belong to the new place Dimension of integration Aspect of belonging Structural integration Material belonging Cultural integration Cultural belonging Interactional integration Relational belonging Figure 1:Dimensions of integration vs aspects of belonging I discuss these aspects of belonging in the following section I discuss these aspects of belonging in the following section. Access to places Belonging is described as ‘comprising place and material objects’ (May, 2013, p.148) and includes the familiarity that we feel in our surroundings, the sense of safety in our communities, how comfortable we are in our homes and public spaces, and is even influenced by our material belongings (i.e.things) (May, 2013). Place is affected by our senses including smell, sight, sound and even taste (May, 2013). Esser described placement as the rights ability to access a place and positions within society, including offer of opportunities (for example a place on a course or a job) (Esser, 2001). This aspect is the most important condition for giving access to forms of capital such as economic and cultural capital (Esser, 2001) as it is linked to the habitus of a person. Both these understandings can affect how we perceive a place and whether we are comfortable (at ease) with a particular place, and also whether we feel accepted or not in the place. Additionally access to place will affect development of cultural knowledge within the place and through interactions with people in the place (Esser, 2001). This is similar to Bourdieu’s theory of habitus: developing cultural capital through socialisation in a field (Davey, 2009). Both Esser and May contribute different understandings of this aspect. May’s definition focuses more on sensory elements of material belonging, and how these can affect the way a person feels about access and opportunity to participate in a place (2013). As an example we can consider a community centre which claims to be inclusive, but has not been built to allow easy access by wheelchair users. Physical elements such as placement of light switches, sinks and table heights are perceived as sensory and affect how the wheelchair user might experience the place as welcoming, and their feeling of acceptance there. Esser focuses on the opportunity for people to exercise the rights to access and participate in a place, or equality of opportunity for access. For 50 example, council services ensuring that there are adequately accessible processes for people to apply for support that is due to them. Based on my knowledge of our participant groups and indicators of integration’s markers and means (3.3.3), it was expected that participants would be more likely to report their experiences of accessing housing, education, work and health and social care. Access to places Access to the places related to housing, education, work and health and social care, is also affected by knowing the ‘rules of the game’. May suggests that ‘collective understandings’ of belonging are built upon ‘negotiated accomplishments’ (2013, p.82) and that the feeling of belonging must be reciprocated (May 2013). However, reports have found that migrants have particular vulnerabilities when accessing places of public services (Kofman, Lukes, D’Angelo, & Montagna, 2009; Policy and Insight Team, 2017). The term public services has been used in this research as a catchall term to describe local authority services including schools, health and medical services, and DWP services. For the purposes of this study, public services staff are customer services staff, caseworkers, social workers, job centre advisors and housing officers. It also includes staff at schools, hospitals and other health services. The government’s audit into racism experienced in public services also showed that people of BAME (Black, Asian or Minority Ethnic) backgrounds were more likely to experience discrimination when accessing public services (Kofman et al., 2009). Incidents of anti-migrant rhetoric, xenophobia and racism, when accessing public services have been reported by migrants (Kofman et al., 2009). There have been recommendations to better support positive interactions in public services settings (Orton, 2012). Research by a London local authority found that discrimination experienced could be due to a lack of migrants’ language skills, the design of systems and the complexity of legal rights and welfare entitlements. Additionally discrimination could also be due to the lack of knowledge and awareness of staff working in public service institutions about migrants’ needs (Policy and Insight Team, 2017). The report recommended training for staff to raise awareness of issues relating to migrant communities (Policy and Insight Team, 2017). Based on previous research, I suggested that engagement in interactions in places that were formal could create opportunities for informal interactions, and I suggested that schools and colleges were examples of these formal places (Graham-Brown, 2015). For many migrants, formal places provide the main opportunity to can gain access to 51 acquaintances and support networks outside of their own ethnic groups (Court, 2017; Orton, 2012). People in these places can support migrants to feel an embodied sense of belonging (May, 2013). Work was identified as one of the places of social interactions earlier in this section. Access to places There are similarities in access to and potential for discrimination in this place as those discussed about public services earlier. Work is also a place where informal social interactions can take place, and relationships can develop between different colleagues. Lave and Wenger’s Communities of Practice concept discussed in 3.4.1 theorises the relationships between professionals with reference to professional development at work (Wenger, 2010) and these are built on the opportunity for meaningful social interactions at work. Work also provides another element that can develop belonging, which is routine. Routine has been described as mundane, draining and repressive (May, 2013). However, routine is also important to providing stability and surety to lives, and providing confidence in what will happen next (May, 2013). This is especially true for migrant women who have moved and experienced uncertainty (Eaves, 2015). In Eaves’ study a participant equated having a job and daily routine with belonging: ‘Maybe in time once I have those things here I might feel belongingness to the UK…’ (Eaves, 2015, p.85). The purpose and self-worth that work provides can be an important source of belonging. Cultural belonging and migrants Culture is usually explained as the particular and distinctive ‘ways of life’ of a group; and social relations, mores, customs and beliefs all make up the meaning and values of the culture (Clarke, Hall, Jefferson, & Roberts, 2018). They are the cultural practices that enable people to socialise into social fields (May, 2013). Language has a crucial role in the process of socialisation to a new culture, enabling the interpretation of the new social culture and learn new cultural practices (Poole, 1992). English and knowledge of UK history is usually listed under cultural knowledge that a migrant should learn to facilitate the integration process (See section 2.3). ESOL classes are places where cultural knowledge is learnt through the language learning 52 activities (Dimitriadou, 2010), but also in the interactions that take place between group members (Court, 2017). It is common for migrants to be treated as a homogenous group of people, trying to integrate with the host community group, which is also often assumed to be a homogenous group (Cantle & Kaufmann, 2016; Stone, 2017). The ‘two-way street’ metaphor reinforces binary assumptions of migrants versus host community. As a teacher, I am often reminded that for the migrant women I have taught, the UK is the first site of hyper-diversity for them (as mentioned in Introduction). They are not only learning how to integrate into the ‘host community’, but also learning how to interact with other migrants who come from countries and cities different from them, which could also be homogenous. The ESOL classroom supports people to learn how to interact with each other through learning about each other’s differences and similarities (for example in ‘Getting to Know You’ activities), norms and behaviours (classroom rules) and learning about linguistic diversity (listening to and understanding each other’s accents). The expectations and knowledge about how to behave is learnt through interactions and relationships with others (May, 2013). Culture is also largely unseen (Schein, 2012), and the culture of a group is only revealed when compared to behaviours, norms and attitudes of other groups (Fortman & Giles, 2005). However, in creating awareness of the cultural differences between groups, there is a risk of not being accepted by the group that is considered the dominant culture (Fortman & Giles, 2005). Cultural belonging and migrants For example, people from migrant groups may not participate in cultural practices of the ‘host community’ such as meeting for a drink in the pub. By being seen not to participate in what could be considered a cultural norm, they could be seen as different. Trust can be affected by perceived differences including cultural difference (Gambetta, 1988). However, culture is more complex than just one culture compared to another as there is no longer one dominant culture in the UK (Weedon, 2004). It is more likely that there are host or settled ‘communities’, which may consist of multiple cultural identities. As a result of ‘transnational mobility’, people are likely to have multiple belongings or form a hybrid cultural identity (May, 2013). Culture cannot be separated from communication as culture is expressed through communication (Fortman & Giles, 2005) and, through interactions with others from different cultures such as people from migrant communities, who already have their own cultural identity, new cultural understandings can be developed (May, 2013). 53 Cultural belonging and systems Culture can also be viewed as the way of structuring society (Baldwin, Faulkner, & Hecht, 2005). The phrase ‘the way we do things around here’ is often used to describe organisational culture (Bower, 1966) and much of the knowledge and practice from corporate culture has been used in organisational design of public services (see for example Gleeson & Knights, 2006; Shaw & Reyes, 2010). As part of cultural belonging, migrant women have to familiarise themselves with how organisations such as those providing services mentioned under material belonging (3.5.1) operate. For example housing services, doctor’s (GP) surgeries and hospitals, education providers and workplaces. These organisations are characteristically bureaucratic (Reimer, 2004) as they are based on ‘relations structured by general rules and principles’ (p.91), and can exclude individuals and groups due to the organisational structures and objectives of the service (Reimer, 2004). Additionally, organisational culture is likely to be influenced by the perceptions and values of the employees themselves. I suggest it is likely that any public perception of migrants (as discussed in 2.2) will have an impact an institution’s organisational culture. Organisational culture as described above also applies to non-profit organisations, businesses and sole traders. Organisational cultural practices can dictate how an employer recruits and selects staff, and manages resources on a daily basis (Schein, 2012). Cultural practices in work settings can differ from experiences from migrant women’s home countries (Eaves, 2015). Employability programmes in colleges or adult learning providers often present employment practices that are by-the-book, and focus on CV-writing and job applications (Department for Education and Skills, 2001). Employability programmes may be designed on the assumption that learners will gain work at larger organisations with standardised employment practices. However research shows that migrant women tend to go into entry-level employment through recruitment practices that may not be based on a formal application process (Eaves, 2015). Migrant women report a lack of confidence to find places to apply for and successfully gain employment and this can negatively affect their cultural belonging in this sphere. Additionally, cultural practices in entry-level jobs, which are of a casual nature, may not be fair to migrants and could negatively affect cultural belonging. A Demos report (Paget & Stevenson, 2014) found that migrants lacking in English skills were at a higher risk of exploitation by employers who did not provide contracts of employment and information about rights at work. 54 Language- not just part of Cultural Belonging Language is usually associated with the cultural dimension of integration (Bosswick & Heckmann, 2006). In frameworks of belonging, language is also often placed within the cultural aspect of belonging (May, 2012). However, I feel that this is not an accurate representation of how language underpins, and is also the key process in, accessing the other dimensions of integration. Esser (2006) linked language and structural integration for access to rights and services; language with interactional integration as crucial in supporting communication to build relationships with the host community; and language learning being a part of cultural integration, as a way of conveying knowledge about the host country. Based on this I suggest that the ability to understand and communicate well enough in the host country language underpins the acquisition of knowledge for cultural belonging. A common language is a ‘key tool for building trust’ (Ipsos Mori, 2007, p.82), and affects the ability to interact to create relational belonging. Language skills also determines the ability to access material belonging through being able to understand and act on rights. Therefore the role of language in a migrant’s process of integration can be understood as wider than simply communicative competency. Language forms the basis of learning about new culture, rights and responsibilities; the method of interacting with others to form social relationships; and it is the key to improving their personal situation in accessing services, or employment. 3.5.3 Relational Belonging Therefore, when they arrive in a new country, migrants are more likely to trust people who are from the same ethnic background as they are and so will build friendships with those people. However, literature suggests that the fundamental reason for migrants seeking out other people like them is trust (Morrice, 2007). At its most basic, trust is ‘a particular expectation we have with regard to the likely behaviour of others’ (Gambetta, 1988, p.216). This expectation leads people of similar ethnic backgrounds and/or experiences to perceive trust in those they share a social identity with (Martin & Tom, 2005). Therefore, when they arrive in a new country, migrants are more likely to trust people who are from the same ethnic background as they are and so will build friendships with those people. As discussed in 3.31, social capital is developed with the people we have strong ties to through bonding social capital (Office for National Statistics, 2001). Studies have shown that bonding social capital can be high amongst ethnic groups because of the immediate resources that the form of capital can provide such as information, emotional support and capacity-building support (Strang & Ager, 2010). However, there is a risk of over-reliance on homogenous groups, at the expense of integration into wider social networks (Morrice, 2007). This has caused bonding social capital to be perceived as ‘bad’ (Hope Cheong et al., 2007, p.31), and for migrants to be perceived as self-segregating and not wanting to integrate (see for example Han et al., 2010; Hope Cheong et al., 2007; Strang & Ager, 2010). However, it is generally accepted that these forms of support are crucial to helping migrants progress in the future (Collyer, Morrice, Tip, Brown, & Odermatt, 2018). 3.5.3 Relational Belonging Relational belonging is about relationships with others. Who we feel we belong (or not belong) with affects relational belonging (May, 2013). The first source of belonging is our family (May, 2013). Friends and other people in our day-to-day community also affects feelings of belonging, though not all relationships affect belonging equally (Baumeister & Leary, 1995). I also attribute Bourdieu’s social capital theories to this aspect. Being part of a group creates social capital that is a strong base for solidarity (Siisiäinen, 2000) and perceived belonging is reliant on concepts discussed in 3.3.1 such as bonding, bridging and linking in establishing trust in relationships (Strang & Ager, 2010). As mentioned, family is usually the first place people feel belonging as they are the primary source of meaning in a person’s life (Stillman & Baumeister, 2009). Socialisation theory suggests that our socialisation through our family develops our cultural understandings of the world (May, 2013), as well as an expectation of how other people who are similar to us will behave (Tanis & Postmes, 2005). This means 55 that as we grow up, we have an expectation of how people of similar backgrounds and/or experiences as us will behave, think and react to us. Friends are also important to a person’s sense of belonging and can sometimes represent the family that one has chosen (May, 2013). Friendships are generally built on similarities rather than differences, such as similarities in class, ethnicity or interests (May, 2013). We are more likely to feel we have more in common with people like us, as our habitus will be similar resulting from the same background, upbringing and experiences (early social fields) (Bourdieu, 2000). Migrants often link with people from their own background when they first arrive in the UK (Hope Cheong et al., 2007). The main reason for this is the perception of greater understanding of their experiences of migration and the likelihood of migrants receiving support from others like them (chain migration) (Morrice, 2007, p.166). However, literature suggests that the fundamental reason for migrants seeking out other people like them is trust (Morrice, 2007). At its most basic, trust is ‘a particular expectation we have with regard to the likely behaviour of others’ (Gambetta, 1988, p.216). This expectation leads people of similar ethnic backgrounds and/or experiences to perceive trust in those they share a social identity with (Martin & Tom, 2005). Acquaintances, strangers and communities Government reports promote social mixing and ‘meaningful’ social interactions (HM Government, 2018; Ipsos Mori, 2007; Social Integration Commision, 2015). There is an 56 assumption that relationships with people we know well are more important to our sense of belonging compared to relationships with acquaintances and strangers (May, 2013). I had previously placed greater importance on social interactions that I coded ‘meaningful’ and I think that this stems from my teaching practice. As ESOL teachers, we are always encouraging our students to practice English with neighbours and friends. This is based on the belief that language interactions in shops or with public transport workers, which are of a transactional nature, would not give sufficient practice to our students to develop their language. However, social capital theory suggests that we should not only focus on strong ties (Putnam, 2000). Granovetter (1973) suggests that weak ties, such as those with acquaintances or strangers, can be just as important to knowledge exchange. Bridging social capital is developed through those with weaker ties (Office for National Statistics, 2001). It is more likely that people with weak ties are different from the people who have strong ties, and therefore have different types of knowledge that both groups can benefit from (Granovetter, 1973). A recent study also revealed that what a migrant might consider ‘meaningful’ as a social interaction may not match our understanding of ‘meaningful’. In Ortaçtepe's (2013) study, his subject took part in three interactions, all with strangers, and he considered two of those meaningful. For his subject, a ‘meaningful’ social interaction was one with reciprocity- one where he was listened to -, in return for listening to the other person (Ortaçtepe, 2013). Therefore a ‘transactional’ English interaction could be taken as meaningful for the individual if the interaction was perceived as reciprocal. Sandstrom & Dunn (2014) researched the power of weak-ties to well-being in community and found that acquaintances affected people’s feeling of belonging. This is because community members learnt expectations of behaviour of the acquaintances they made in the community, be it a neighbour of shopkeeper. Rather than conceptualising ties as a binary of strong versus weak, there should be a ‘tie continuum’, which would begin with people that we interact with most regularly and face-to-face, all the way to strangers (May, 2013). Acquaintances, strangers and communities Previously I had not considered strangers as having an impact on a person’s feeling of belonging especially if social interactions with those strangers were limited.. However, strangers in a person’s locality, whom they do not know, but they encounter on a daily basis, begin to become familiar (May, 2013). This contributes to trust in the local area, as there is a development of predictability and familiarity that trust is built on. 57 Furthermore, the categorization of who is a stranger and who is an acquaintance is unclear. In my previous research, some participants called their neighbours friends, and for the others, their neighbours were called strangers (Graham-Brown, 2015). Research into diversity and trust from US and Canada showed that research participants in diverse neighbourhoods who talked to their neighbours regularly were more trusting than those who did not talk to their neighbours regularly (Stolle, Soroka, & Johnston, 2008). Their findings were that trust was challenging in diverse neighbourhoods where there were not enough social interactions (Stolle et al., 2008). Therefore, the types of everyday, ordinary and routine interactions by people in local communities or neighbourhoods created a sense of belonging for those people (May, 2013). May also suggests that ‘community’ is an alternative group to ‘family’ and ‘friends’ and a way people organise themselves to ‘embody a sense of local belonging’ (2013, p.122). The life story aspect of belonging The importance of a person’s first socialisation, to their subsequent continuous process of socialisation is significant and accounted for in the framework. Miller (2009) and Hedetoft (2002) note the importance of history in the formation of identity and belonging. Life stories are seen as not static records, but narratives that change as people experience and express their memories (Bennett, 2014). This aspect of belonging accounts for the fact that migrants moving to the UK are not empty slates, with no prior experiences or conceptualization of their identities. At the same time it recognizes that historical or autobiographical experiences are not linear and that people’s memories of such experiences may change depending on the present situation of the individual (Kraus, 2007). I have chosen to use the term life stories rather than life histories, based on the understanding that life stories are depiction of events in a person’s life based on how they see their life at that particular moment (Miller, 2000). Additionally, using life stories takes into account that belonging is experienced in relation to time and future trajectories (May & Muir, 2015). 3.5.4 Temporal belonging As mentioned earlier, belonging is related to time. Rather than being a linear process of historical points in time, temporal belonging is based on the relationship between memory and self and time (May, 2016). Other researchers use different expressions to describe this temporal aspect. Antonsich (2010) refers to it as the autobiographical aspect, and Kraus relates it to story-telling or narratives (Kraus, 2007). Online relational belonging In my previous research, it was noted that online methods of communication instigated by the tutor, enabled participants to communicate with each other outside the class (Graham-Brown, 2015). The communication became a way of extending conversations and discussions that were started in the class. Since then other forms of online communication such as Whatsapp groups have been used at ELATT to support out-of- class communications and relationship-building. Using Whatsapp people can feel like they are in two different places at the same time, thus over-coming the issues of geography and time (May, 2013). This in turn has created a world where people could live together even when apart (May, 2013) and this has created a sense of community for some users (Church & de Oliveira, 2013). Aharony's (2015) study of students who had moved away from home showed that they used Whatsapp to communicate with existing friends to ask for support on things they did not know. Furthermore, they relied upon friendships that had already been made using Whatsapp rather than starting new relationships (Aharony, 2015). Whatsapp communication was found to maintain existing social capital in Aharony’s study (2015). Whatsapp communication has been found to consist of mundane ‘day-to-day’ ‘tosh’, where the conversation was on-going (O’Hara, Massimi, Harper, Rubens, & Morris, 2014, p.1136). The nature of Whatsapp groups is that they also enable ‘ongoing group relations’ of groups that already see each other often (O’Hara et al., 2014, p.1137). Online communities can be an invaluable for belonging and support (May, 2013). 58 Developing a sense of belonging takes time Past, present and future trajectories are also considered in this aspect of belonging. As a temporal experience, belonging is experienced in and through time (May, 2016). I take this to mean that time and experience affect how we see ourselves. Temporal aspects have sometimes been thought of as a linear progression (May, 2016). However, researchers who have looked at time and belonging have cautioned against viewing the trajectory as entirely linear (Bastian, 2014; May, 2016). When considering life stories affecting participants socialisation, these can be re-conceptualised or re- understood (e.g. by improved self-understanding and reflection) by participants differently at different times based on present experiences (Kraus, 2007). Past, present and future trajectories are also considered in this aspect of belonging. As a temporal experience, belonging is experienced in and through time (May, 2016). I take this to mean that time and experience affect how we see ourselves. Temporal aspects have sometimes been thought of as a linear progression (May, 2016). However, researchers who have looked at time and belonging have cautioned against viewing the trajectory as entirely linear (Bastian, 2014; May, 2016). When considering life stories affecting participants socialisation, these can be re-conceptualised or re- understood (e.g. by improved self-understanding and reflection) by participants differently at different times based on present experiences (Kraus, 2007). Routine (discussed in relation to work in 3.5.1) and familiarity (in 3.5.3) are built upon the ability for something to be experienced over a temporal period. To develop familiarity and routine it is necessary to experience repetition. In turn this repetition 59 creates an expectation of future experiences. Therefore unlike material, cultural and relational belonging, temporal belonging is a vehicle for the entire socialisation process. 60 60 3.6 The framework for belonging The framework for belonging illustrated in Figure 2 below, has been designed to enable exploration of the experiences of migrant women over the period of the study, and to examine the relationship between their social interactions and belonging. p g g Figure 2: Diagram of framework of belonging Using Bourdieu’s theories of habitus and social fields to explain belonging (based on May. 2013), the aspects of belonging described above are all part of the socialisation process and contribute to the feeling of belonging in each particular social field. There can be multiple belongings (as discussed in 3.5.2) and people could feel one aspect of belonging and not another, all aspects contribute to the overall sense of belonging to the new place. Temporal Belonging Material Belonging Relational Belonging Cultural BelongingSocialisation into places (including Figure 2: Diagram of framework of belonging Temporal Belonging Material Belonging Relational Belonging Cultural BelongingSocialisation into places (including Figure 2: Diagram of framework of belonging Using Bourdieu’s theories of habitus and social fields to explain belonging (based on May. 2013), the aspects of belonging described above are all part of the socialisation process and contribute to the feeling of belonging in each particular social field. There can be multiple belongings (as discussed in 3.5.2) and people could feel one aspect of belonging and not another, all aspects contribute to the overall sense of belonging to the new place. Using Bourdieu’s theories of habitus and social fields to explain belonging (based on May. 2013), the aspects of belonging described above are all part of the socialisation process and contribute to the feeling of belonging in each particular social field. There can be multiple belongings (as discussed in 3.5.2) and people could feel one aspect of belonging and not another, all aspects contribute to the overall sense of belonging to the new place. 61 61 3.7 Research Aims and Questions: The aim of my research was to better understand the experiences of migrant women who are learning English, their social relationships and their sense of belonging. My research question is: My research question is: What is the relationship between migrant women’s social interactions and their perception of their own integration? To address this question, I asked the following sub-questions: To address this question, I asked the following sub-questions: 1. What are participants’ integration experiences? 2. What are the places that migrant women participate in social interactions? 3. What is the nature of their social interactions within these places? 4. What are participants’ feelings about their sense of belonging in these places? The following chapter is an explanation of the methodology used to research questions above. The following chapter is an explanation of the methodology used to research questions above. 62 62 4. Methodology In this chapter I explain my methodological approach for my research. In the first part, outline the design, and describe my methods of data collection and the sample for research. I present my ethical considerations and end with a discussion about the reliability and validity of my research. In the latter part of the chapter, I describe the analytical approach, methods and stages used to derive findings. 4.1 Design In order to best understand the perceptions of participants of the study and the development of their social relationships whilst they attended their community ESOL course, it was important that the study was qualitative, in-depth and the design that was chosen fully supported listening to authentic voices of participants and their perceptions. I used a narrative inquiry design to capture life stories of participants and to gather data on participants’ perceptions over time. Narrative inquiry as a research design has been described as a way of ‘collecting, analysing and re-presenting people’s stories as told by them’ (Etherington, 2004a, p.75). It is a general design that captures lived experience over time and takes into account the interaction between personal experience and cultural context (Ehterington, 2004). Bruner suggests that narrative inquiry, particularly using autobiographical narratives, is a way of interpreting and reinterpreting experience, the narrative of which has been created in the mind (Bruner, 2004). Etherington (2004b) also suggests that the narrative approach can support us to better understand how socialisation impacts the creation of identity within local and wider sociocultural contexts. The narrative approach appeared to be a good fit because of the alignment with the social constructivist philosophy, and reference to socialisation and identity forming. The research project involved six women over a 13-month period, whilst participants attended community ESOL courses at ELATT and the immediate period after their courses had ended. There were two main methods of data collection: narrative interviews and oral diaries. Analysis of data included narrative analysis, thematic analysis and classic content analysis approaches. 63 4.1.1 Research questions In order to answer the research questions in 3.7, I added the following questions: 1. What are participants’ integration experiences? a. What are participants’ prior integration experiences? b. What are participants’ experiences of being new arrivals? c. What are participants’ experiences at the end of the research? 2. What are the places that migrant women participate in social interactions? a. Description of places of social interactions 3. What is the nature of their social interactions within these places? a. Types of social interactions b. Players in the places c. Activities within the places d. Relationships within the places 4. What are participants’ feelings about their sense of belonging in these places? a. Material belonging b. Cultural belonging c. Relational belonging d. Temporal belonging 64 64 4.2 Methods of data collection It was important for me that this research project was democratic based on my social constructivist stance and taking into consideration the background and experiences of the research participants. As discussed in the Introduction, migrant women whom we support at ELATT tend to have low levels of confidence. Their previous experiences of interviews would have been with people in positions of power such as with Home Office officers, council staff or prospective employers. Wengraf (2001) discusses the impact of past interview experiences of participants on the quality of subsequent research interviews and data collected. Therefore it was important for me to ensure that as the researcher, participants did not feel I had power over them, and that their responses were not influenced by their perceptions of what I wanted to hear, or their memories of any other previous interview scenarios they had experienced. These were also part of my ethical considerations discussed in 4.4 of this chapter. I searched for specific guidance for conducting interviews with people who had experienced significant change in their socio-cultural environments, such as through migration, and also supplemented the interview sessions with an additional independent method of data collection, which was oral diaries. I had used and developed this method in my previous research. These methods are discussed in 4.2.2 and 4.2.3. 4.2.2 Narrative interviews Narrative interviews were carried out over a 13-month period. Each participant had an initial biographical narrative interview method (BNIM) interview. Following this, bi- monthly semi-structured interviews were scheduled. Interviews began in December 2015, with the final interview being in January 2017. My initial project plan was to complete four interviews with project participants over a 9-month period from October 2015 to July 2016. However, each project participant encountered personal issues, which meant that I had to revise this plan, including the frequency of data collection. There were 14 interviews and 5.25 hours of oral diaries. Wengraf’s Biographical Narrative Interview and/or Interpretive Method (BNIM) was based on the earlier narrative interview works of Rosenthal and Schutze (Apitzsch & Siouti, 2007). Breckner used this method in studies of migration and belonging (see Breckner, 2002) and although the experiences particularly pertained to the lives of people migrating and settling in Germany, I felt that this method would be suitable for this research project because of the lived experiences of the participants and the impact on their socialisation experience. 65 BNIM has a rigid structure and Wengraf had developed in-depth guidance for carrying out interviews for data collection, and a framework for interpretive analysis (Kalekin- Fishman, 2002; Wengraf, 2001). BNIM consists of a three-session interview process (named subsessions) (Wengraf & Chamberlayne, 2006). The process is briefly summarised below: • Subsession 1 and 2 are usually in one main interview, with subsession 3 to take place in the subsequent interview, if necessary (for example if there was not enough data from Subsessions 1 and 2).The guidance suggests that the main interview (subsession 1 and 2) should take around 2-3 hours (Wengraf, 2008). • For subsession 1, it is imperative that the SQUIN (single question aimed at inducing narrative) question is used and that the interviewer does not ask any other questions apart from those that would carry on the narrative. • In between subsession 1 and 2 there should be a break for the interviewer to select items from notes made, that will be probed in subsession 2. • In subsession 3, further questions arising from the earlier subsessions can be explored using more traditional semi-structured interview strategies. (Wengraf, 2008) I encountered difficulties with this method in the first interviews that I carried out. The guidance and literature around interviewing suggested that it was not necessary to pilot this particular method (Wengraf, 2008). 4.2.2 Narrative interviews However, recognising that the participants of my research were students studying ESOL, and could probably find it difficult to tell their narrative without preparation, I had shared the SQUIN with them prior to the interview, to enable them to understand the question I would be asking. My SQUIN mirrored Wengraf’s exemplars and is below: “I’d like you to tell me about your life and your journey till now. I’ll listen and take notes but I won’t say much and I won’t interrupt you. So please just talk and tell me about your life history, events and experiences that have been important to you up till now.” In spite of the preparation, the first issue I encountered was a lack of length of narrative forthcoming from my participants. In other researcher’s examples (Buckner, 2005; Davey, 2009; Roseneil, 2012; Shoderu, Kane, Husbands, & Holly, 2012; Wengraf & Chamberlayne, 2006) participants produced an average one hour’s data. In two of the research projects (Buckner, 2005; Shoderu et al., 2012) data produced was large and required several researchers to deal with. All but one of my participants only produced short narratives, some without going into ‘particular incident narratives’ (PIN). Not all hort narratives, some without going into ‘particular incident narratives’ (PIN). Not all 66 participants kept to what Labov would describe as coherent sequences (Patterson, 2008) and in some cases, their responses seemed not to respond to the SQUIN. This meant that in some cases, I had to explore general personal historical narratives before being able to focus on PIN in the second subsession. participants kept to what Labov would describe as coherent sequences (Patterson, 2008) and in some cases, their responses seemed not to respond to the SQUIN. This meant that in some cases, I had to explore general personal historical narratives before being able to focus on PIN in the second subsession. 67 67 Participant Narrative interviews 1a Time Elma Yes 36:58:00 Flora Yes 27:00:00 Asma Yes 15:00:00 Lucy Yes 34:34:00 Noor Yes 35:00:00 Samira Yes 17:00:00 Figure 3: Interview 1 times Participant Narrative interviews 1a Time Elma Yes 36:58:00 Flora Yes 27:00:00 Asma Yes 15:00:00 Lucy Yes 34:34:00 Noor Yes 35:00:00 Samira Yes 17:00:00 Figure 3: Interview 1 times Figure 3: Interview 1 times Participants also became exhausted during the process and two participants became upset as the stories they were re-telling were of particularly traumatic life experiences and were upsetting. Figure 3: Interview 1 times 4.2.2 Narrative interviews One participant had to be referred to our safeguarding process as the information she disclosed was of a concerning nature (as per the project’s ethics process, and my organisation’s policies). This procedure then affected her availability to continue with the subsequent interviews. All participants struggled to go in-depth with their narratives, which prompted me to reflect on their language level and ESOL needs. Previous studies using BNIM methods that I had researched above appeared to have proficient English speakers. My participants fed-back that they were tired from speaking for so long in English as it required much more thinking and effort than they had anticipated. Two participants felt it was difficult to talk about themselves and their experiences in such detail, as they were not used to it. I researched an alternative method and identified Hollway and Jefferson’s (2000) Free Association Narrative Interviewing (FANI). It was suitable for people who may have difficulties producing narratives, have difficulty keeping to coherent narrative sequencing and particularly suited to subjects that required longitudinal research to be able to research their narratives in depth (Hollway & Jefferson, 2008). 68 Hollway and Jefferson developed FANI when interviewing what they termed ‘defended subjects’: their participants were people who had been affected by crime and the criminal justice system, and found it traumatic to talk about their previous experiences (Hollway & Jefferson, 2017b). They too had started their research piloting the BNIM method and found difficulties with eliciting narratives from their participants (Hollway & Jefferson, 2004). Rather than focus on individual sessions they focused on collecting data over the period of the research, and they were able to gain data on the whole narrative (Hollway & Jefferson, 2004). In this adapted method, after each interview, the researcher listens to the recording, and makes notes of open-ended questions to ask in the next session. In order to ensure standardised topics, an interview frame, based on the research question and any prior data is used. Researchers need to be aware of adaptations and revisions to their research design over the period of their study, and ensure there is a clear record of revisions or adaptations to avoid distortion at analysis stage (King & Horrocks, 2010). I ensured that I kept detailed records to exhibit any revisions made. 4.2.2 Narrative interviews I used this method of interviewing and preparation for future interviews for each participant and found I was getting more narrative from participants. I also recognised that any non-sequential telling of their stories, was meaningful in itself, and noted these as part of my field notes and initial listening. FANI is based on psychosocial analytical methods, and recognises that for some individuals, the recollection of meaningful narratives is based on unconscious links rather than factual sequences (Hollway & Jefferson, 2004). Therefore the method is particularly suitable when researching identity and perception of interview subjects (Hollway & Jefferson, 2004). Additionally, the non-sequential telling of stories resonated with the temporal aspect of belonging discussed in 3.5.4, whereby people do not necessarily remember events in sequence, but based on how they currently feel or perceive their memories. 4.2.3 Oral diaries As mentioned in 4.2 I also used oral diaries to gather data about participants’ social interactions in English and used these in preparation for exploratory interview questions in the free association narrative interviews. Oral diaries are a method of participatory data collection that involves participants making recordings about their experiences. The diaries were kept over a period of 1 year. Although paper diaries and video diaries have been used previously in research with ESOL students (see for example Norton, 1995 and Callaghan, 2011) there are relatively few research projects using oral diaries to gather ethnographic and 69 biographic experience over time. Some biographic narrative methods suggest using diaries and observations as additional methods of data collection for triangulation, for example in BNIM, where the method is also an interpretive method (Wengraf, 2008). This was not the reason for using oral diaries in my research, as I am not attempting to triangulate the data or prove a form or ‘truth’ in the experiences of my participants. Instead, my research focus on participants’ perceptions meant that oral diaries supplemented the reflections produced in the narrative interviews, or served as a record of PIN (particular incident narratives) for further exploration. For example, I found out about Elma’s child’s accident from listening to her oral diary, and was able to explore her experiences in her next interview. They also supported participants as an aide memoir so that when they came to their interviews, they could remember significant events and PIN that had happened in the time since the previous interview. Based on my previous experience using oral diaries, I gave training sessions to participants and a laminated guide of oral diary questions. The questions were designed to support participants to record the factual parts of any interaction that took place (e.g. date, time, who was there) before moving onto more reflective questions such as how they felt about the interaction and how they felt about the other person. The list of questions are shown below: 70 70 Oral diary questions 1. When did the interactions take place: a. Today’s day and date b. When did you use English to communicate today? 2. What was the interaction about: a. What happened? Say a bit about the situation b. Who did you speak to? c. What was the conversation about? What did you say? What did the other person say? d. Were there any communication problems? 4.2.3 Oral diaries 3. How did you feel? a. Did you feel that the other person listened to you? Did they give you time to speak? Did they help you to communicate with them? b. Were there any communication problems? 4. About the other person/party: a. How are you related to this person? i. If they are a friend, are they a good friend? ii. If they are a family member, do they support you with your life? iii. It they are a teacher/key worker/other service person, how do they help you? iv. If they are a shopkeeper/TfL worker/library assistant, do you know their name or see them regularly? b. Do you speak to them regularly? How often do you speak to them? c. Does this person help you? d. Do you help them or give them your time? Figure 4: Oral Diary Questions Figure 4: Oral Diary Questions One participant found it easy to reflect and be critical about the interactions they had. The majority of participants were able to follow the questions broadly and produce rather meaningful diary entries but not answering all questions. One participant did not follow the instructions at all, but instead chose to record her personal feelings about her life, which in itself was quite a valuable resource for questions to explore in the narrative interviews. I collected oral diary recordings from participants on average every fortnight. This enabled me to conduct initial analysis to form semi-structured interviews in time for the next interviews. Additionally, there were also ethical reasons for this, which are discussed in 4.4. Overall both methods of data collection worked well to produce data to answer my research question. 71 71 4.3 Sample This research is of women who came to the UK on spousal or family reunion visas and were still considered new to the UK and have been here under 10 years (newly arrived according to the Home Office criteria). The sample was both purposive and opportunistic. Purposive sampling is where people are selected to ensure that participants meet certain characteristics (Cohen et al., 2005). The sample was purposive to ensure participants reflected the wider composition of migrant women from non-English speaking countries. Statistics from 2013 showed that women from India, Pakistan and Bangladesh formed the largest group of applicants (Home Office, 2017) for spouse and settlement visas. Therefore it was important that women from these countries listed participated in the research. The non-English speaking majority countries with the next largest numbers were China, Thailand, Sri Lanka, Afghanistan, Nepal and Iraq (Home Office, 2017) and I was confident that the groups of ELATT were representative of these groups and it was likely that participants would be from some of these countries. The women who took part in the research and their countries of origin: Name (chosen by participants) Country Elma Somalia via Egypt Flora India Asma Pakistan Lucy China Samira Afghanistan Noor Morocco via The Netherlands Figure 5: Participants and their countries of origin Figure 5: Participants and their countries of origin Participants were also at similar English language level and were able to participate on the research project without requiring interpreters. Participants were students who had achieved at least Entry-Level 3 English or ESOL. This meant that they could: • Speak to communicate information, opinions and feelings on a range of familiar topics, in formal and informal situations. • Speak to communicate information, opinions and feelings on a range of familiar topics, in formal and informal situations. • Listen and respond to information and narratives on a range of familiar topics, and use strategies to predict meaning and clarify understanding on some unfamiliar topics. 72 • Engage in discussions with others, making relevant contributions and responding to the contributions of others. • Engage in discussions with others, making relevant contributions and responding to the contributions of others. Functional descriptors for Entry Level 3 and Level 1 Source: Welcome to the UK LLU+ (Dudley, Kelly, Kirsh, Moon, & Tranza, n.d.) Functional descriptors for Entry Level 3 and Level 1 Source: Welcome to the UK LLU+ (Dudley, Kelly, Kirsh, Moon, & Tranza, n.d.) Functional descriptors for Entry Level 3 and Level 1 Source: Welcome to the UK LLU+ (Dudley, Kelly, Kirsh, Moon, & Tranza, n.d.) By mitigating the need for translation services I avoided the issues that translation can cause, such as incorrect interpretation of messages, or influencing the response with their own opinions (Esposito, 2001). Translators would have also been an added cost. Additionally, the aim and design of the thesis was to support empowerment and participation and using translation services where participants might be relying on a stranger to express their opinions was contradictory to the epistemological values of the study. There were also several other reasons for choosing participants at this level of English. Based on pilot and previous research (Graham-Brown, 2014, 2015), participants at this level were also able to: • Participate in out-of-class interactions with confidence because they would be confident in the use of certain language structures. • Confidently participate in semi-structured interviews that could last up to one hour without using interpreters, but be able to use tools to aid independent translation if necessary. • Discuss their experiences negotiating the immigration system, as this is the minimum language level for applications for residency. Figure 5: Participants and their countries of origin Sampling was also opportunistic (or convenience sampling) as it relied on ELATT students being available and willing to take part in the research (Burton, Brundrett, & Jones, 2008). This has affected how representative the sample was of the population of migrant women, as the intention was to include women from India, Pakistan and Bangladesh. Of the twenty women invited to participate, nine were of Bangladeshi origin but none of them was able to participate in the research project. However, as the makeup of ELATT students reflected Home Office statistics, participants still were a representative sample of wider immigration groups. Please see Appendix 6 for a more detailed table of the participants’ profiles. 4.3.1 Challenges with this participant group One challenge was the issue of attrition. I originally intended to recruit five women so that a minimum of three women would participate until the end of the research period. 73 In November 2015, six women applied to join the research and completed informed consent forms. After the initial introduction to the research meetings, only five of the six participants participated in interviews. Additionally, after initial interviews, I realised that each participant was facing difficult personal situations and therefore all were at risk of leaving their course at ELATT at any time, and that this could affect the number of participants on the research project. Between April and June I recruited a further three participants but only one of them continued. In November 2015, six women applied to join the research and completed informed consent forms. After the initial introduction to the research meetings, only five of the six participants participated in interviews. Additionally, after initial interviews, I realised that each participant was facing difficult personal situations and therefore all were at risk of leaving their course at ELATT at any time, and that this could affect the number of participants on the research project. Between April and June I recruited a further three participants but only one of them continued. Apart from experiencing precarious personal situations, it was also difficult to schedule the interviews with participants. Many had busy lives and the only time they would come to ELATT would be for their English class, after which they would either have to leave promptly to collect their children from childcare or school, or return home to care for their relatives, or go to work. The in-depth and individual interviews were difficult to schedule and carry out within the time periods I had set myself. Participant Number of interviews Elma 4 Asma 3 Flora 3 Lucy 5 Samira 4 Noor 3 Figure 6: Table of number of interviews with participants Participant Number of interviews Elma 4 Asma 3 Flora 3 Lucy 5 Samira 4 Noor 3 Figure 6: Table of number of interviews with participants Participant Number of interviews Elma 4 Asma 3 Flora 3 Lucy 5 Samira 4 Noor 3 Figure 6: Table of number of interviews with participants 4.4 Ethical Considerations The main ethical issue was one of power relations, which is especially with reference to the insider researcher role (Costley, Elliott, & Gibbs, 2014). Participants were aware of my role as head of department and this may have affected the decision by some women whether to take part in the research or not. Based on experience of previous research projects, I delivered the introduction to research, and the idea of informed consent in stages. This ensured that when communicating the idea of informed consent to prospective participants, participants were comfortable to decline to participate if they were not interested, and to know they had the right to withdraw subsequently. As discussed earlier, some participants agreed to take part but did not make themselves available for interview. I also ensured that I delivered information about the research in a lesson format so that they could understand the terminology used in the informed consent forms. I allowed them time to read and think about the research project (including hand-outs that they could take home) and ensured that they understood that all responses were fully voluntary. If at any point in the research they felt uncomfortable to respond to questions, they were told they had the opportunity to decline and or withdraw. The methods of data collection attempted to mitigate issues of imbalance of power between researcher and research participant. I recognised the risk that participants might respond to research questions in a particular way so as not to appear to give an incorrect answer to the researcher (me), a senior manager in the organisation. My role could reinforce power differences between the researcher and participant (Hammersly & Traianou, 2012) and participants could potentially be in a vulnerable position having disclosed personal information about themselves to the researcher. Apart from the duty of care for the participant, there was also a risk that these power differences could cause inauthentic research responses (Hammersly & Traianou, 2012). Therefore, it was imperative that participants understood that there was not an expected answer, but they could answer the question, as they felt comfortable to, without the risk of this affecting their position as a student at the organisation. As discussed in 4.2.3, apart from interviews, to ensure that this research process was participatory, and data was as authentic as possible, oral diaries were chosen as an additional method of data collection. Figure 6: Table of number of interviews with participants Although there were challenges with using narrative interviews with this client group, the data that I was able to gather about the lives and experiences of these women is rich and in-depth. As someone they trusted, they were honest about their feelings and perspectives with me (please see discussion in 4.4 about ethical considerations vis-à- vis vulnerable participants). Based on this research, I would recommend the method of interviewing used in the research, however, would caution future researchers to consider the challenges and put in place strategies to support the researcher and participants. 74 4.5 Reliability and Validity It is generally accepted that there are additional challenges in proving robustness of research and ability to generalise findings in social science and qualitative design (Gerring, 2001). Robson suggests using a scientific attitude towards research design to ensure that research is robust: meaning that the research is systematic, sceptical and ethical (Robson, 2011). Systematic research design involves ensuring that certain concepts are adhered to (Gerring, 2001) and both Cohen (Cohen et al., 2005) and (Denscombe, 2009) provided clear frameworks which I used to create the research design. Typically, social science researchers will have the themes of accuracy, accountability, generalisations and proof to consider when dealing with data, analysis and conclusions (Webster & Mertova, 2007). Validity of research centres on the validity of data and accuracy of analysis in the research. Yin (2009) lists three types of validity in empirical social science research: construct validity, internal validity and external validity. Construct validity is concerned with how data is collected and analysed for the concept being measured (Yin, 2009). Denscombe poses these questions: ‘Has the research asked the right questions? Are the data sufficiently precise and detailed?’ (Denscombe, 2009, p.141) To answer the first question: whether the correct questions have been asked, the conceptual framework of the research (discussed in Chapter 3) shows that critical evaluation of the context and existing theories have formed the basis of the research question and design. Furthermore, the interview framework used is described in 4.2. To answer Denscombe’s second question, most social science research guidance suggest using a variety of data collection methods to enable triangulation of data to verify the accuracy of the data (Cohen et al., 2005) and the sufficiency of the data (Yin, 2009). In this study, the narrative inquiry method used was to deal with data that was in-depth and entirely based on individual perspective (as described in 4.1). Therefore triangulation of data did not seem an appropriate approach. Indeed narrative researchers have argued that the nature of narrative inquiry is at odds with the attempt to use multiple sources of data to verify a biographical narrative of lived experience (Webster & Mertova, 2007). Instead, they suggest emphasis should be placed on the process of narrative method used to gather data (described in detail in 4.2) and the analytical framework and tools (described in 4.6) (Webster & Mertova, 2007). 4.4 Ethical Considerations With this method, I recognised that the likelihood of disclosure of confidential situations was higher. Part of the rationale behind regular reviewing of oral diary entries was to ensure any disclosure would be acted upon in a timely manner and appropriately. British Educational Research Association (BERA) 75 guidelines were used but in all situations, organisational Safeguarding Policies took priority (British Educational Research Association, 2011). As discussed previously, I had to refer one participant onto ELATT’s safeguarding procedures after the initial interview, and we supported her throughout her time on the research project. I ensured that informed consent was fully understood by participants, and other people who were important to the participant such as their spouses and family members. Previous experience had shown me that family members of migrant women are involved in the decision-making process of whether they will participate in research. In my previous research, I had made a leaflet explaining the research project, including informed consent to use to communicate with family members and I produced this for this research too. I also added a further question and answer session, although no one took the offer up. The safety of data once collected, including confidentiality issues, was also considered. Participants are not referred to by their real name in any saved document or file; pseudonyms chosen by participants themselves have been used from the first session. Additionally, I have tried to remove or replace any data that could give away participants’ identity- e.g. borough names, job details. All data files are either stored on the university assigned drive, ELATT’s secure servers or in ELATT’s cloud, which is subject to regular cyber-security testing. 76 76 4.5 Reliability and Validity The former supports proving the accuracy of the data (construct validity) and the latter ensure findings presented are robust (internal validity) (Webster & Mertova, 2007). To answer Denscombe’s second question, most social science research guidance suggest using a variety of data collection methods to enable triangulation of data to verify the accuracy of the data (Cohen et al., 2005) and the sufficiency of the data (Yin, 2009). In this study, the narrative inquiry method used was to deal with data that was in-depth and entirely based on individual perspective (as described in 4.1). Therefore triangulation of data did not seem an appropriate approach. Indeed narrative researchers have argued that the nature of narrative inquiry is at odds with the attempt to use multiple sources of data to verify a biographical narrative of lived experience 77 77 ‘Truthfulness’ is also part of ensuring validity (Denscombe, 2009). However, the usual mechanisms to test for truthfulness also did not seem to suit the narrative inquiry design. Webster and Mertova suggested new criteria for reliability and validity in narrative inquiry based on Huberman’s research (2007). Other researchers such as Clough, Bold and Czarniawska also dismiss the usual tests for reliability and validity as not suitable for narrative research (Clough, 2002; Bold, 2012; Czarniawska, 2004) and also present alternative concepts. Most of the concepts address validity and reliability by thinking about the accessibility by the reader of the research, rather than a search for an ‘ultimate truth’ which, according to the epistemological stance of narrative inquiry arguably does not exist (Silverman, 2013). To ensure I had tested for reliability and validity of this study I used a combination of Bold’s and Webster and Mertova’s concepts: access; honesty, verisimilitude, trustworthiness and authenticity; and transferability (Webster & Mertova, 2007). 4.5.1 Access Access is concerned with a) the access by the reader of the research to the participants’ world and access to the process by which knowledge has been constructed between the participant and researcher; and b) access to the data (transcripts), research and field notes which has formed the findings of the research (Webster & Mertova, 2007). Therefore, access means setting the scene for the research well, so that the reader is able to have access to the cultural context of the participant. The methodology, including research design and the detailed account of how the research was carried out, along with the conceptual framework for analysis enables the reader access to the process of knowledge construction in my research. To ensure integrity, it was important that risks and challenges were also presented, as these limit the risk of giving positive findings when the data may not show this. More importantly, in narrative inquiry, the constraints, challenges and limitations are part of the whole context of the narrative and may give important information about the research and participants. In part 4.2 and 4.3, I explained these constraints (e.g. opportunistic sampling), limitations (e.g. oral diaries limited responses) and challenges (e.g. participant experiences; BNIM interviews). Access is concerned with a) the access by the reader of the research to the participants’ world and access to the process by which knowledge has been constructed between the participant and researcher; and b) access to the data (transcripts), research and field notes which has formed the findings of the research (Webster & Mertova, 2007). Therefore, access means setting the scene for the research well, so that the reader is able to have access to the cultural context of the participant. The methodology, including research design and the detailed account of how the research was carried out, along with the conceptual framework for analysis enables the reader access to the process of knowledge construction in my research. how the research was carried out, along with the conceptual framework for analysis enables the reader access to the process of knowledge construction in my research. To ensure integrity, it was important that risks and challenges were also presented, as these limit the risk of giving positive findings when the data may not show this. 4.5.2 Trustworthiness, verisimilitude and authenticity These criteria are particularly addressing the ‘reliability’ of the research as in narrative inquiry it is highly unlikely that conducting the same research again will return the same results (Moen, 2006). For an alternative test for trustworthiness the researcher needs to demonstrate the trustworthiness of their research by showing that they have accurately represented the complex participants’ stories, and that the narrative constructions from the research are truthful to those participants (or the original constructors of the narratives) (Korstjens & Moser, 2018). One strategy is to ensure there is credibility from prolonged engagement and persistent data collections (Korstjens & Moser, 2018). A more usual test for trustworthiness is in the confirmation by participants of the truthfulness of the representations of their narratives (Webster & Mertova, 2007). All participants were invited back to meet with me at the end of the research period to confirm the interview data. Bruner (1991) suggested verisimilitude in narrative research as an alternative to tests for falsification in scientific procedures. Verisimilitude is the appearance of being true or plausibility (Webster & Mertova, 2007). Bold asks the question: ‘Are the findings consistent with other findings and my own understanding? (Bold, 2012, p.5)’ Narrative is a familiar and conventional form and our memories are organised in narrative (Bruner, 1991). Therefore a test for verisimilitude is one of plausible narrative construction, and this will be based on the reader’s own reference of narrative. Using independent readers in the writing process of this thesis has supported the test for verisimilitude. To achieve authenticity Webster and Mertova advise to ensure enough information is provided, and to pay attention to sufficient narrative coherence (2007). As part of ensuring the authenticity and verisimilitude of this research, two colleagues have read the complete research, as critical others and experts in the field. Additionally, one migrant woman from a separate programme read the report to give feedback on plausibility. 4.5.1 Access More importantly, in narrative inquiry, the constraints, challenges and limitations are part of the whole context of the narrative and may give important information about the research and participants. In part 4.2 and 4.3, I explained these constraints (e.g. opportunistic sampling), limitations (e.g. oral diaries limited responses) and challenges (e.g. participant experiences; BNIM interviews). Additionally, participants’ life stories were created using pen-portraiture, which is a method of analysis but also for presentation to enable the reader to gain an understanding of research participants’ contexts (Howatson-Jones, 2011). 78 78 4.5.3 Transferability One of Bold’s tests for what I understand as ‘external validity’ is whether findings are applicable in similar contexts (Bold, 2012). In-depth qualitative research tends to be specific and therefore there is not usually an attempt to make claims that findings are generalised (Clandinin & Huber, 2010). However, Webster and Mertova suggest that transferability of findings should be considered. My research has given insight into the experiences of six migrant women on their pathways to integration. Although I am not able to generalise findings across the entire group of migrant women who are learning 79 English and settling in the UK, I am able to confidently share the findings regarding social interactions in places and identify transferable insights into the experiences, for other women who are in similar situations as my participants. So far in this chapter, I have detailed the methodology of this research. The research design is a narrative inquiry and I have discussed the methods of data collection, including challenges, limitations and constraints, and ethical issues. I have also given careful consideration to issues of validity and reliability of the research study. My analytical framework and data analysis is explained next based on the questions in 4.1.1. 80 80 4.6.1 Analytical approach The formal data analysis process was completed over approximately 13 months. In actuality, data analysis began as early as after each participants’ first interviews as I used the first BNIM interviews to form the questions I would ask for the following interviews. As with Hunter’s experience (Hunter, 2010), the analysis process was messy, with no clear demarcation between early data collection, and the ‘writing up’ process, and went through many complex phases with different iterations of categories, themes and theory development. Hatch (2002) stated that: “Data analysis is a systematic search for meaning. It is a way to process qualitative data so that what has been learned can be communicated to others. Analysis means organizing and interrogating data in ways that allow researchers to see patterns, identify themes, discover relationships, develop explanations, make interpretations, mount critiques, or generate theories. It often involves synthesis, evaluation, interpretation, categorization, hypothesizing, comparison, and pattern finding. It always involves…“mindwork” ...Researchers always engage their own intellectual capacities to make sense of qualitative data. (Hatch, 2002, p.148) In order to gain answers to the research questions in 4.1.1, data was analysed in three stages: In order to gain answers to the research questions in 4.1.1, data was analysed in three stages: In order to gain answers to the research questions in 4.1.1, data was analysed in three stages: • Part A: analysis of the narrative interview transcripts; o identifying places that migrants participated in social interactions; o describing the places of social interactions; o describing social interactions and relationships within them; • Part C: analysing participants’ feeling of belonging The analytical processes and methods I used are summarised in the table below: 81 81 Research Questions Analyse for: Analysis method used Priori data/codes RQ1 Sequencing of life story: 1. L1 Socialisation (Life stories prior to migration) 2. SLS Migrant (Life stories as a migrant in UK/London) Narrative analysis (FANI) combined with thematic analysis (Braun and Clarke (2006) 6-phase guide) Basic a priori codes: Education and prior language learning. Other coding is inductive. RQ2 1. Identify places that migrant women participate in Classic content analysis (Typological analysis) Literature review and prior research 1. Places 2. Description RQ2 2. describe places that migrant women participate in Thematic Analysis (Braun and Clarke) Literature rev/ prior research RQ3 3. Identify social interactions and relationships within the places Classic content analysis (Typological analysis) Literature rev/ prior research 1. Interactions 2. Name 3. 4.6.2 Participants’ experiences prior to migration As discussed in 3.5.4 it was important to gain information about participants’ life stories prior to migration and as new migrants to enable an understanding of their integration process, and feelings of belonging. The FANI method supported out-of-sequence narration based on the importance participants give to those memories, or how they recall memories (Hollway & Jefferson, 2017b). The narrative analysis process I used was based on Hunter (2010)’s use of Rosenthal and Fisher-Rosenthal’s Stages in Practical Analysis (Rosenthal & Fisher-Rosenthal, 2004, p.261). The stages are broadly: analysis of biographical data; reconstruction of story into pen-portrait; thematic analysis. The method of narrative analysis requires the researcher to hold the whole story during the process of analysis to be open to changes in coherence and contradictions (Hollway & Jefferson, 2004). Based on principles of gestalt (‘the idea that the whole is greater than the sum of the parts’ Hollway & Jefferson, 2017a, p.61), the first stage was to understand each participants’ whole story. In order to do this I had to listen to the data repeatedly, making extensive electronic notes. The value of this process was that I was able to gain an in-depth understanding of each individual’s life story prior to the start of the research process and at every stage, and so was then able to recognize the changes reported by participants as they participated over the research period. Hollway and Jefferson suggest making links utilizing theory and reflexivity to start interpreting the whole context (Hollway & Jefferson, 2017a). Based on the conceptual framework I was highlighting data relevant to the theory and then reflecting upon the data I had identified, making notes before the next stage. The next stage of data analysis was to produce pen portraits to use later in data presentation, and to enable the participants’ whole story to be present through the analytical process (Golsteijn & Wright, 2013). The production started with re- sequencing narrative interview texts so that each participant’s text was in sequential order, and after this further re-writing text into third-person narrative or biographical narratives. This was to enable thematic analysis to take place across life-stories of the participants (Hollway & Jefferson, 2004). After this stage, I used thematic analysis. Braun and Clarke have called thematic analysis a foundational form of qualitative data analysis, especially suited to ‘complex, diverse and nuanced’ research (Braun & Clarke, 2006, p.78). 4.6.1 Analytical approach Description RQ3 4. Nature of the social interactions • What are the other players or experts in the places? • Relationships within the places? • Activities within the places? Thematic Analysis (Braun and Clarke) Literature review: context/players/com municative acts/ language use/power relationships RQ4 Impact of social interactions on 1. Material Belonging 2. Cultural Belonging 3. Relational Belonging What are the participants’ feelings about their sense of belonging? Thematic Analysis (Braun and Clarke) From Literature review of belonging Figure 7: Table of the analytical process I used MS Excel spreadsheets to organize and manipulate all my data. I ensured that codes were recorded alongside quotes from the narrative interview texts to allow for a h i i l i i i if Figure 7: Table of the analytical process I used MS Excel spreadsheets to organize and manipulate all my data. I ensured that codes were recorded alongside quotes from the narrative interview texts to allow for a return to the original interview transcripts if necessary. 82 4.6.2 Participants’ experiences prior to migration The stages are briefly: 1.Familiarisation with data; 2.Generating codes: using a priory coding and inductive coding; and then categorising 3.Generating themes: using identification and pattern recognition; 4.Reviewing themes: interpretation using conceptual framework of 83 belonging, data manipulation and further interpretation; 5.Defining and naming themes; 6.Producing text for the report. To capture these complex and diverse experiences of participants, the biographical narratives were divided into Part A1- prior to moving to the UK, and Part A2- just after arrival in the UK, to enable thematic analysis of participants’ experiences. After this stage, data was coded and categorised, then analysed and organised based on the patterns to enable reporting as per Braun and Clarke (Braun & Clarke, 2006, p.79). This involved analysis of Part A1 text for codes and themes of importance, starting with limited a priori codes from existing literature as described next. There is little in current literature about pre-migration factors that could influence a person’s future integration (Morrice et al., 2019). Most literature on integration list English language ability, work experience and level of higher education being influential factors in future labour market participation (Heckmann, 2005; Rubin et al., 2008). Prior education level, literacy level and work experience are also thought to influence future language learning ability (Allemano, 2018; Esser, 2006). Therefore, apart from the level of education, prior language learning and work experience, the remaining headings were based on inductive thematic analysis of the text. The resulting data were organised into these categories: The resulting data were organised into these categories: g g g • Basic information (e.g. country of origin, ethnic backgrou • Family situation including role of spouse • Socio-economic background/situation • Prior language-learning experience • Prior education/schooling • Jobs and Work experience • Basic information (e.g. country of origin, ethnic backgroun • Family situation including role of spouse • Socio-economic background/situation • Prior language-learning experience • Prior education/schooling • Jobs and Work experience Additionally there emerged a section related to identity, which was organised into these headings: • Visible identity • Independence and efficacy • Emotional/ feelings I subsequently prepared the pen portraits for presentation. The narratives were not all equal in length and content as some participants had been through more life experiences compared to others. However, keeping to the common headings enabled a degree of standardisation between the narratives. 84 84 4.6.3 Participants’ experiences as new migrants The data for the period of when participants had just arrived in the UK were coded and then organized under broad categories derived from the integration types listed in the literature review (structural, cultural, interactional, identificational). These consisted of the initial reactions, feelings and barriers experienced when the research participants all had just moved to the UK and since. The categories were: The categories were: The categories were: • Initial emotions • Support from immediate family/husband • Citizenship/rights knowledge • Language-specific barriers • Other barriers • Support received • Personal motivation • Initial emotions The text from each group was then manipulated and grouped together to enable further analysis of their perceptions and experiences to identify themes. 4.6.4 Places of social interactions In order to identify and name places that migrant women participated in social interactions, I used typological analysis, whereby a deductive analysis was applied based on predetermine codes (Hatch, 2002). Part of classic content analysis (CCA), instead of generating themes, the number of times each code is used is counted (Leech & Onwuegbuzie, 2007). CCA typically starts with a priori codes (Leech & Onwuegbuzie, 2007), and for this research I started with codes that had come from the my earlier research (Graham-Brown, 2015) (see Appendix 8 for these). After this stage, analysis continued with the inductive codes (Leech & Onwuegbuzie, 2007), adding codes as I analysed both the narrative interview texts and oral diary texts. I added to these codes when I found additional places that I had not listed in my a priory codes. I identified approximately 61 places (in Appendix 8) that subsequently underwent a process of rationalisation and reduction. I also identified which places were most important (most accessed or participated in) by each participant through using a diagrammatic exercise. 85 Places of social interaction Number of women who reported most participation in the places Sphere represented ESOL class group including via Whatsapp 6 Local Community Hospitals and GPs 3 Public Services Schools and/or nurseries 4 Part Local Community, part Public Services Home (including temporary home) 3 Private Local authority services 4 Public Services Ethnic community 4 Local Community Local community or religious organisation 3 Local Community Work 1 Work Business 1 Work Figure 8: Places of social interactions Based on the recurrence of participation in the place from the data, and importance given to the places in the data, I drew ‘diagrams’ or ‘maps’ of the places for each participant (see Appendix 9). Using these diagrams, I sorted places into groups either based on similar categories (e.g. nursery and childcare centre). The analysis for Part B2 and B4 was using thematic analysis, using Braun and Clarke’s (2006) method (see 4.6.2). For Part 2B, I analysed each place or group of places to produce a description of the places and the interactions within those places. The descriptions were using a combination of common knowledge of the places (for example the basic description of the local housing office), together with thematic analysis of participants’ data about those places. 4.6.4 Places of social interactions After compiling the descriptions of the places, I went through a process of renaming and regrouping certain places that I had initially marked separately. For example, I put ‘Community Group’ and ‘Ethnic Group’ together because of the description being similar when I was analysing the data. Where a place of participation was not physical but online or on the phone I chose to use the main group rather than starting up a separate group. For example, where Flora’s interaction with the council housing department was both in person and on the 86 phone, these have been grouped together under local authority services. This is because my reading of the data was that characteristics of the place carried on being true even when Flora was not physically in the council’s service centre. Phone, Whatsapp or other forms of online communications were seen as a medium for participation in the place, rather than being seen as a separate place. S See section 3.5.3 for a discussion of online spaces as an extension of places. 4.6.5 Identifying and describing social interactions To analyse parts B3 and B4 (identifying and describing social interactions and relationships within the places) I selected the main places identified in 4.6.4 for each participant and proceeded to organise data into these sections per individual and places: 1. Context 1. Context 2. Social interactions that took place- this identification was using typological analysis as described in 4.6.4 3. Players 4. Communicative acts 5. Language used 6. Power relations After this stage, I used thematic analysis (Braun and Clarke (2006) described in 4.6.2). After this identification and pattern recognition, I applied a descriptive process to ensure that each social interaction, player, communicative act and language use was described well. Following this was the interpretation stage where I theorised the significance of these patterns (Braun & Clarke, 2006) using the conceptual framework from Chapter 3. For example looking for patterns of participation in domains identified as places of markers and means listed in 3.3.3, players in the field and the different power relations between the players (3.5.3), and the significance of language use (3.5.2). This process of description and interpretation of themes was applied to the remaining stages. Once I had identified data and coding for each cell in the spreadsheet, I combined the different cells into a new table, which pivoted the themes identified earlier by places of social interactions rather than by individual participant. This process of description and interpretation of themes was applied to the remaining stages. Once I had identified data and coding for each cell in the spreadsheet, I bi d th diff t ll i t t bl hi h i t d th th id tifi d li combined the different cells into a new table, which pivoted the themes identified earlier by places of social interactions rather than by individual participant. The themes underwent a further process of interpretation using the conceptual The themes underwent a further process of interpretation using the conceptual framework. At this point I made a change to number 6- Power relations, and instead used this section to focus on social relationships within the places. p p g p framework. At this point I made a change to number 6- Power relations, and instead used this section to focus on social relationships within the places. framework. 4.6.5 Identifying and describing social interactions At this point I made a change to number 6- Power relations, and instead used this section to focus on social relationships within the places. 87 4.6.6 Participants’ perception of belonging Data from the final interviews with participants was analysed for their perceptions of their own experiences about changes since the beginning of the research project. Participants’ feelings about participating in the places Participants’ data was analysed for their feelings about participation in places, and categorised based on codes of relational belonging, material belonging and cultural belonging. Additional self-perception from a temporal (time-change) point-of-view was also recorded. Based on the interpretation of belonging being a feeling of ‘at ease’, positive evaluative expressions such as increase in confidence, feeling happiness, feeling comfortable and relaxed were coded as expressions of belonging. Negative evaluative expressions such as sadness, insecurity and pain were coded as not belonging. Data was organised for patterns and themes similar to the process described in 4.6.2. Themes from Part A were compared to themes from Part C in a process of interpretation, synthesizing, evaluation and hypothesizing in order to generate theory for discussion of the aspects of belonging. Participants’ perceptions and aspects of belonging Data manipulation was crucial in recognizing and identifying patterns. I organised the emergent themes based on the different places of social interactions and presented this in an iteration of the findings chapter. There were themes which were expected based on review of literature and previous research during the conceptualization of the framework. From the aspect of material belonging, it was anticipated that the data would confirm the importance of access to a community. From the aspect of relational belonging, the importance of friends and relationships with classmates was an anticipated theme. Within cultural belonging, it was expected that data would show the value of social interactions to learning about diversity and new cultural knowledge. In the temporal aspect of belonging, it was anticipated that social interactions would affect memory and changes in self over time. The process of interpretation, synthesizing, evaluation and hypothesizing mentioned earlier revealed new themes from the data, including the social interactions in employment and job-searching activities, access to public services, the importance of acquaintances and strangers and the affect that repetition and routine have on perceptions of belonging. 88 Further reorganization using tables and diagrams enabled me to reconsider the themes based on aspects of belonging such as in Figure 9 below. Highlighted themes indicate new themes from analysis of the data. Aspect of belonging Local community sphere Public services sphere Work sphere Material Belonging Access to a community Access to services Experiences of access to work Relational Belonging Importance of friends as replacement for family - Whatsapp as a tool for extending communications Building trust - The importance of weak ties The value of a customer-facing role Cultural Belonging Learning about diversity Language of bureaucracy Experiences of employment Temporal belonging: Rather than being located in one sphere, the temporal aspect is experience across spheres. Memory and changes to memory over time (previous experiences and life stories) Repeated experiences over time Routine and familiarity Figure 9: Participants' perception of their own belonging Data tables also supported me in drawing conclusions from the discussions, as it was easier to see the findings from a ‘bigger picture’ point-of-view. The process also enabled me to identify themes, which were not significant enough to include considering the limits on the length of the thesis. In this chapter I presented the methodological framework for data collection and management. 5. Findings and Discussion In this chapter, I present findings from the research. The chapter is in three parts: • Findings from participants’ narratives and discussion of the life story aspect of belonging (RQ 1) • Findings from analysis of the social interactions that participants reported, places in which interactions took place, and the nature of those social interactions (RQ2 and RQ3) • Discussion of themes related to aspects of belonging, which suggest the relationship between social interactions and belonging (RQ 4) Participants’ perceptions and aspects of belonging I detailed the stages of analysis and methods of data analysis used in order to formulate findings about the relationship between social interactions in English and sense of belonging. The process required careful staging and slightly differing analytical tools for the different research questions. In the chapter 5, I present my findings and discussion. 89 10 Religious clothing worn by some groups of Muslim women. 5.1.1 Participants’ life stories Participants’ experiences as newly arrived migrants in the UK, their situation at the start of the research, and their situation at the end of the research period is presented below as a part of their life story. Themes related to participants’ life stories and aspects of belonging, derived from narrative and thematic analysis, are presented in 5.1.2. Appendix 6 has a table of participants’ profiles that can be read in conjunction with these stories. As discussed in 3.5.4, life stories are important to the temporal aspect of belonging. Life stories are seen as not static records, but narratives that change as people experience and express their memories (Bennett, 2014). Apart from the experiences that participants bring with them when they moved to the UK, the experiences they have encountered as a newly arrived migrant, and throughout the period of the research, are likely to affect the perception of their belonging. Belonging is experienced in relation to time (May & Muir, 2015) and the framework shown in Figure 2, shows the temporal aspect of belonging as the basis within which all other aspects are experienced. In order to convey the pathways that participants experienced in their integration journey, these texts of life stories, derived from pen- portraiture have been used. As discussed in 4.6.2 pen-portraiture is part of narrative analysis and can also be used in the final written thesis to support dissemination of participants’ ‘perceptions, experiences and feelings in a lively, authentic, meaningful and accessible way’ (Campbell, McNamara, & Gilroy, 2011, p.15). This method is particularly suitable for ‘voiceless populations’ (Golsteijn & Wright, 2013, p.308) and so 90 meets the thesis aim to give a voice to migrant women. The abridged life stories are a tool for the reader to be able to learn about participants’ stories. Elma Elma met her British Somali husband in Egypt. She came to join him with their one- year-old child in 2012. When she arrived in the UK, she, her husband and her daughter lived in her mother-in-law’s house. She soon experienced controlling behaviour by her husband; he restricted her movements and made her fear an Islamophobic attack if she went outside in her abaya and hijab10. He told her they could not afford fees for her to study English and he did not give her enough money for basic expenses. She later discovered that he had another family. He threatened to have her deported if she complained about him. One day she left the house and met a woman who spoke Arabic. The woman advised her to go to a local Somali community group. She started studying English and got a job through the community centre. After her second child was born, she decided to leave him. Unfortunately, there were complications with her visa due to application errors by her husband. She left to live with her aunt and later went to social services. She was supported to get temporary accommodation for her and her family and to change her immigration status. She was provided short-term accommodation in Brixton and stayed there for five months. After that, social services moved her to Bromley. She tried to apply for school for her daughter in Bromley, but she was told they could not give her a place as she was still in temporary accommodation. At the start of the research project, Elma was living in temporary accommodation in Bromley, taking her daughter to school in Clapham (approximately 10 miles away), and then on three days of the week travelling to Hackney to drop her son off at the childcare provider before coming to ELATT for classes (an additional 9 miles). She was studying Level 1 ESOL qualifications. She also completed the Community Guides training, a programme to encourage and develop people to become area guides for other women who were new to London. She lived alone in a B&B with her two children and she received £200 per week to live and provide for her children. She was working on her application for Indefinite Leave to 91 91 Remain. She was being supported to apply for child maintenance payments from her husband. Elma At the end of the research, Elma reported a large increase in confidence. She attributed this to both an increase in cultural knowledge through her development in English and through the learning she had gained from friendships she had made and strengthened over the life of the project. She also commented on the role her ESOL course played in her English language development, friendships, learning cultural practices and supporting each other: “…When I…chat with the others, I felt like, ‘Oh everyone has a problem, but in a different way…” She had to overcome several unusual and serious situations such as the hospitalisation and surgery of both her children, and then her emergency surgery and hospitalisation from appendicitis. In these situations, she was reliant on friends she had made from her daughter’s school and old neighbours from her previous accommodation. She said she trusted her friends with her children and vice versa, and regularly had her friends’ children over for sleepovers. She said that the most important thing to her feeling comfortable was to know that if something happened to her, her children would be safe. She also gained her permanent right to live in the UK and subsequently needed to complete a large number of forms for different agencies to apply for benefits such as child benefit and housing support. In the past she had paid for help to complete these forms. Her friends encouraged her to try to complete the forms herself. When she achieved this on her own, she said she felt that she could now do anything. She felt that she could understand the service staff at the agencies better. Although there were still issues to overcome such as her daughter’s school location, she felt confident that she could get the situation resolved. “I feel like, oh now you improved English, you can do everything…I’m going to have a bright future.” Flora Flora’s family arranged her marriage as was their custom and she married a British Indian man. When she came to join her husband in 2009, she and her husband lived in a shared house. Her husband worked full-time and over the weekends, but they always spent one full day together every week visiting places in London. 92 92 Flora found voluntary work in a community centre. After a year, they liked the quality of her work and she started to work two-days a week, paid. She carried on working for a few months. In 2011, Flora received her Indefinite Leave to Remain. After that, she started volunteering in a school and started a course for Supporting Learning in Schools. At the time, she and her husband were trying to have a baby but encountered difficulties and they were advised to undergo treatment. Flora had to give up work as she suffered from ill health. They had a daughter, born in 2011. In 2014, Flora and her husband decided that she and her daughter would go to India before their daughter turned three. Unfortunately, her husband died suddenly and unexpectedly whilst Flora and her daughter were away in India. After her husband’s death, they were made homeless and had been living in hostel accommodation. They had a small single-bed room with shared bathroom and kitchen facilities. Her husband’s family had cut contact with her, only getting in touch on her daughter’s birthday and special occasions. Some of her husband’s friends were still in contact and they visited them when they could. She was receiving regular counselling support for her bereavement. At the start of the research, Flora and her daughter were living in the hostel in Newham. She attended an ESOL course at ELATT and attended a counselling session every Thursday. She also started a course to prepare her to become a teaching assistant. At the end of the research period, Flora reported improvement in her feeling about her language ability. She said she felt better about her language, and more trusting of her classmates. She felt they were friends and that she was comfortable with them. However, she still felt a lack of confidence to start conversations. However, she still felt a lack of confidence to start conversations. Flora She also reported more confidence to speak more at the GP or the bank compared to before when she would not express herself fully. “…I have to express myself, I have to tell it is right or wrong, so…I have to try…If he or she don’t understand then they can ask me again and I have to explain…so that hesitation is gone now.” Her housing situation had not changed and she was feeling increasingly frustrated with her situation. However, she did not report an increased ability to complain about the situation. She felt that she was spoken to by the services, rather than being updated on their situation or included as part of the conversation. 93 Flora reported that her daughter’s happiness was important for her, and she felt that her daughter was happier. She had been able to give her daughter opportunities to experience celebrations, to visit places on family days and have holiday experiences. Her existing family friends played an important role in supporting Flora to be able to do these things with her daughter, as they would drive them or go together with them. Flora continued to report being sad and down from the sudden death of her husband and continued attending counselling sessions. Lucy Lucy met her husband through an introduction by a friend. After getting married, she came to the UK in 2010 by herself, leaving her daughter behind. Her daughter was still completing her secondary school study. Lucy lived with her husband and his daughter (her stepdaughter) in London. Her husband worked as a kitchen porter and so had a low income. He did not give her enough money to run the household and so she supplemented this with her savings. She wanted to start working as quickly as possible but was unable to find jobs teaching Mandarin (a form of Chinese) at schools that would accept her level of spoken English. She relied upon the ethnic Chinese community to get teaching work, but unfortunately was regularly taken advantage of by people paying her less than minimum wage, or expecting her to work for free, as a favour. Lucy also had difficulties with her stepdaughter who bullied her verbally, and did things to her belongings. In their arguments, her stepdaughter was racially abusive to her and her husband also made racist comments. Lucy started studying ESOL and the Life in the UK test. She passed the test. She started studying on a teaching assistant course in Camden and came to ELATT to study ESOL. She also had made friends with an older English man and some English university students she had met whilst using public transport. They had informal agreements to practise language together as a language exchange- Chinese in exchange for English. At the start of the research, Lucy was still looking for job opportunities and trying to get as many qualifications as possible. Her daughter had moved over to join her and was studying at a UK university. Although they were not living in the same house, she saw her daughter more regularly. She was learning English at ELATT and studying on another vocational course at a different provider. She was also practising English with 94 an English friend and still regularly saw the older man who had conversations with her on her commute. Lucy reported an increase in her confidence using English. She said she was more confident to talk to different people. She felt that her pronunciation and listening skills were better, meaning that she could understand people and they could understand her better. Lucy However, she still felt a lack of confidence to converse in more complicated or in-depth interactions. Lucy gave the example of conversations with people on a personal level, where she perceived they did not have interests, backgrounds or education level in common. In addition, she reported lacking in confidence to participate in job interviews. Lucy reported many trust issues with making friendships. She said that she felt alone but at the same time reported that she did not want to befriend people as she had been let down by previous friendships. She felt that the lack in confidence in people had led to a lack of confidence in herself. She also felt that her ESOL classmates were not able to be her friends because they did not share the same ideas or education levels and interests. “… but you know, (to) make friend…you really need the same idea and the same life,…you can’t change.” “… but you know, (to) make friend…you really need the same idea and the same life,…you can’t change.” “… but you know, (to) make friend…you really need the same idea and the same life,…you can’t change.” Lucy had been looking for work for the period of the research project and had recently become friendly with a lecturer from UCL. The lecturer was trying to help her secure some part-time teaching at the university. Lucy said she had become more selective about work she accepted, after the job she worked over the summer, where the employer treated her badly. She now only accepted tutoring jobs for children whose parents had been to prestigious universities. Samira Samira married her husband through an arranged marriage and came to the UK in 2011. When she first arrived in the UK, her husband was helpful and did a lot to help her learn about how to travel, go to the GP, go to the shops and other activities. She described him in a positive light as a person who supported her well when she first came to England. In 2012, her husband got depression and he was also diagnosed with a form of PTSD. She had to do quite a lot of the household management and took charge of the day-to- day running of the household including visiting the council offices to complain about her 95 housing situation. They had two children and at the start of the research, she was pregnant with their third child. At the time of her first interview, she was living in a one-bedroom flat. All four family members were sleeping in one room. All her neighbours were older people, living individually in one-bedroom flats. It was a quiet area, mainly reserved for elderly people, and the residents complained about her children. The flat was on the third floor of a council block without a lift and this required her to carry her buggy up flights of stairs. Samira started ESOL classes in 2011 when she first came to the UK. She started an Entry 2 class at, an education charity in Hackney. Samira also had interrupted learning because she stopped when she got pregnant with her first child. She re-started learning at a Hackney women’s project, but when the charity closed down she was referred to ELATT. At the start of the research project, Samira had been working hard to get a better place to live. The family was still living in over-crowded temporary accommodation. She was studying on a childcare course and a Level 2 English course at ELATT. Samira reported a large increase in confidence at the end of the research. She felt that she could solve problems, she could now find answers online and that she could now do things for herself, and even helping others with their problems. She had become impatient with the delays to her housing situation and found out about local councillors from other people in her groups of friends. Samira She wrote to the local councillor who investigated her case and with further follow-up from Samira, she was moved to semi- permanent accommodation not long after she had her baby. She also had to deal with the birth of her child alone, as her husband was at home with their children. She applied to change her son’s school, passed her driving test and applied for allocated parking from the council. These tasks all involved bureaucratic processes such as form filling or procedures to follow and Samira said she felt good that she was able to do these tasks independently. She attributed her increase in confidence to the support she received whilst on her course at ELATT, the increased language skills, knowledge about rights and cultural knowledge, and the support of the people from the groups she knew. She felt that her friends were important as a replacement for family. She felt that friends could give different ideas and perspectives for solving problems and she had made many friends from various classes and groups that she had participated in. In her 96 new home, she regularly hosted neighbours and said that she quickly felt comfortable in the new area through interacting with the neighbours. “That is why…I’m feeling, happy with my friends, when I’m (in) touch…messaging…I’m feeling better. That is why it’s important to…have more friends. If I don’t have friends maybe if I…have some problem I can’t solve, I can’t share with no one. That’s why I want to-, I make friends.” Samira reported regular and confident use of social media and online communications with friends. She said that keeping in touch via Whatsapp had greatly helped the relationships to grow from regular social interactions. The group of classmates would only meet each other during class times and interactions would not have extended out of the class. At ELATT, she said that the teachers’ encouragement for students to participate in a VLE (virtual learning environment) and Whatsapp group contributed to classmates interacting with each other on non-class days. Asma Asma’s family arranged her marriage to a British Pakistani man. She came to join him in 2014. Asma lived at her in-laws’ house with her husband when she first arrived. She was expected to help with house chores and with caring for her father and mother-in- law. Her husband worked full-time so she was at home with her in-laws alone much of the time. She was expected to be home all the time and had her movements monitored by her mother-in-law. She had to ask her husband for money and did not have any income of her own, as she was not working. She was not used to being reliant on someone else for money. She started studying at ELATT and made friends with classmates. She also started looking for work. At the start of the research project, Asma was suffering from stress and not coping well. She was depressed. She studied at ELATT on three sessions a week but had to leave promptly after her sessions. Asma had to leave her studies for several months because of her personal situation. Asma came back to the research project near the end of the data collection period. She had overcome all of the previous issues, which had caused her to suspend her learning for a period. She was no longer experiencing controlling behaviour at her in-laws’ home. She reported that the support from her wider family and cooperation from her husband meant that she was able to take the time she needed to evaluate her 97 situation, and make changes for herself. She reported trying to talk about difficult situations more at home. She was also able to access health services she needed. Asma reported that her friends from her ESOL group were supportive and helped her overcome her personal challenges. When she went away, they kept in touch via Whatsapp. situation, and make changes for herself. She reported trying to talk about difficult situations more at home. She was also able to access health services she needed. Asma reported that her friends from her ESOL group were supportive and helped her overcome her personal challenges. When she went away, they kept in touch via Whatsapp. Asma reported that her friends from her ESOL group were supportive and helped her overcome her personal challenges. When she went away, they kept in touch via Whatsapp. Asma “Now I, I can talk…I’m sharing with my friends everything. Before that…you know, I keep in me, everything I keep in me so…at the end of the day…I’m crying. Crying all the time.” Asma identified that gaining a job made a great difference to her increase in confidence, because of the social interactions that she was having with people. “When…I started my work. Every day I meet with different people, I make friends, so I speak with them and I’m more confident, becoming more, more, more confident every day.” She reported feeling brave and confident, and that ‘happy times were back’. Noor Noor and her husband moved to London together in 2013 because they feared religious intolerance in The Netherlands. She did not know many people in the UK. She had a lot of support from her husband because he spoke English fluently. She used online sources for information. For example if she wanted to visit a mosque, go shopping, or visit a government department, with the help of her husband. She found it difficult to access services or get a job, as the processes were different to those in The Netherlands. She felt that she had more freedom in the UK, as she was comfortable to wear Islamic dress. At the start of the research project, Noor was studying on an ESOL course with ELATT. She was looking for opportunities to start to provide a service to help women with alternative therapies. Her aim was to be to get closer to God. During and at the end of the research period, Noor reported that she was more relaxed and comfortable with her classmates and she felt that her increased confidence in class, positively affected her confidence out-of-class. However, she still felt concerned about trusting people too quickly. She also felt that although friends could be relied upon for help in place of family, they could never truly replace family members. She felt that it was easier to interact with people who were of the same religion, as they would 98 have something in common but that she was happy to meet with people of different religions although it would depend on the personalities of the people. have something in common but that she was happy to meet with people of different religions although it would depend on the personalities of the people. Noor had been offering alternative therapies services to women in her mosque community and she felt good that she was able to do this. She felt confident to offer this treatment wanted to continue improving her skills. Noor still felt less confident to access different places because of the way she dressed. She felt that certain places were not accepting of her being dressed in the ‘niqab’ religious clothing. She felt comfortable living in the areas that she knew and had become familiar with. Watching the news, she felt concerned about the rise of Islamophobia in London. Noor However, she said that she felt more comfortable in London compared to her previous home in the Netherlands, where she felt persecuted for her choice of religious dress. She said she felt that English people treated people well and were polite. “Yeah, there is a bit of change. I’m more, like, confident because you have no one here, you are in the…environment, and…you know the places. Yeah, you’re more confident.” 5.1.2 Participants’ experiences as newly arrived migrants affecting their belonging As discussed in the literature review, an individual’s starting point in SLA is influenced by factors such as their previous experiences of learning, their school attainment, previous work experiences, literacy and skill in first language, age and other influences (Duff, 2007; Kings & Casey, 2013) (see section 3.2.1). In second language socialisation this has been described as an individual’s first language socialisation (Pavlenko, 2004). Therefore, a person’s previous socialisation will also affect their integration process and progress towards feeling belonging. The life story aspect (see section 3.5.4), was identified as one of the aspects of belonging, and is discussed in relation to the participants of the study and the experiences up to the point that they started on my research project. The following themes emerged from the data analysis of life stories. Each of these themes is related to the aspects of belonging discussed in 3 5 3.5. 99 Previous experiences of life changes- temporal belonging Some participants presented as more independent and more able to access help more than others. For example, Elma and Samira talked about their pathways with more confidence and described several challenging situations that they had overcome. Elma was able to leave a controlling husband and Samira was able to support her family and care for her husband’s health by herself. Both women had refugee backgrounds. They had early experiences of loss of family members, and of having to look after siblings and working. It may be that their previous experiences of learning to live in another country or to be in charge of their family units gave them transferable skills to settle in the UK. As discussed in 3.5.4 temporal belonging is the relationship between memory and self and time (May, 2016). Therefore, I suggest memory of experiences overcome in the past can affect a person’s efficacy. Efficacy is the feeling that a person has control over outcomes (Stillman & Baumeister, 2009). Therefore if a person has a memory of a previous positive outcome, they could feel efficacy in future. In the situations of Elma and Samira, the life story aspect could have affected their resolve to overcome situations they encountered, and I think this could be interpreted as resilience and resourcefulness. Additionally, data suggested that memories of experiences in the past could influence how a person might react in the future, the level of confidence with which they do so. Elma’s previous impression of the UK, formed when she lived in Egypt, gave her confidence to leave her house in London when her husband instructed her not to. Elma’s confidence to reach out to a stranger who spoke Arabic may have been influenced by her recognition and familiarity with people she had grown up with in Egypt through her temporal belonging. Noor’s confidence with her new area was based on previous familiar experiences of a similar European country (The Netherlands). However, for participants whose prior experiences were limited to places that were different to the UK, and those without familiar systems, they felt less confidence and ease in their new area from the lack of life stories to contribute to their temporal belonging. 11 See Appendix 3 for ESOL descriptors Previous experiences of learning English- cultural belonging All participants had come to the UK within the previous six years and had learnt English to at least a basic level in the countries that that had previously come from. This was through learning English at school as a subject (Flora, Asma, Elma and Noor), studying English to a higher level at university (Lucy) or learning a basic level of English through 100 popular culture and family members (Samira). Elma had experience of learning an additional language as a young person when she and her family fled to Egypt. Some participants had continued to learn English (through studying ESOL) with other colleges/providers before and had progressed (Samira, Lucy, Elma), whereas others were new to an ESOL class as they had either recently arrived (Asma), or had not joined a course before (Noor and Flora). In spite of participants’ levels being broadly similar, working towards Level 1 in ESOL (B2 CEFR)11, there seemed to be a lack of confidence in their own language ability. “When I arrived in UK, I faced…one challenge. Language, this language, English.” Asma “When I arrived in UK, I faced…one challenge. Language, this language, English.” Previous pattern of social interactions outside of class- relational belonging Apart from Samira and Lucy, remaining participants reported low participation in social interactions outside of their class at the start of the research. Noor and Flora expressed a reluctance to make friends as they felt that it took a long time for them to trust other people. Lucy had friends whom she had met in public places such as bus stops, trains and at the library. She participated in ‘language exchanges’ with the friends, in which she taught them Chinese, and they helped her practise English. Asma was isolated due to her family situation, and Elma was unable to maintain friendships, as she regularly had to move accommodation. These reasons for not taking part in social interactions were influenced by more than just perceived language levels. Barriers such as difficult personal situations or precarious living arrangements affect a person’s ability to participate in social interactions. As discussed in 3.5.3 family and friends play a large role in giving meaning to a person’s life (Stillman & Baumeister, 2009) and being part of a group creates social solidarity (Siisiäinen, 2000). Participation in social interactions with others affected participants’ early feelings of relational belonging. Of the participants, Samira reported having the greatest number of friends such as parents she knew from her ‘Stay and Play’ group and nursery, and friends made on the ESOL classes she had attended in the past. Although she did not have a local ethnic community group, she felt confident that she had support from others. In contrast, Elma received support from her own ethnic community and with this support was able to leave her husband. When Elma was moved she lost the regular support of her ethnic community group. In 3.5.3 it was discussed that migrants usually search for those from the same ethnic background due to trust (Tanis & Postmes, 2005) and perception of support (Morrice, 2007). However, based on the experiences of these participants, the nature and quality of that support might not be good. Lucy reported seeking out people from her home community group (the Chinese community) but did not receive the support she needed. Asma, Noor and Flora were reliant on their husbands for support and the outcomes were mixed. Therefore although support from own ethnic community group is important for newly arrived women, it is important not to assume the support would be suitable and appropriate. Asma As discussed in 3.5.2, the knowledge of English and confidence to use the language plays a significant role in cultural belonging. Participants were developing their language at a level that should have allowed them to access places, and to the cultural information contained within those places, but they all expressed a lack of confidence in their language ability. The ways that they had previously learnt English may have played a part in their lack of confidence. Participants who had not studied English in the UK might not have learnt the language within the context of everyday use. In my professional role, I have encountered students who are able to pass reading and writing tests, but unable to use English in interactions as they had learnt the language using online methods, or classroom worksheet activities. Of the participants, Samira, Elma and Asma exhibited the most confidence to interact in English early on in the research. These three participants had reported learning English by talking to others such as family members, through popular culture on television or radios, or through use of local English in their country of origin. Therefore, the knowledge of English that these participants had gained was developed through interactions with others, and not just through classroom lessons. As discussed in 3.5.2, culture and communication cannot be separated (Fortman & Giles, 2005). Therefore if migrants had learnt language as a subject, separate from cultural elements unique to the UK, it is likely that their cultural belonging was low. Participants’ sense of belonging was affected by their confidence using English in the UK, and some participants felt more confidence than others. 101 Previous pattern of social interactions outside of class- relational belonging However, the knowledge that they are able to get help and support may affect their confidence levels. Relational belonging is affected not just by the relationships with others in the community (whether of a similar background or different) but the support that newly arrived migrants feel that they can get from those relationships. 102 Home environment situation at the start of the project- material belonging If belonging is a ‘feeling at ease with oneself and ones surrounding’ (Miller, 2009), then it is likely that safety and security will also impact a person’s feeling of belonging (Sumsion & Wong, 2011). In Ager and Strang’s indicators of integration (mentioned in 3.3.3), safety and security was listed as one of the domains to facilitate integration, as if a person does not feel secure and safe, they may not be able to feel integrated (Ager & Strang, 2004). In the belonging framework, safety and security is related to place, one place in particular being the home. All participants reported issues with housing, or not feeling unsafe/uncomfortable at home. Three participants were living in temporary accommodation: in hostels with inadequate cooking/living facilities (Flora), or in temporary housing that was overcrowded (Samira) or temporary housing that was not suitable for their children’s schooling situation (Elma). Lucy experienced bullying at home and Asma experienced controlling behaviour. Noor reported issues with tenancy and house sharing. Material belonging is defined as being able to access places and be comfortable in those places (section 3.5.1). It is likely that the lack of being comfortable with their home affected their material belonging. In my study, material belonging was related to places outside of the private home environment based on my research focus, which was social interactions in English. Although participants may have used English language at home, it was difficult to ensure that they would only report interactions in English. Therefore, the focus was on social interactions in places outside of the home. However based on the data, the affect of home to material belonging, and to an overall sense of belonging is an area that could be further researched. Prior experiences of support from public services- material belonging As newly arrived migrants, they had received a lot of support from organisations, local healthcare services and local authority services. There were several interactions reported with professionals and workers of this sphere. The interactions were considered social interactions as they were situated in the social world (discussed in Section 3.6.2). In these interactions, the data suggested there was some imbalance of power based on the roles of the people providing the service (for example a housing officer appeared to have absolute control over whether someone is awarded housing). These interactions showed that there was access to support by participants and also indicated that they had gained sufficient cultural knowledge to exercise their rights to get the support. However, support they received did not seem effective. Participants reported being able to ask the particular case worker/ health worker for support but 103 none of the participants escalated their requests when their cases were not resolved. Participants could therefore be seen to have material belonging to a degree, as they were able to access the services that could get them help to solve their problems. Barriers to social integration My research did not intend to study the barriers to integration that people face. However data in the above section confirms existing research about barriers to integration (Ager & Strang, 2008; Dimitriadou, 2010; Morrice, 2007). The examples from my research are housing issues (Flora, Samira, Elma, Noor), confidence using language (all), childcare and caring responsibilities (Flora, Asma, Elma, Samira), family situations (Lucy, Samira, Elma), lack of work opportunities (Lucy, Asma, Noor) and health and wellbeing (Asma, Flora). 5.1.3 Participants’ early sense of belonging Participants’ early sense of belonging was affected by their social interactions in places that they were already participating in. Literature about needs of newly arrived migrants is usually focused on functional needs such as where to access services such as housing and ESOL classes (Baynham & Simpson, 2010). The analysis of migrant women’s early experiences shows that all migrant women were already accessing services with a good degree of confidence. However, there were differences in the perception of belonging of the participants. As discussed in 3.5.4, Miller (2009) and Hedetoft (2002) note the importance of history in the formation of identity and belonging. This is part of the temporal aspect of belonging shown in Figure 2. Participants with previous experiences of migration and life-changes and those with more experiences of interactions and support from groups of people were more confident in the relational, material and cultural aspects of belonging. The next sections presents analysis of places in which social interactions take place, the nature of those social interactions, and how those social interactions affected participants’ sense of belonging from the different aspects of belonging. 104 5.2 RQ2: What are the places in which social interactions take place? The places of social interactions identified in the data have been categorised under four main spheres: Local community: • ESOL class group (informal such as the non-teacher led conversations) • Schools and nurseries (informal elements such as meeting parents at school gates) • Ethnic community or religious community (for example a Somali community group) • Local area community (for example a local park group) Public services: • Hospitals and healthcare services (such as GPs and health visitors) • Schools and nurseries (formal elements such as paperwork and meetings) • Local authority services (such as the Housing Department and Jobcentre Plus) Work: Work: • Employer workplaces • Job-searching and prospective work • Job-searching and prospective work Private: Private: • Home • Home The ESOL group Participants were all part of the same ESOL course. As part of the course, the teacher encouraged students to continue communicating with one another outside of class time. Students would interact with each other before and after class, and during their break times. The teacher further enabled out-of-class communication by introducing a virtual learning environment (VLE) platform, and suggested they form a Whatsapp group. Some students in the class had been on previous courses with ELATT and others had only recently joined the course. Ethnic and religious community Elma received support from a Somali group, which helped her find English classes, and then later with form-filling and advice. The group also had a social element, organising celebrations for Somali diaspora living in South London. Elma only periodically visited after she was moved further away from the area. Noor was involved in religious activities at the Local London Mosque, attending the mosque daily, and more than once a day. She described a serious environment, somewhat formal place, where people were focused on worship. However, there were other spaces within the place, which were more relaxed. There were study groups, which also had a social element. She met other women and was able to ask for advice if she needed it. She also described being invited to join people for tea, coffee or meals before or after services. Schools and nurseries (represented in both the Community and Public services Three participants had children in school and nursery. The majority of the parents’ participation in school/nursery was in two places: at the school building with school administration itself (management, teachers and policy) and at the ‘school gates’ with other parents. The formal place was included in the public services sphere (5.2.2 below). Samira and Elma both had two children, one child in school and the other attending nursery. Samira also attended additional activities such as ‘stay-and-play’ groups at the nursery. Flora had a daughter in school. Participants did not describe places in this sphere as their physical Participants did not describe places in this sphere as their physical Participants did not describe places in this sphere as their physical school/college/community buildings. In the ESOL group, the non-teaching and learning part of the ESOL class was described as the place in which the group interacted in the 105 absence of the teacher, or teacher-instructed activity. As mentioned, this was in the time before and after the class or during breaks when students were in the student café and the ESOL course provider’s common areas, and when the group was not attending classes in the formal classroom deli pattern. Participants also described activities taking place outside of the provider space such as when members of the group met up in parks, cafés or in houses. There was also an online aspect of the group, which was not physically bound as it was using apps like Whatsapp and the teacher-led VLE. 5.2.2 Public services sphere The places represented in the public services sphere are local authority services such as the housing office, hospitals and GP surgeries, the Job Centre, the Citizens Advice Bureau and schools (e.g. in formal teacher meetings and with administration). Based on the similarity of descriptions and types of social interactions, the places are presented jointly as a sphere. 106 The spaces of the public services sphere were described as highly formal and there were clear division of spaces that were authorised for physical access to those with permission. There were many instructions to follow, usually stated on signs and posters. The spaces were not designed to be comfortable and are usually sparse and cold. Hospitals and healthcare services, Local authority services Four participants described several interactions with hospitals and GPs and four participants reported interactions with authorities responsible for housing, immigration, social services and advice services. Elma and both her children had serious illnesses and accidents causing hospitalisations. Samira was caring for her husband who had PTSD. Additionally, she was pregnant and gave birth during the period of the research. Asma and Flora had support for depression. Elma, Noor, Flora and Samira needed housing related support. Samira and Elma also had immigration support needs. Schools and nurseries At their children’s schools or nurseries, participants did not necessarily report this place as the physical school building although the classroom and office were mentioned. Elma and Samira also talked about the local locations of schools. They had both been moved by council/social services and they were unable to move the their children to more local schools. While the school gate and PTA meetings were described as quite relaxed and informal, dealings with the school office and meetings with teachers (parent-teacher meetings about children’s performance) were described in a more formal and transactional way. 5.2.3 Work This is a formal sphere. Two participants (Asma and Lucy) reported several interactions in the work place and in the act of looking for work. Both participants experienced different employment types. They reported similarities and differences with work in their home country or previous experiences of work. Both Asma and Lucy were able to get casual work, for which working hours were not guaranteed and subject to change with little notice. Asma left her first job because she was unhappy with the shifts she was given. She quickly found another job in the same type of job but with better shifts. Asma worked in small beauty salons, for individual owners. Lucy was paid at much less than minimum wage for teaching jobs and Asma never received an employment contract. Lucy also accepted a short-term role of a nanny on a yacht for a six-week period during the summer holidays in 2016. 107 Based on their experiences of looking for work, the place ‘job-searching’ was identified. The place was incorporated into the ‘work’ sphere, as there were similarities with work place. Although job searching did not have a physical space, I interpreted the job- searching activity as a place based on the cultural, physical and material elements of the activity, and the interactions between employers and participants. Lucy attended interviews for teaching jobs, and later tried to start a language school and language classes of her own. Home This is a private space, usually in the homes of the husband’s family, except in the cases of Flora, Samira and Noor, when they first moved to the UK. Later Elma also moved out of her husband’s family accommodation. All participants except Noor had come to the UK to join their husbands, after a period of separation, some waiting up to two years for visa approvals. Additionally, they all had not spent much time with their husbands. Therefore, additional to learning about their new life in UK, they were also learning to live as a married person. Only Lucy talked about the difficulties of living with someone when they did not seem to have things in common with each other. The multiple socialisations into roles and place were likely to also affect the women’s perceptions of belonging. 5.2.4 Private This is an informal sphere and includes the homes of participants and family member or close friends’ homes. 5.3 RQ3: What is the nature of the social interactions? In order to understand the nature of the social interactions, four sub-questions were applied to the data analysis: • Who are the people within the places? • What are the activities that take place within the places? • What types of social interactions take place within the places? • What are the social relationships within the places? Players Participants described their involvement in schools/nurseries in two areas: school gates where they dropped off their children and the parent teachers’ association (PTA) membership and meetings. The data shows ‘the school gate’ as a tangible concept. This is the usual coming-together of parents dropping-off at school in the morning or collecting their children at the end of the school day. This was described as a time when parents talk to other parents and children. The PTA meetings were held in a meeting space within the school grounds. The research participants described their ESOL group as a place with a diverse mix of students- of the 11 students in the cohort there were 8 country nationalities represented with different ethnic backgrounds. Membership of the group provided a lot of opportunity for interactions in the common language they were learning- English- with people who were from different backgrounds and spoke different languages. Participants’ abilities and previous learning experiences were diverse meaning that the group consisted of people with different perspectives. Places excluded from research Two places of social interaction presented complications at the analysis stage. The aim of my research was to examine the social interactions that participants took part in using English. However there were two main places reported in the narrative interviews, which were problematic due to the lack of clarity of the language that was being used, or where participants reported there were multiple languages being used, including English. One of these, the home, was mentioned in earlier in 5.1.2. The second place was ethnic community organisations including religious organisations. A recent report on Indicators of Integration suggested relationships with people who shared similar cultural backgrounds, practices and faith (usually close family members) contributed to a sense of belonging (Ndofor-Tah et al., 2019). Therefore, I think there is potential to explore these places in other research. 108 Additionally, in early analysis of data, it appeared that women with more support from husbands and families were more likely to develop confidence in English and take part in more social interactions. Bearing in mind the point made earlier about socialising into new roles as married people, as well as being a new migrant, it appears that there are challenges to integration for this group of women, that has not been researched previously. Samira and Flora reported that when they first arrived in the UK, their husbands taught them how to use public transport, encouraged them to join activities in the area and supported them to practise English. Samira’s husband taught her English phrases and encouraged her to join ESOL classes. Flora’s trajectory was positive in her first two years in the UK when her husband was still alive. She started a paid job and secured a role volunteering as a teaching assistant. Based on this early analysis, it seems that this would be an interesting area for other research in the future. 109 Activities and practices In the school setting, all participants reported lots of opportunity for interaction with other parents through the school relationships, either at the school gates, through the PTA meetings or other organised activities. Elma reported a steady increase in friends made through her daughter’s friendships at school. Samira was also successful in starting relationships with other parents through her involvement in stay-and-play and early years groups. Flora did not have the same relationships with other parents, although she described many opportunities to interact. The main activity of the ESOL group was interacting with and supporting each other. The activities in this place are discussed in the section on social interactions within the places. 110 Social Interactions and Relationships: ESOL group Many of the early social interactions reported were about getting to know each other as they started to form relationships with each other. Using English, they described and talked about themselves by giving and receiving personal information. They also talked about their lives and personal situations, and future aspirations. Part of these introductory interactions were likely to be teacher-led as part of building rapport with students and promoting collaborative classroom behaviour. These early teacher-led activities extended to subsequent out-of-class interactions. The places were generally described as a democratic place of participation. Participants described that some people had more experience in some areas, and others in different areas; therefore everyone brought knowledge to the place. Participants reported that talking to people from different countries was challenging and talking to other classmates was a way to practice speaking and listening to each other’s accents: “To communicate with other people from different countries…English is the main language that we use, so we only speak in English.” Elma “To communicate with other people from different countries…English is the main language that we use, so we only speak in English.” Elma “Sometimes to understand the accent is difficult” Samira “Sometimes to understand the accent is difficult” Samira “Sometimes to understand the accent is difficult” Samira All participants reported misunderstandings related to accents that they encountered in daily life and the exposure to different accents in the ESOL group helped them develop better listening skills. For four of the participants the only language of communication they could use was English as none of the other students in their class shared a common language. The other participants reported a conscious decision not to use their own languages to include other classmates in their conversations: “We speak English because, you know, Nana, Mehra...they don’t understand.” Samira Large common language groups sometimes made students from other backgrounds/language groups feel less included - Elma noted that some students who spoke the same language would stay within their language groups in the break and possibly meet up with each other without involving all the other students. In contrast, same-language groups felt more inclusive for Flora- Bengali speaking students were easier for her to communicate and feel comfortable with. For Asma, Muslim and Asian students were easier for her to communicate and feel comfortable with, and to share experiences and get advice from. However, Samira noted that there was a conscious effort to ensure the non-Urdu speakers were able to understand, so they talked in English, which was the common language. 111 Perception of differences between people or groups was a barrier to communication for some participants. For four of the six participants, the UK was the first place they lived where they met people from cultures and backgrounds different from theirs. In the ESOL group, Lucy talked about not understanding Muslim students because they did not have the same experiences as she did. She had previously only mixed with people who were university colleagues in China. She said she felt like she and the Muslim students had nothing in common. Flora (in the early stages of her course) and Noor reported that they self-segregated by keeping to themselves because they felt they could not trust others. Participants also recognised that they were able to practise grammar and accuracy, whilst they practised speaking: “(My classmates) they are very helpful…If I stuck somewhere…they trying to complete my sentence. “Sometimes to understand the accent is difficult” Samira So…I learned a lot with my friends.” Asma Practising fluency within a safe group was important to allow them to internalise the language they had learnt resulting in confidence to use English in other situations and with other people outside of the ESOL group. Participants used online platforms to extend communication. Participants used online platforms to extend communication. “We did Whatsapp chatting…if she want to know something or I want to know something.” Flora Whatsapp and the VLE were used by the participants to text and record speeches to communicate with each other in English. Elma, Samira, Flora and Asma reported that this was a method of practising English with each other outside of class. However, not all students participated in the Whatsapp group equally. The students who participated in the Whatsapp group seemed to be composed of the students who were from South Asia and the Middle East. The Chinese and Spanish speaking (non- EU) students did not participate in the same way. Lucy did not participate in the Whatsapp group however, she also reported a lack in confidence using digital communication and that she did not have anything in common with the other students in the class. Apart from practising English, social interactions were also about developing knowledge about life in the UK and supporting each other. They talked about their new life in the UK. They described and compared experiences of living in the UK, cultural differences between them or that they had experienced, and discussed laws and common practices. 112 “I learned from my (classmates)…about the cultures and the people…In this country it’s okay if you…put your finger like this (shows a gesture) and…it means…‘Come here.’ So it’s not a big deal but…in my country it means very bad.” Elma Participants reported sharing information about popular topics such as celebrations in the UK, learning about each other’s cultural practices, and information related to driving tests and Life in the UK tests. Although the topics in the class supported participants to learn a great deal about citizenship and life in the UK, the discussions continued outside the classroom and involved participants sharing personal experiences additional to those discussed in the class session. “She tell me, ‘I have too much problem, I can’t go outside by myself and I can’t read the form, fill the form…the council, doctor and my husband as well doesn’t help me.’ She requested, ‘Can you help me, please?’” Samira (talking about another student asking her for help.) They also practised asking for, giving and receiving advice from each other. Participants used online platforms to extend communication. As a follow-on from the previous type of interaction, those who had experienced situations, such as passing the Life in the UK Test, would give advice to others in the group. Participants reported discussing challenges they faced in their lives and giving and receiving advice to support each other. Whatsapp communication enabled them to send messages to each other for information or advice outside of the days they met in class. This meant that as well as feeling supported at any time when they were away from the group. In the ESOL group, people who had been through more experiences and knew more about systems of public administration e.g. benefits, driving tests, housing, seem to have been listened to by others more. Participants like Samira, who had passed her driving test, were perceived by others to be more knowledgeable mainly from personal experiences and expertise in navigating systems. “I can communicate with the society now.” Elma Social interactions within the ESOL group played a role in three main ways. For participants, this was the first site of authentic language practice and hearing the different accents that English is spoken in. As mentioned in 5.1.2, participants’ level of English should have enabled them to feel confident. However, the diversity of English that they encountered in London is likely to have affected the confidence to use English. Practising and learning from each other supported the development of confidence to use English out-of-class. Social interactions also were the vehicle for learning about new culture and citizenship- 113 related topics. Social interactions enabled migrant women to receive and give support to each other, and share experiences. On days when they did not meet, online social interactions enabled them to continue practising language, giving and receiving support and sharing experiences. As shown in the framework diagram (Figure 2) and discussed in 3.2.1 and 3.4.2, the process of socialisation is contingent on learning to use new language, through the use of the new language in the field (second language socialisation). The social interactions in the ESOL group highlighted in the findings shows this process of socialisation, including the development of language and cultural knowledge that affected participants’ identity. Social Interactions and Relationships: The School Gate In the school setting, similar ‘getting to know you’ and daily small talk interactions as with the ESOL group took place but with a focus on participants’ children’s relationships and activities. There were also similar interactions related to helping each other. The data showed a variety of interactions which were informal and related to friendship-building for example taking part in gossip, speaking about what happened to them that week before/after school/ESOL class, and the opportunity to share funny stories or frustrations. Elma said she eventually found the confidence to approach other parents at her children’s school because of her confidence from the ESOL class. Samira was more confident and talked about several friendship groups she had made from previous playgroups and nursery that her son had attended. Flora however, reported limited confidence to start conversations with other parents. The school gate was an intimidating place for Flora as she found starting conversations with other people difficult even in her own language. There were two other parents who spoke either Bengali or Gujarati, her home languages. She reported a slow development building relationships with them and none with English-speaking parents at the school gates. “I just stand there, watch my girl play. I couldn’t say anything.” Flora For Elma and Flora, low confidence in English language was a barrier to building social relationships with other parents who were fluent speakers. At the school gates, participants reported being invited to children’s birthday parties as soon as their children started Year 1 at school. Flora was reluctant to attend birthday parties but took her daughter as it was expected. She reported feeling unable to mix with other parents at the parties. Earlier in the research, Elma reported being afraid to talk to other 114 parents. She felt that she needed a better command of English and she saw them as having a higher status than her because of their language ability. parents. She felt that she needed a better command of English and she saw them as having a higher status than her because of their language ability. Some participants reported use of Whatsapp to communicate with each other. As with the previous section, the use of online social relationships supported the interactions to continue past the school gate, and endure. “We have a Whatsapp group and…we contact each other, we message each other and we meet, we bring our children together, we have picnics sometimes together.” Elma “They are missing me, they ask when I am coming to see them” Samira. Samira’s friendship group (from her child’s previous nursery and Stay and Play group) contacted her on Whatsapp to ask her to meet up, suggesting that those relationships were still maintained although her son had started school. Elma participated in the PTA for her daughter’s school after another parent encouraged her: “At first I was like ‘No, no, no,’ because I was afraid. I was, like, ‘If I go there, I can’t speak…English well, so what shall I tell?’ Then she said to me, ‘This is your child’s school as well. You…need to know what’s going on.” Elma “At first I was like ‘No, no, no,’ because I was afraid. I was, like, ‘If I go there, I can’t speak…English well, so what shall I tell?’ Then she said to me, ‘This is your child’s school as well. You…need to know what’s going on.” Elma Prior to this, she had no knowledge of what a PTA was. Through interactions with other parents she found out about the PTA and became aware that she has the option to attend it. Players The roles of the medical professionals and administrative staff in hospital and GPs are defined. It was clear that staff had important and critical tasks to do and so it was a formal environment with little opportunity for meaningful social interactions. Participants saw doctors as possessing a lot of knowledge, nurses less so. In this sphere, administrative workers were the ones that participants communicate with the most. These were hospital receptionists, admissions team staff or caseworkers in local authorities and the Home Office. There were also other customers or patients in the sphere. However, communication between these groups was not common or encouraged due to privacy. “I just stand there, watch my girl play. I couldn’t say anything.” Flora Later in the research, Elma reported the most number of friends that she had made were from her daughter’s school. She said that they all spoke different languages so they all communicated in English and that they met up and did things together outside of school. Samira also reported many relationships with other parents made from the nurseries and schools that her children attended. Social interactions in the school setting supported newly arrived migrant parents to take part in what seemed like frivolous chat (gossip, the activities of the week) but this served to socialise them into the group of parents and to build relationships and trust with them. Social interactions also support them to learn about culture related to schools’ and other parents’ expectations of them in the UK school system. Similar to social interactions in the ESOL Group, the school gate also supported the socialisation of participants. Part of the process was the experience of routine and repetition as per the discussion of temporal belonging in 3.4.1. Belonging develops when there is a ‘comfortable and familiar’ feeling in a place (Yarker, 2019). 115 Activities and practices The activities of the school are formal: there were expectations concerning attendance and punctuality, completion of work and parents’ participation in homework and projects. Elma and Samira, who were requesting new school places for their children, were dealing with a formal process. The place was also described as requiring lots of participation by parents through helping children with their homework, helping children with activities set by the school e.g. dressing up for World Book Day or volunteering on class visits. There was an expectation to be involved in the school learning activities at home. In formal public services places, the access to specialists, advisors and managers were protected or restricted by reception staff or lower level advisors. There were systems for how to get appointments or to be seen if they walked in. In some cases, it was to take a number and wait, in other cases appointments needed to be made on the phone or on advisor invitation. All housing workers and advisors also used rules and policies with regards to housing in their responses to participants and participants accepted those responses without complaining. Only one participant had escalated her case to the local councillor in Hackney. The other women (Flora and Elma) had been told to wait, and they did. Although bureaucratic, it was not always clear from the council or other provider websites how the process of getting an appointment worked. Noor recounted a day she and her husband waited in a queue only to find out that everyone in that queue had taken a ticket to see an advisor, and by the time they realised this many more people had joined the queue and they had to go back to the end of the waiting time. She said 116 that processes and procedures were vague and therefore they were reliant on what the advisors said. Social Interactions and Relationships: local authority services Council administrators, advisors and workers seemed to hold the power in other places within the public services sphere. Participants were always in need of something from the council, were disadvantaged by bureaucratic systems and process, and often reported confusion and that the systems did not make logical sense. The felt they were not always given a clear answer. In spite of this, participants reported formal service, which was polite and they felt that their workers/advisors were trying to be helpful. Many interactions were related to listening to instructions or processes. Flora had to attend a meeting in the Job Centre: “…so she is my allocated officer, Antoinette. Yeah, that day she just want to know any change in my circumstance or not. Because they have my old details, so…that day she wanted to know any change or not. Then she told me… if anything change in-between, so I have to tell her. Otherwise I will lose next appointment.” Flora When she arrived, she found that the purpose of the meeting was to inform her that she needed to let them know if there was any change in her circumstances. The interaction was described as one sided, with little discussion or opportunity to negotiate her position. Any questions were to clarify the instructions, and often they were closed questions. Other forms of interactions were requests made by participants for basic services, for which they had the right. Elma requested a school place closer to their temporary home with the council: “I tried to apply school for my daughter…near where I live, but the school says that… ‘Because you live in bed and breakfast, we’re not allowed to give a school for your child, unless you have a private house, or a temporary house, or a normal house, or council house’” Elma These exchanges were more complicated and usually meant that participants had to have an understanding of the way council bureaucratic systems worked. Participants’ developing English language skills created an additional barrier to ensuring they understood the process correctly. When Elma finally got a permanent house, she found she still could not get a place for her daughter at the local school: “I tried to apply…where we live now but we couldn’t get it. They said, ‘There’s a long waiting list and you are…number 26.’ There’s another 25 kids…waiting in…the same class as my daughter’s year. Social Interactions and Relationships: schools/ nurseries In schools, teachers and especially head-teachers have clear power and responsibility because of their positions. Requests for attendance by teachers at parents’ evenings were seen as compulsory along with any other homework set/activities. Teachers talked to parents about children’s progress, issues or problems. All participants with children said they talked to teachers in English. The parents’ evenings were the main way of having these discussions at school. With the children who were still in nursery, there was the opportunity on a daily basis to speak to the nursery nurses and/or teachers when they collected their children. Generally, parents reported that teachers updated them about issues with their children: “I said, ‘How is my son in school? He is okay?’ And she said… ‘his understanding is okay, but he need a teacher to speak with him, because the language he can’t speak properly’…(She said) ‘Every Tuesday from two o’ clock to three o’ clock, you have to bring him and stay with him.’ I told her, ‘It’s okay, I’m coming with him,’ Samira These interactions seem to be less about discussion, but more about accepting what the teachers had already decided was the appropriate action for their children. Samira’s description of her interaction gives the impression she had not been involved in discussing any alternative intervention for her son. Flora also reported lack of involvement about her daughter: “(My daughter) she didn’t tell anyone, so they didn’t notice the whole day she was in the wet dress. After that, she get cold,” Flora As it was formal, it was also difficult for parents to question actions taken by teachers. When Flora’s daughter didn’t tell her nursery teacher about her wet dress, the teachers said that it was her daughter’s fault. Flora described incidents where her daughter came home from school with cuts as she had fallen over and the teachers had not noticed this. Flora felt that she was not able to question teachers’ responsibility over things that happened in school time. Participants also had to communicate with school administration about absences, illnesses or accidents. For example, Elma regularly had to tell the school that she was not able to collect her daughter on time and to give permission to another parent to take her daughter. These interactions were formal and transactional. 117 Social Interactions and Relationships: local authority services She’s going to be Year Two in September…there’s 118 another 25 kids that are in Year One and they want to go to same school. So…they said…maybe in September, hopefully, like a year.” Elma Participants were disadvantaged by bureaucratic systems and process, and often reported confusion and that the systems did not make logical sense. The felt they were not always given a clear answer. Participants also requested for further clarification. Flora wanted to know why other families in the hostel had secured accommodation when she had not. Participants generally did not complain and did not report knowing how to complain: “I accept it because they said no they are not going to send me any temporary or anywhere, I have to wait, I asked how (long) is the (wait for the) house…Then they showed a list, one of the lists, their retailer list.” Flora Flora and Elma reported that they felt there was nothing they could do about the system and they just had to accept the decisions made by their key worker or social workers. There was a sense that there was something not right with the system but they were not aware of their rights to make any changes. Unlike Flora and Elma, Samira escalated her requested more suitable accommodation as her living situation was over-crowded. When she was unsuccessful with the council team, she escalated this request to a local councillor. She reported that she had heard about her local councillor through someone: “That is why I went so many time to councillor to tell them, because I had too much problem here, I have three children in one bedroom.” Samira She was able to get a meeting with the councillor at the surgery and then he sent her a letter inviting her to meet with him to discuss her options of moving to a larger property. Social Interactions and Relationships: health services Interactions in medical and health services were related to listening to instruction, and also responding to questions about the health area being discussed. There were two places where there were highly positive interactions reported. When Samira was pregnant, interactions with her health worker, nurses and doctors were positive. Flora also reported receiving positive support from her bereavement counsellor. She felt that he tried to understand her when she was trying to explain complicated emotional experiences and could not find the right words. Samira’s family planning nurse used Google to help her understand the implications of different birth control interventions. 119 In hospital, Elma said that the character and manner of the nurses were a determining factor for how she felt on the ward: “So…the doctors were kind and very nice. I was happy with what they say and how they explained to me and how they were dealing with me…but the nurses, some of them…were rude, very rude!” Elma “So…the doctors were kind and very nice. I was happy with what they say and how they explained to me and how they were dealing with me…but the nurses, some of them…were rude, very rude!” Elma She reported that she was not treated well by some nurses, and that other patients agreed with her when they talked. However she did not complain about it and formed the opinion that nurses were rude for no reason. In general participants reported doctors and specialists were more sympathetic and supportive in social interactions, helping them to understand their health situation or treatments. However, they found that nurses and administrative/support staff were not so helpful. Elma described a situation where her toddler fell into a road: “I said (to the ambulance controller), ‘Please can you come, he’s just a baby, and he’s bleeding, and I think he broke his hand,’ but they said, ‘No, Sorry, we can’t come, but you, you can call 111, or you can just take a taxi or something.’...I didn’t have money with me…I was shocked actually…” Elma There were also a limited number of interactions with other people within the place, who were not staff such as other clients, reported. Elma had limited interactions with other patients about the nursing staff. However, it was not usual to talk to other customers or clients in these places. Social Interactions and Relationships: health services In hospital/medical settings participants reported that they did not have enough information from medical professionals. Although doctors were described positively for the way they communicated with participants, Elma and Samira said they had a lack of sufficient information about their situations. Samira said she did not really know what was happening with her husband’s condition despite attending his appointments with him and reporting that she was confident she could understand his doctor. Social interactions in the public services sphere reveal a lack of agency by participants and I suggest that this lack of agency is due to the social interactions that they experienced. Relationships were of a formal nature and participants reported an awareness of hierarchy of the people they were interacting with e.g. teacher, head teacher, doctors and nurses. In the school and the local authority settings, school staff and caseworkers’ interactions were instructional, giving little room for participants to respond with queries. From the descriptions of the interactions, it seemed that 120 participants’ own experience and knowledge was not valued. For example not engaging with parents about their own child development. Although the interactions were not negative, some of them are described in ways that suggest the public service staff were indifferent and dismissive. Therefore the interactions were not all positive either. Social interactions in the public services sphere reveal a lack of agency by participants and I suggest that this lack of agency is due to the social interactions that they experienced. Relationships were of a formal nature and participants reported an awareness of hierarchy of the people they were interacting with e.g. teacher, head teacher, doctors and nurses. In the school and the local authority settings, school staff and caseworkers’ interactions were instructional, giving little room for participants to respond with queries. From the descriptions of the interactions, it seemed that participants’ own experience and knowledge was not valued. For example not engaging with parents about their own child development. Although the interactions were not negative, some of them are described in ways that suggest the public service staff were indifferent and dismissive. Therefore the interactions were not all positive either. Social interactions within more formal places, where there are strict boundaries and expectations of behaviour affect participants’ socialisation process. Social Interactions and Relationships: health services As discussed in 3.4.4, interactions within spaces give social meanings to a space (Dourish, 2006) and so participants’ interactions within these places affect a person’s development of identity and belonging. Players Asma had three jobs over the time of the study. She generally worked for sole traders (her boss), and had some colleagues. In her employment, the boss was the decision- maker. She also had many clients, whom she talked about positively. Lucy found that in schools there were several staff with hiring responsibility and it was not always clear to her who was in charge of hiring. She would communicate with one contact and then later discover that there was someone else reviewing her application. With community centres and libraries, Lucy said it was easier to know the person to talk to if she had attended the library or community centre as a client. Lucy also had prospective business partners. She communicated with one in Chinese, she spoke to the other in English. 121 Lucy also met private clients and children, who were her students. Later as a nanny on a yacht, she was involved in the daily care of two children. Activities and practices Employability skills (the ability to write an application form, an application email, respond to job descriptions and pass at an interview) were important to Lucy’s job sector. Lucy reported that she felt that employability skills were almost more important than the skills of the job itself. For the jobs that Lucy was applying for, schools had minimum standards of English language competency even though the job did not involve using English to teach. She was able to read/write and complete forms/letter to a high standard of language but the fluency, accuracy and pronunciation of her spoken language let her down. She did not perform well in interviews and believed that this was why she was unsuccessful. Asma’s process of application was of a different nature, which was through her contacts and asking for work: “I got one job…at salon. So I work there only one month…I wasn’t happy that time with my work…I left that work and…then my one friend, she giving me some number…for…the other salon manager. So I called there, she said, ‘You can come for a trial.” Asma After starting, she found different employers had different activities/expectations. For her first job, Asma was expected to carry out the shifts that she had been scheduled without complaint and she left the job. She was able to gain another one shortly after. There were also expectations of how she would talk to clients and make them feel comfortable and welcomed. In trying to start her business, Lucy had to make presentations to potential customers. She had two different business partners - a Chinese trainee teacher whom she had met through a class at ELATT, and a friend who had recently returned from being abroad and was unemployed. It was difficult for Lucy and she was only successful where she had sought venues with Chinese-speaking contacts. In other venues, her presentations were not successful. The trainee teacher friend later decided to open a business without her while the other decided to go to university and left Lucy to continue without him. When Lucy worked on the yacht as a nanny, her work activities included being involved in quite personal spaces with the family. The family extended their holiday without 122 consulting with Lucy, as this was considered normal for the family. Lucy negotiated for them to let her leave. Social Interactions and Relationships: work The interactions within the work and employment place were mostly formal, especially at the stage of looking for work. These interactions took place in both text and spoken from: For example, completing forms and CVs online, talking in interviews including presenting qualifications, information about previous experience, and responding to questions, queries and requests for information. In Lucy’s experience, schools have a lot of power in decision-making and in setting the recruitment standards. Lucy reported she was frustrated by the fact that there were many people teaching Chinese without adequate teaching qualifications or skills. The processes meant that at school-level she thought they were not able to adequately assess Lucy’s teaching abilities. Lucy had to conform to the systems and meet required standards to have the opportunity to gain employment in a school. In Asma’s experience, interactions at work took place when communicating with clients or customers. For example, greeting customers, asking customers what they wanted, making conversation/small-talk with customers having treatment and asking for and receiving payment: “I’m relaxed because…I meet different people (clients) every day and I talk with different people and… I speak to them…something…I, every day I learn new things so this is good, really…good for me.” Asma Asma reported that she enjoyed this small talk and that it helped her develop her confidence to communicate well. She enjoyed communicating with customers and felt comfortable. Therefore, it seems that she perceived there were positive relationships with customers. There were also interactions about work conditions such as changes in hours and other requests. Asma was able to discuss issues with her employer and was able to choose to leave the job she wasn’t happy with. Although the employer has more power in the employment relationship, in Asma’s case she was confident that she would find another opportunity and she did. Lucy’s business ideas involved constant communication with community centre staff about her proposals. The interactions were of a persuasive nature to convince the centres to give her work. When Lucy worked as a nanny, she had to work all day into 123 the evening, caring for the children and teaching them Chinese. Social interactions were of an instructional nature. Social interactions in the work sphere differed for the two participants, and based on the different activities. Social interactions enabled Asma to get entry-level work, and then to build on her confidence dealing with customers once she had work. Social Interactions and Relationships: work However for Lucy, her social interactions in English with language schools and community centres did not help her achieve her goals, which was to get skilled work. 124 5.5 What are participants’ perceptions of their belonging Participants’ situations at the end of the research project showed that all participants had increased their confidence in using English, they had all increased their confidence to interact with other participants in the ESOL group. Five of the six participants had supported each other. Three participants had improvements in their housing situations, two gained jobs and one was volunteering. One was no longer experiencing controlling behaviour at home. All participants reported increase in confidence in their life in the UK. At the end of the research, it was expected that participants would experience belonging in different places, and different degrees (3.5.2). As shown in the summary (5.4) above participants were able to overcome many issues in their lives and reported increased confidence to use English in different spheres. Elma and Samira reported the greatest positive feelings related to sense of belonging. Both reported feeling comfortable at home and in other spheres such as public services and local community. They reported a greater ability to advocate for themselves and others. Their data also showed an increase in social relationships. Asma reported an increase in happiness and confidence and attributed this to the improvement of her relationship with her family at home, and working. Participation in social interactions at work had made her feel more confident in her area and more positive about her future. Noor, Flora and Lucy reported less positive sense of belonging. Noor felt at ease in most places such as the mosque, the jobcentre and in her local area. However, she reported low trust of people who were not like her and fear of Islamophobia in public areas and on public transport away from her local area. Flora’s confidence was affected by her housing situation, which did not improve, and this affected her self-confidence to solve problems for herself and her daughter. She was still lacking in confidence to speak English outside of her ESOL class group and her therapy sessions. However, her data showed an increased participation in leisure activities with her daughter with greater happiness reported after the activities. Lucy was affected by her experiences trying to find work. She perceived unfairness in recruitment processes. She also reported low trust of people who were different from her. She continued to report making new acquaintances with ethnically Chinese people in London, in spite of many of her previous relationships being of an exploitative or unequal nature. 5.4 Main summary of findings Main findings indicate that there is a relationship between social interactions in English and my research participants’ perception of their own integration. Participants’ experiences of integration prior to starting the research were varied. They were affected by previous experiences of life changes and migration, their previous experiences of learning English, the social interactions outside of their ESOL class that they were already participating in, their home situation at the start of the project and the existing support they were already receiving. Although not the focus of this research, barriers to integration were also identified. Participants participated in social interactions in places within four spheres: Local community, public services, work and home. Within the local community sphere, participants took part in social interactions with people in their ESOL group and the parents at their children’s schools. They built relationships with other classmates and parents outside of the ESOL provider and school setting. The nature of the relationships was informal and supportive, with social interactions such as giving advice, making social conversation and helping each other with language practice. Participants also used online communications to extend their social interactions. Within the public services sphere, participants took part in social interactions with staff working in schools or nurseries, and with staff who worked in local authority and health services. The nature of social interactions was formal and instructional. Participants made requests for support and were given procedures to follow. Finding indicated that public services staff were perceived to hold authority and participants’ main activity was asking for support and assistance. Two participants were involved in the work sphere. The social interactions for one participant were with employers and customers and the other with prospective employers and prospective customers. The nature of the relationships was different with the different types of employers and customers. 125 5.5 What are participants’ perceptions of their belonging She had found a group of university tutors, who were Chinese, and reported some promising work opportunities through these connections. 126 All participants reported an increase in confidence in English from social interactions with others either within the ESOL group or with other migrants and service staff outside of the group. However all participants reported low social interactions with ‘English people’ or host community members, although this did not seem to directly affect their sense of belonging in all places. The following is the discussion of research findings of participants’ perceptions of their own belonging in the four aspects of belonging. Access to a community As discussed in 3.5.1, interactions in formal settings could create opportunities for interactions in informal settings, with schools and colleges suggested as one such formal setting. Data from this current research shows that ESOL groups and children’s schools were an important place for participants to access and participate in interactions. This research illustrates the importance of formal places such as education establishments in providing opportunities for migrants to participate in social interactions and create relationships. In this situation, participants were not friends but they were developing relationships based on their shared experiences. In section 3.5.3 the term ‘community’ is used to describe people who come together based on shared experiences, social ties or shared spaces. Samira and Elma’s relationships with other parents are examples of this (see 5.1.1). Although participants described the people from their ESOL group and school gates as their ‘friends’, based on their descriptions I feel that community is an appropriate way of describing the relationships. The examples of the ESOL group and the ‘school gate’ show the importance of a physical space as a starting point to building social ties and local support networks for migrant women. ESOL classes were where migrants study English, and the interactions among the ESOL group members outside of their formal class learning also appeared to be of great value to the migrants’ perception of language development and sense of belonging. Flora’s main opportunity for regular social interactions in English was with other parents through the school (see 5.3.1). Children’s school and nurseries are also recognised as sites for language learning (All Party Parliamentary Group on Social Integration, 2017), with many local authority and community learning providers holding family learning classes in such places. Data from my research suggests that as well as language learning, access to social interactions at ‘school gates’ and other parent-to-parent engagement activities supported participants’ access to a community. Participants who participated in social interactions 127 at their children’s school and with their ESOL group reported an increased feeling of belonging to a group or community. Access is also related to knowing the ‘rules of the game’. As discussed in 3.5.1 May suggests that ‘collective understandings’ of belonging are built upon ‘negotiated accomplishments’ (2013, p.82) and that the feeling of belonging must be reciprocated (May, 2013). Elma participated in the PTA for her daughter’s school after another parent encouraged her. Access to a community She described how she went to the meetings and just listened, until she felt she understood ‘the way to talk’ in the meetings. When felt confident, she said she started talking in the meetings. Elma’s example of accepting the invitation to join the PTA, and then attending meetings until she felt confident she could participate is an example of socialisation into a new place. Her subsequent increase in confidence to fully participate in the PTA meetings is an indication of her sense of belonging in the place. Therefore as suggested in the framework (Figure 2), social interactions developed participants’ material belonging, through access to a group of people including sympathetic interlocutors. Access to services As discussed in 3.5.1, migrants have difficulties accessing public service places. From a sensory perspective, the places in the public services sphere are formal and have physical boundaries, which can indicate where people are allowed to access. The restrictive physical space can be seen as intimidating. Although child-friendly, schools and nurseries were seen as formal places. There were signing-in procedures, locked doors that prevented unauthorised access and rules about access and safety. From an equality of access perspective, the practices in public services places described by participants show that access to specialists, advisors and managers were protected and restricted by reception staff or lower level advisors. As described in section 5.1.2, all participants were already confident to access several places within the public services spheres with routine issues such as housing and general medical needs. However, during the research, circumstances that were more unusual took place and in these situations, lower levels of confidence to access less familiar types of public services were reported. One area where there appeared to be lack of confidence for participants was to complain when standards of service were not met and/or to challenge when insufficient information was given to them. Both Elma’s children had accidents and required emergency attention. However, there were different standards of service experienced 128 in both situations. When the school rang for an ambulance for her daughter’s playground accident, an ambulance came. However when Elma rang for an ambulance for her son’s fall in the road she was told to take a bus. She was sent to the wrong hospital and waited, before being sent on to a different hospital. She attempted to ask for information but was told to wait. They experienced an estimated wait time of 6 hours. She said she felt at the mercy of the ‘people in charge’. Three participants had ongoing housing issues (described in 5.1.1). Flora, who had been living in a hostel with her four-year-old daughter reported being made to feel that her request for better accommodation was not fair and reasonable,: “…Because they said first to me, they are going to move me…within four months from there to a temporary house. Access to services But I have waited eight months there but they didn’t do anything so I went to ask them, and they said, ‘No,’ they are not going to send me any temporary house.” Flora “…Because they said first to me, they are going to move me…within four months from there to a temporary house. But I have waited eight months there but they didn’t do anything so I went to ask them, and they said, ‘No,’ they are not going to send me any temporary house.” Flora Using policies and processes, the housing officer justified the long waiting time. There was a lack of transparency about why some people in the hostel had gained housing when she had not and this caused her to feel not worthy of permanent housing. Participants seemed to think that they could not change anything and just accepted what advisors had told them. Elma and Flora’s responses above showed that they felt there was nothing they could do apart from to wait as they had been instructed. This could also be seen as an indication of a lower cultural knowledge- that is the rights that they had as residents. Only Samira escalated her complaint to the local councillor but only after visiting the council many times and being given the information about how to contact the local councillor by another community organisation: “I went so many times to council, but they…didn’t do nothing. Then I found someone to help me and give me the councillor number. I went to the councillor, I told them, ‘Please, I am pregnant, another baby is coming, where I put bed for her?” Samira In Samira’s example, the persistence she showed could have been affected by her self-efficacy she had gained as a result from her previous experiences (discussed in 5.1.2). My research participants reported feeling powerless and lacking in confidence in these places and I suggest these were related to the interactions that they had there with staff. Such interactions reduced Flora and Elma’s confidence to access services. As discussed in 3.5.1, the lack of English language skills is likely to have played a part in 129 the lack of confidence. However, negative interactions with staff in public services places also contributed to a lack of participants’ confidence in their ability to deal with difficult situations. Access to services Whereas, the participant who had positive interactions with their caseworkers or services staff built confidence to feel more confident about bureaucratic processes. Samira reported a higher degree of confidence to escalate complaints because of the support and advice from advisors and friends. For migrant women on my research, interactions with public services staff affected their feeling of material belonging. Experiences of access to work As described in 5.2.3 Lucy experienced challenges to gaining work, whereas Asma was able to gain work three times. Lucy perceived that she was discriminated from high-skilled jobs in teaching Chinese that she was applying for. In her experience, she found that English-speaking people had the advantage even when the job involved being able to teach a different language. She also claimed that those fluent in English with low or no Chinese teaching qualifications were employed and she was disparaging of this practice. Migrants are often discriminated from higher skilled work because of the perception of their language level (Paget & Stevenson, 2014) and this could be due to the lack of fluency or incorrect pronunciation. Lucy was able to be self-critical and identified that it was her spoken English language fluency that prevented her from being successful. She had been shortlisted for interview through her written applications. Asma was in work at the end of the research; she had been looking for any type of work and took on lower-skilled jobs. The method of application was in person and through personal connections. She had also changed jobs to an arrangement that suited her better. The ability to get work quickly and then change jobs could be from the confidence she gained from working or because of her previous work experiences (owning her own business in Pakistan). For these two participants, access to work was affected by the requirements of the employers of different job types. For the jobs Lucy was looking for, employers were looking for candidates with specific minimum requirements. Asma was able to gain employment using social connections although the jobs were not secure. Asma also reported a large increase in confidence from working. She said this was due to the increased social interactions she was having with clients and she identified the 130 act of working as affecting her confidence. For Lucy, being unable to gain work affected her negatively: “…You just feel very sad, you don’t want to do anything… so I, I feel I’m not good (at English). You have good quality and nobody know you…” Lucy “…You just feel very sad, you don’t want to do anything… so I, I feel I’m not good (at English). You have good quality and nobody know you…” Lucy Lucy had been offered interviews from written applications she had made. Experiences of access to work However, when she went for the interview, she did not pass because of her spoken English skills. When she approached non-Chinese speaking community centres to talk about setting up classes, the results were similar. It seemed that introductions were not sufficient to gain work in higher skilled jobs. A higher level of formal language proficiency was required. However, when Lucy opted for an entry-level job role, she got a job through her contacts. When on the job, she found she was able to communicate her demands to terminate her working contract, indicating that social interactions play a greater role in getting jobs and negotiating job conditions in the entry-level job market. The affect of lack of work on a person’s confidence has been noted in 3.5.1. The statement above shows the impact the experiences of not getting work has had on Lucy’s confidence. These two participants had previously worked all their adult lives; with them reporting that work was important to them. I could see that work played a significant role in their self-perception of their identities. The experiences of these two participants lead me to suggest that work or lack of work affected their feeling of material belonging. The importance of friends as a replacement for family As discussed in 3.5.3, family is the first place we feel we belong. However, for newly arrived migrants, family might not be available or close. Three participants did not have friends from their own ethnic community. Participants were creating new relationships during the research programme. Samira had built up a network of other parents from her son’s nursery and school. These parents sent her messages saying they missed her as she had moved. This in itself is an indication of belonging to a group. Of the participants, Samira presented as confident and was able to overcome many challenges over the period of the research. She regularly reported feeling good and happy, and confident that she could do anything she needed to. As described in 5.1.1 she successfully complained to a local councillor to demand the council rehome her and her family only weeks after giving birth, and made Home Office applications independently. 131 Samira reported that she valued friends a lot, as she did not have anyone from her home community for support, and that she viewed friends as replacement for family: “Because I don’t have family here…if I need some help or something…friends are (good).I have friends, they can help me. And also if I’m bored or feeling sad I can see Samira reported that she valued friends a lot, as she did not have anyone from her home community for support, and that she viewed friends as replacement for family: “Because I don’t have family here…if I need some help or something…friends are (good).I have friends, they can help me. And also if I’m bored or feeling sad I can see them.” Samira Elma too relied on friends for help, especially with childcare when she and her children were in hospital for emergencies. She was part of a group of about seven parents from her daughter’s school and they regularly helped each other out. They were all from a variety of backgrounds with only two parents born in London and one with an English background. The importance of friends as a replacement for family Apart from support networks, participants also identified that their friends helped them to not feel ‘bored’, ‘sad’ or ‘lonely’ contributing to their feelings of happiness: “…the more I tried, the more they helped me, and the more they come closer to me.” Elma As discussed in 3.5.3 friends are also important to a person’s sense of belonging and can sometimes represent the family that one has chosen (May, 2013), and that communities can be built on similarities such as shared experiences or interests. In the case of the research participants, it is the local school/ESOL group that has drawn them together to move from classmates and acquaintances to becoming more like friends. As well as giving and receiving support such as advice and sharing concerns with each other, their developing friendships supported them to feel confident that they had support for their children. Elma said that she was no longer worried about her children if something happened to her. She was confident that her friends would step in to help and this made her feel calmer and more confident. I suggest that for participants who had developed a good network of friends, they felt a stronger sense of relational belonging. Social interactions with friends and other sympathetic interlocutors supported the socialisation process, and increased participants’ relational belonging as suggested in Figure 2. Whatsapp as a tool for extending social interactions I was struck by how close the participants reported their ESOL group had become, regularly exchanging messages, organising meet-ups out-of-class in local cafes, or each other’s houses. Past ESOL classes I had taught and the research participant group from my earlier studies had not maintained the friendships they had made during 132 their course of study with us. Reasons for friendships not enduring are similar to findings by (Dimitriadou, 2004) in her thesis. She found that women with families tended to be more restricted by family commitments and geographical issues. The women on our programmes are often mothers and some travel from other boroughs to Hackney. The difference that I noted between the former examples and my research participants was the increase in the use of Whatsapp for communications, which I had not seen previously. As discussed in 3.5.3, online communities can be an invaluable tool for belonging and support. My research participants, who used Whatsapp, said that they were able to remain in touch even though they were not available to join in physical meet-ups. The group was also a site for asking for advice and support from each other when they were not able to meet face-to-face. Participants used Whatsapp to maintain relationships in periods of absence. In Elma, Samira and Asma’s experience, although they were moved by their housing services or had to have a break in study for maternity the Whatsapp group created enabled them to continue the existing relationship they had with the ESOL group. They were still able to participate and benefit from personal support and updates about their ESOL course. Aharony's (2015) research suggests that Whatsapp maintained existing social capital (3.5.3). For my research participants, the use of Whatsapp allowed them to maintain the social capital they had accumulated in the group over time, through remote ways of continuing to share their experiences. Close relationships mean frequent interactions (May, 2013). Although research participants reported using Whatsapp for planning where to meet and to talk about homework, most of the more frequent interactions reported were of social chat and sharing news with each other: “When we’ve been to class, usually we talk, or otherwise we have a Whatsapp group, maybe if anybody have any gossip or anything. Whatsapp as a tool for extending social interactions So that time we chat on Whatsapp group…” Flora Even when the participants had seen each other physically in class, they would still message the group later that day. O’Hara et al’s research shows that Whatsapp is not just a replacement but also an extension for groups that already see each other often (O’Hara, Massimi, Harper, Rubens, & Morris, 2014). The addition of Whatsapp seemed to support the building of relationships between participants who were actively using the mode of communication, creating closeness between them, even when they did not see each other on a daily basis. 133 The data from this study suggests that participants benefitted from using Whatsapp to engage in social interactions in English with their peers in the ESOL group, and that these interactions deepened their social connections to form friendships. These participants reported greater support networks and more friendships contributing to their relational belonging. Therefore, the additional opportunity to interact with others continued to support the socialisation process, and affecting participants’ perception of relational belonging. The value of weak ties As discussed in 3.5.3 interactions with people who were considered weak ties, such as acquaintances or strangers, can contribute to the development of trust in the local area. Talking to other people they encountered in the places that they lived enabled participants to learn expectations of behaviours of people around them. Samira and Elma reported many interactions with shop workers and neighbours and Asma with customers. Noor regularly interacted with strangers in her religious community. This contributed to their trust in the local area, as there was a development of predictability and familiarity that trust is built on. Participants who reported taking part in interactions with others in their local area and daily lives also reported they were confident in their areas: “Yeah because…you know…(the)…more you…live and the more you learn…the language and…you have a connect(ion) with society…the more that you trust and the more that you feel confident…” Elma Concerning increasing their confidence in an area and their sense of belonging, it appears that interactions with strangers can increase familiarity with people in the neighbourhood and area, and increase migrants feelings of trust in the local area. However, concerning creating relationships with people from the ‘host’ community, the weak ties would need an amount of time for any relationship to develop. I suggest it is possible to feel a sense of belonging when some aspects of belonging have good levels of confidence, whereas others do not, as exhibited in Elma’s situation. Public services staff providing services to participants could be classed as acquaintances or strangers. For some participants, they had been assigned workers to their cases. For example Flora had a housing caseworker, and Elma has a social workers. The familiarity of these workers helped participants to feel safe and trusting, in spite of the situations that they were in, which in some cases were highly stressful. Perceptions by participants of the way they were treated also affected their trust in the service. If they were perceived to be polite, friendly and supportive, participants were 134 more likely to feel comfortable. Flora said that her housing officer was kind and spoke to her ‘nicely’. Elma built up a good rapport with her social worker that they were able to joke with each other. In these situations, prior experiences of the interactions with service staff supported participants to know what to expect. The value of weak ties However, where a participant had to encounter a different place or person with no point of reference, it was unlikely that there would be any weak ties. For example, Lucy found it difficult to approach some community centres, as she had never participated in social interactions with the people working in those centre, and was unsure of how to approach them. However, as she had visited her local library often, she felt confident to approach the library coordinator to offer to deliver Chinese language classes there. For my research participants, prior experiences of social interactions with strangers, usually public services staff, affected their feeling of relational belonging. However, there was a lack of reported opportunities to take part in social interactions with host community members and this could affect relational belonging and the ability for migrants to take part in social mixing. Language and interactions: developing trust in others In 3.5.2, the relationship between language and belonging was discussed, emphasizing the role of language in building trust. Participants talked about the importance of trust in enabling them to develop social relationships with people they met. In the ESOL group, they felt that trust was first developed with other ESOL classmates. As discussed in 3.5.2 English language skills play a large role in confidence for migrants to participate in social interactions and all participants made the link between their confidence in English and levels of confidence to talk to other people. Elma also linked interactions in English with the development of trust, which she identified was due to the ability to ‘understand’ the other person: “.…the key thing is the language, because if you can’t…communicate with the others and you can’t understand them, they can’t understand you…You don’t feel confident at all. Yeah, you can’t trust and…they can’t…” Elma “.…the key thing is the language, because if you can’t…communicate with the others and you can’t understand them, they can’t understand you…You don’t feel confident at all. Yeah, you can’t trust and…they can’t…” Elma The definition of trust in 3.5.3 is that it is a particular expectation regarding the behaviour of other people. Without the ability to communicate and understand each other, it would be difficult for newly arrived migrants and others to build trust in each other. However, trust is also built upon prior experiences of others’ behaviours. Both Lucy and Noor reported that they found it difficult to build trust with people who were different from them. Noor attributed this to the discrimination she had experienced from 135 non-Muslims in The Netherlands, where she had previously lived, and Lucy talked about her lack of prior experiences of meeting people who were different from her: “Before…(I was at) school, then work at school (university), so I never have chance (to meet other)…people…I always at school…so I don’t have…many time (to) work with the people, like a team…” Lucy “Before…(I was at) school, then work at school (university), so I never have chance (to meet other)…people…I always at school…so I don’t have…many time (to) work with the people, like a team…” Lucy She felt that if she did not have anything in common with a person, she would not be able to build trust with them. Lucy reported that her friendships were mainly with Chinese-speaking people. Language and interactions: developing trust in others Her interactions with English-speaking people were typically with university-educated people who became friends with her to participate in informal language exchanges. However, her interviews suggested that the language exchange friendships replicated a teacher-student archetype, which was a familiar archetype for her. Lucy’s experiences match the existing literature that people are likely to trust people they feel they share a social identity or similar experiences with (section 3.5.3). Nonetheless, Lucy still reported an increase in confidence to participate in social interactions with others, even if these were not often, and only with selected people. Participants reported that the ESOL group provided a safe space for participants to practise social interactions in English and build their confidence. All participants reported that their ESOL Group had supported them to gain confidence to interact with others. My research suggests that participating in social interactions enables people (migrants and host community) to comprehend each other, and to develop knowledge and awareness of each other’s behaviours, which can support building trust between different people. Trust is identified as an important element in building relational belonging, as it is related to the confidence in others’ (May, 2013). My research participants reported developing confidence from their increase in language skills, and this affected their confidence to take part in social interactions, which I suggest affected their feeling of relational belonging. With exception of Lucy’s case, almost all participants’ reported social interactions in the local community sphere with other migrants who had moved to the UK, like them. This meant that there is likely to have been a high level of bonding social capital, e.g. meant that there is likely to have been a high level of bonding social capital, e.g. support for each other. However, these participants were missing opportunities to gain bridging social capital- that is relationships with people who were different from them. As presented in the introduction, migrant women reported that they find it challenging to have social interactions in English with people from the host community. They are also perceived to not want to get involved in their communities (2.1). When considering As presented in the introduction, migrant women reported that they find it challenging to have social interactions in English with people from the host community. They are also perceived to not want to get involved in their communities (2.1). The value of customer-facing jobs In my previous research (Graham-Brown, 2015), one inconclusive finding was that opportunity to work could either be a barrier or a support for participants to increase confidence using English. I suggested that customer-facing roles increased participants’ social interactions in English. Again, this was based on the experiences of only two participants. The current thesis findings for one participant who was in work is similar. Asma reported that she experienced a quick and significant growth in confidence and attributed this to her work. Specifically, she identified that this was due to the opportunities she had to speak to customers on a daily basis, and the social interactions she participated in with customers from different backgrounds. As described in 5.4.1 she had just returned from Pakistan after a period of absence because of her personal circumstances. She was depressed and had low confidence before going to Pakistan and so the change in her confidence in work and life was substantial. For this participant the customer-facing role with regular social interactions affected her feeling of relational belonging. Language and interactions: developing trust in others When considering 136 social cohesion (see 2.2 and 3.3.1), the involvement of the host community is crucial to enable social mixing, so can people ‘get along’ with others in the community. In the absence of meaningful social interactions, May’s research suggests that weak ties can also support the development of relational belonging (May, 2013). For research participants socialisation took place in many places, through different types of interactions and with different people who played different roles. All of these interactions contributed to participants’ perception of their relational belonging, as well as material and cultural belonging in different spheres. This is represented in Figure 2 by the stacked circles that show that all forms of belonging are developed as socialisation takes place. Learning about diversity As defined in 3.5.2, culture can be seen as ‘ways of life’ and describes how people from social groups act and think. Cultural integration is dependent on migrants’ knowledge and understanding of life in the UK, their rights, responsibilities and shared values. My research participants’ social interactions supported the development of knowledge of life in the UK, rights and responsibilities. Where the specific knowledge was not learnt in their ESOL class, for example passing the UK driving test, content of 137 the Life in the UK test (history, law and procedures), processes for children’s school applications and visa applications, participants shared their experiences and taught each other. Apart from the knowledge described above, social interactions also supported participants to develop values of diversity, tolerance and inclusion of others. The social cues that the group practised are an example of how social interactions with the ESOL group reproduced learnt social values and behaviours that they then practised in daily lives. For example, the act of not speaking their own language with those who shared a similar first language so as not to exclude others was a real world practice of inclusive behaviour. As described in 3.5.2, for most of my participants, London was the first time they had lived in a super-diverse city. All participants reported instances where they did not know how to behave with people from different backgrounds, including having communication difficulties with those who had different accents. Earlier in the research, Asma described her frustrations at being unable to understand a bus driver with a Jamaican accent. Later when she started working, she participated in social interactions with customers who spoke with the Jamaican accent and built confidence to interact with them. Elma also described important norms and behaviours that she learnt: “For example in my country when people do something for you…we don’t use ‘thank you’. Before, when someone did something nice for me…I just walk away. One day I was on a bus with my buggy, and a lady got up and moved her buggy to the side so I could put my buggy. I just did that and sat down. Then she said ‘thank you’. And I was like, Oh my God, I should have said ‘thank you’ because in this country they always say ‘thank you’. Learning about diversity So I said ‘Thank you’ and the lady said ‘Yeah yeah yeah’...” Elma Elma had learnt about formal language and polite phrases to use in her ESOL class, but it remained a conscious act that she had to remind herself to do, and over time would internalise. Through regular interactions, she was able to practice the accepted norms. The incident also revealed the type of cultural misunderstandings that can happen between groups of people, who do not share similar cultural conduct and behaviour. These cultural misunderstandings could affect trust between groups. Participants in my research reported learning cultural norms from their interactions and relationships with others. Other examples are Asma with her customers, Samira with her neighbours and other parents, and Noor with her mosque congregation. These learning experiences increased their sense of cultural belonging. Elma had learnt about formal language and polite phrases to use in her ESOL class, but it remained a conscious act that she had to remind herself to do, and over time would internalise. Through regular interactions, she was able to practice the accepted norms. The incident also revealed the type of cultural misunderstandings that can happen between groups of people, who do not share similar cultural conduct and behaviour. These cultural misunderstandings could affect trust between groups. Participants in my research reported learning cultural norms from their interactions and relationships with others. Other examples are Asma with her customers, Samira with her neighbours and other parents, and Noor with her mosque congregation. These learning experiences increased their sense of cultural belonging. 138 Participants reported that they learnt about cultural practices whilst practising speaking and listening in their ESOL class and their ESOL group. Through their narratives, they also described socialisation into social groups such as the PTA, neighbourhood groups and workplaces using social interactions to make sense of, learn and reproduce cultural practices and behaviours, norms and attitudes. Social interactions were important for participants to know the ‘rules of the game’ in the different places that they participated. As hypothesised in the literature review, social interactions strengthen cultural belonging, through the development of new cultural understandings. As represented in Figure 2, the combination of aspects of belonging affect cultural belonging. The language of bureaucracy Participants had to understand systems and processes for applying for support in situations such as housing, immigration and welfare benefits. They also had to remember facts for their complicated cases, and report these to advisors/case workers. The skills involved were listening to advisors/case workers in order to understand these bureaucratic processes. The skills they developed also included sequencing complicated instructions. Samira was applying for a parking permit with the council: ‘I filled the form for the appointment for parking. I went to council, I take the form…(but) I sent my license paper for a photo card. And now I’m waiting for the photo card to come…’ Samira As discussed in 3.5.2, bureaucracy in public services can be viewed as part of cultural knowledge, and familiarity with systems and processes can increase participants’ confidence to deal with public services. Bureaucratic systems of a country can be taken for granted by people who have been socialized into those systems. The example above of the stages that exist in an application process can confuse migrants who are new to these systems. Elma, Samira and Noor reported that they have been able to overcome bureaucratic issues. For Elma, the interactions with her case worker were helpful to support her understand and work through the requirements of her housing situation: “(When I got my document in the post)…I went to Lambeth Housing, and then I said, ‘Now I got my…‘Leave to remain. So I need house.’…My caseworker laughed at me, and he said, ‘Nothing’s gonna happen in one day.” Elma “(When I got my document in the post)…I went to Lambeth Housing, and then I said, ‘Now I got my…‘Leave to remain. So I need house.’…My caseworker laughed at me, and he said, ‘Nothing’s gonna happen in one day.” Elma In 5.4.2 earlier, I had discussed the importance of positive interactions with public service staff in supporting participants to feel confident about accessing public In 5.4.2 earlier, I had discussed the importance of positive interactions with public service staff in supporting participants to feel confident about accessing public 139 services. Elma always reported positive interactions with her caseworker. As with other cultural practices and expectations, social interactions help support and build the understanding of the culture. Experiences of employment Lucy and Asma’s experiences of work culture were limited to entry-level jobs: Asma in beauty therapy and Lucy as a short-term nanny on a yacht. For Lucy the nanny job was different to how she had previously applied for jobs, and her prior experiences of working. There was no application process or contract and this unsettled her. Agreements were made on text message. Asma also did not mention a contract, only a day’s trial and then a subsequent job offer. Asma, who had less prior work experience compared to Lucy, was able to get work with ease. The cultural ‘way of getting work’ that Asma pursued, could have been similar to her previous job-searching experiences, and this could explain why she was highly confident in communicating with her employer, including making decisions about whether to stay in the job or go and look for a new one. In her nannying job, Lucy reported she was treated as a general servant and expected to work long shifts. Asma was able to use the casual status of her work to her advantage and left when she was unhappy with the shifts she was given. In the experiences of these two participants, work practices of employers affected the cultural belonging experienced in this sphere. The language of bureaucracy I suggested that bureaucratic processes are also a type of everyday culture that we interact with and that social interactions supported migrant women on this project to build their confidence to deal with the bureaucratic processes. Elma’s social interactions with her friends also helped her to increase confidence to fill in forms for the Home Office and housing. Prior to this she had paid for someone to fill the form for her. Elma, Noor and Samira reported being able to complete complex applications for different government functions independently resulting in successful applications. Research participants’ social interactions supported them to access cultural knowledge and learn cultural practices, and this developed their cultural belonging. 5.5.4 Temporal belonging As discussed earlier in the chapter (5.1) participants’ experiences as newly arrived migrants and at the start of the research, affected their temporal aspect of belonging. I also suggested that the temporal aspect underpins the whole socialisation process and therefore how participants develop and experience material, cultural and relational 140 aspects of belonging (section 3.5.4). From the data, I suggest that temporal belonging is experienced in three main ways: repetition, routine and through memory. In each of the aspects, we can see how participants have experienced belonging over time. In the material aspect of belonging, Elma’s example of repeatedly attending PTA meetings and watching until she felt confident to participate is also time-related. The observation of activity at the PTA gave her knowledge of what happened, and the next times she attended the meetings, she continued to build recognition of the activities. In each of the aspects, we can see how participants have experienced belonging over time. In the material aspect of belonging, Elma’s example of repeatedly attending PTA meetings and watching until she felt confident to participate is also time-related. The observation of activity at the PTA gave her knowledge of what happened, and the next times she attended the meetings, she continued to build recognition of the activities. Within the relational aspect of belonging, the development of relationships with others is also contingent on time and repetition. In the same PTA example above, apart from the activities, Elma would also have been developing relationships with other parents in the groups, and so building an expectation of how they would behave, developing trust in them. Similarly, in the cultural aspect of belonging, learning and experiencing new cultural knowledge is also affected by how often the activity is repeated. As discussed earlier, temporal belonging is the relationship between memory, self and time (May, 2017). Elma and Samira’s experiences prior to moving to the UK seems to have affected their material belonging. For other participants who did not have prior experiences of moving, the understanding of how a new public services place operates is likely to be affected by the memory of interactions in another public services place within the sphere. I suggest that there is a transferability of expectation of how things are done and the social interactions that will take place. The third way temporal aspect of belonging affects sense of belonging is through routine and familiarity. 5.5.4 Temporal belonging As discussed in 3.5.3 and 3.5.4, routine can support increase in confidence in the work sphere and local community sphere. As seen with Asma, the routine of being in work supported her to develop confidence and ease in the work sphere. Social interactions at work helped her build confidence to speak in English. In the local community sphere, routine interactions with acquaintances and strangers in the local community can create familiarity (May, 2013). Therefore as suggested in 3.6 the temporal aspect of belonging is a crucial part of the process of socialisation towards gaining a sense of belonging. 141 5.6 Discussion on significant findings Social interactions allowed participants’ access to a community of people. Social interactions supported migrant women to build trust in people who were different to them, and created their own community of friendships and support. Participation in social interactions with this community supported women to feel less lonely or isolated, and contributed to their increased confidence. Whatsapp is a powerful tool for extending social interactions and strengthening relationships and contributed to the development of the group’s social relationships. This finding is significant because of the possibility for migrant women to maintain relationships created over the period of study as discussed in 5.5.2. Despite participants’ reported social interactions being with other people who were different to them, those interactions were almost always with other migrants, settled or newly arrived, in London (as discussed in 5.5.2). Overall participation in social interactions was lower than I had anticipated at the start of the research, but participants reported the lowest participation in social interactions with ‘White English’ people, those my participants would consider host community. This finding is significant because of the emphasis on social mixing (discussed in 2.3) by the government. Research also points to the role of the ‘host community’ in supporting migrants to integrate (section 3.3.1), and this should include host community participation in social mixing to enable community cohesion. The lack of host community participation in positive social interactions with newly arrived migrant women will also affect whether they fully develop a sense of belonging, and not just partial belonging in some aspects of belonging. Another finding was that social interactions with acquaintances and strangers in participants’ local community supported the development of trust in local area and belonging. This is also a significant finding because of the perception that only ‘meaningful social interactions’ (Casey, 2016) could support integration and sense of belonging. It suggests that more than can be done to encourage participation in social interactions by migrant women with people they encounter daily in their communities. This would support language development and enable relationships to develop. Social interactions supported migrant women to learn cultural knowledge, practices and behaviours outside of classroom sessions. These gave them access to cultural activities (for example the PTA), and the ability and confidence to manage bureaucratic processes in their own lives. Findings indicate that cultural knowledge is more often learnt from social interactions with other people (see section 5.5.3). 5.6 Discussion on significant findings This finding is 142 interesting because there is an expectation that newly arrived migrants will learn most of the cultural knowledge they need within an ESOL class. A suggestion could be to encourage volunteers to support newly arrived migrants with learning cultural knowledge and practising social interactions. As suggested in 3.5.2, language was not just part of cultural knowledge. Findings indicated that language was the method of interacting with others to form social relationships to build confidence to interact in English. Language formed the basis of learning about new cultures and knowledge about rights and responsibilities. Language was also the key to improving personal situations in accessing services and employment. Finally, time-related aspects such as repetition and routine affected all aspects of belonging. Findings indicated that routine affected development of trust in different places, and development of trust to build relationships. The findings have highlighted the importance of stability and security of housing, education and work for migrant women who are settling in the UK. Time also impacts on the length of time it takes to develop cultural aspects of belonging, for example English language proficiency. Considering the annual cuts to funding for ESOL, the findings from this study suggest that migrant women should be enabled to learn English over a stable and longer period of time, and that an investment into longer-term programmes to support integration could be more effective. A recent large-scale longitudinal project found similar findings (Collyer et al., 2018). 143 6. Conclusion “I’m feeling very good because...before when I came here...my speaking was not...good and understanding...my listening...was not good…. I want to make friends but I can’t speak with them... Now I make, make a lot of friends... I had before eight, nine friends. Now...I have fifteen, fifteen more friends.” Samira This is your new home, this is where you live and this is where you belong now…” Elma This is your new home, this is where you live and this is where you belong now…” Elma The aim of the study was to research the relationship learning English and integration, focusing specifically on the relationship between social interactions in English and migrant women’s sense of belonging. Participants described a multitude of personal challenges such as housing issues, family issues and medical emergencies and so I was aware that it would not be possible to claim that all participants had achieved ‘joy, contentment, happiness or fulfilment’, described by May (2013) as products of having a sense of belonging. However, as people have multiple belongings, and belonging itself is a state of social change (May, 2013), participants did report feeling at ease, confident and comfortable in several places of social interaction in their lives, and so I suggest these indicated their sense of belonging. The relationship between participants’ social interactions in English and their feelings of belonging was positive. The study set out to contribute to academic knowledge about how social interactions affected participants’ perceptions of their own belonging. Recognising the limitations of this small and qualitative study, I am aware it is not possible to extrapolate results. However based on the findings, I suggest that social interactions supported participants to build trust, developed social ties, and share and gain cultural knowledge resulting in a greater sense of belonging, characterised by being comfortable to participate in places in their local community, trusting of others in those places, and self-reported confidence. 144 6.1 Limitations of the study The first limitation of the study is that it is not possible to apply generalisations to the population of migrant women due to the small number of participants of the study. The aim of the study was to investigate the relationship between social interactions and belonging and this required an in-depth research design, which at this stage could not be achieved with a larger sample size. However, as a recommendation for further research, it may be appropriate to focus on one aspect of belonging and explore the opportunity to research this with a larger sample of women. A second limitation was on the number of social interactions recorded. Although I had a large amount of data, I was expecting to have more social interactions reported. For two participants issues relating to their personal situations caused interruptions in study and these personal situations also affected their ability to participate in social interactions. However, a recurring issue from my previous research is the lack of sufficient opportunities for participants to take part in social interactions out of their classroom sessions as reported by my participants. It is not possible to know exactly how much the low number of reported social interactions were due to the reporting mechanism, meaning the oral diary method, which is reliant on participants’ self- reporting, or the genuine lack of opportunities to participate in social interactions. If there are future research projects examining this issue, it may be appropriate to examine how to best enable reporting of social interactions to get the most accurate data. This limitation is also part of the recommendations. The final limitation is in presenting the volume of narrative data of participants’ life stories. Some participants have lived through and experienced many situations, and these have been shared with me in rich details in their narrative interviews. It is a limitation that a researcher cannot always present research participants’ lives in enough detail and richness, whilst maintaining a focus on the research aim. Finally, there were many other areas of interest in the study, which I was not able to explore due to the tight focus and word count of this thesis, and I have listed them in 6.3 under recommendations for further research. 145 Professional practice As mentioned, one of the rationales for this research (section 2.4) was to inform future programme design at ELATT. There are some implications for ESOL professionals, which we will use to adapt and revise our delivery, but also to disseminate to others in the field. Firstly, by understanding the lives of our students outside of the classroom better, specifically about their participation in social interactions in English, we can think about how they are developing their language and belonging within their communities. This knowledge could support better design of course content and practice activities for students outside of their ESOL course. This research, although limited in generalisability, suggests a positive relationship between participation in social interactions in English and a migrant’s sense of belonging. The challenge for ESOL providers is how to enable opportunities for ESOL students to take part in social interactions in English, outside of their classes. Secondly, encouraging students to use online communications such as Whatsapp to maintain social interactions with each other outside of class times could support students to take part in online social interactions in English, and develop social relationships in order to support each other. Practitioners should also consider necessary development of digital skills within the ESOL classroom to support the use of online social communications out-of-class. Finally, providers specialising in ESOL for migrants could engage with public service organisations to offer training and support for their staff that provide services to migrant clients. Support could be so that the staff can better understand migrant-related issues, especially how to better support migrant clients with low levels of English language skills in their interactions. Contribution to professional knowledge As described in section 2.4 (academic rationale for research), there has been a lack of research about the relationship between learning English and integration. This thesis findings have shown that learning English, supported these research participants to develop confidence to take part in social interactions, which in turn positively affected their feeling of belonging. Participants’ experience also showed that taking part in social interactions supports a person to develop trust in other people in their area or 146 community. It is hoped that policy-makers can use this research along with other recent research on the impact of language and integration to make decisions on future funding for ESOL provision and integration projects. Additionally, it is hoped that this research will give insights to policy-makers about the experiences of migrant women to counter any misinformation about the willingness of women to learn English and integrate. Finally, the role of ‘host and settled communities’ is made clearer in findings from this research. Participants reported limited social interactions with people they deemed ‘English’ and ‘UK people’, instead relying upon networks of other migrants. It would be interesting to explore how to involve everyone who has a part to play in the integration process of migrants who are settling in the UK, such as host community groups. 147 6.3 Recommendations for further research There are a number of recommendations for further research based on the limitations of this study and interesting phenomena discovered during the analysis stage. Firstly, the ability to generalise the findings are limited by the small sample size. One recommendation would be to explore how to increase the sample to a more representative size, without losing the narrative element of the research. Based on data from some places, I think there is potential for further exploration in a different piece of research. I was specifically investigating social interactions in English by participants but in two places participants were not able to be clear about what language was used, possibly because of code-switching between languages. Given the increased interest in multilingualism and the impact of using first languages in learning English, further research about the value of multilingual places in supporting migrant women’s integration could be carried out. Another area of interest is in the value of entry-level work to a person’s development of language and belonging. Further research into the experiences of working ESOL students is necessary to establish whether being in low-paid work supports or inhibits language development. Additionally, it seemed from early data analysis that women who had more supportive home environments at point of arrival in the UK, were more likely to have more confidence in English and take part in social interactions in English. It would be interesting to explore the effect that the home environment and support from family members can have on a migrant women’s progress in English and personal development, and what type of support is most successful. Finally, there is opportunity to further research language socialisation in the UK context. 148 Bastian, M. (2014). Time and community: A scoping study. Time & Society, 23(2), 137– 166. https://doi.org/10.1177/0961463X14527999 Bibliography Ager, A., & Strang, A. (2004). Indicators of Integration: final report. Retrieved from https://webarchive.nationalarchives.gov.uk/20110218141321/http:/rds.homeoffice. gov.uk/rds/pdfs04/dpr28.pdf Ager, A., & Strang, A. (2008). Understanding integration: A conceptual framework. 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Retrieved from https://www.jrf.org.uk/report/immigration- social-cohesion-and-social-capital-what-are-links 167 Appendix 1 EU Common Basic Principles Appendix 1 EU Common Basic Principles Appendices 168 Appendix 1 EU Common Basic Principles Appendix 2 Example course content for ESOL community course Appendix 3 ESOL Functional Skills Descriptors Appendix 4 English Language Levels ESOL and CEFR Appendix 5 Participant difficulties Appendix 6 Participant Profiles Appendix 8 Codes: Places of social interactions Appendix 9 Diagrams of places of social interactions Appendix 10- Types of Social Interactions 169 COMMON BASIC PRINCIPLES FOR IMMIGRANT INTEGRATION POLICY IN THE EUROPEAN UNION: COMMON BASIC PRINCIPLES FOR IMMIGRANT INTEGRATION POLICY IN THE EUROPEAN UNION: The explanations provided are intended to give direction to the common basic principle. The description is indicative, by no means exhaustive and will be further developed in the future. 1. Integration is a dynamic, two-way process of mutual accommodation by all immigrants and residents of Member States. Integration is a dynamic, long-term, and continuous two-way process of mutual accommodation, not a static outcome. It demands the participation not only of immigrants and their descendants but of every resident. The integration process involves adaptation by immigrants, both men and women, who all have rights and responsibilities in relation to their new country of residence. It also involves the receiving society, which should create the opportunities for the immigrants' full economic, social, cultural, and political participation. Accordingly, Member States are encouraged to consider and involve both immigrants and national citizens in integration policy, and to communicate clearly their mutual rights and responsibilities. 2. Integration implies respect for the basic values of the European Union. Everybody resident in the EU must adapt and adhere closely to the basic values of the European Union as well as to Member State laws. The provisions and values enshrined in European Treaties serve as both baseline and compass, as they are common to the Member States. They include respect for the principles of liberty, democracy, respect for human rights and fundamental freedoms, and the rule of law. Furthermore they include respect for the provisions of the Charter of fundamental rights of the Union, which enshrine the concepts of dignity, freedom, equality and non- discrimination, solidarity, citizen's rights, and justice. Members States are responsible for actively assuring that all residents, including immigrants, understand, respect, benefit from, and are protected on an equal basis by the full scope of values, rights, responsibilities, and privileges established by the EU and Member State laws. Views and opinions that are not compatible with such basic values might hinder the successful integration of immigrants into their new host society and might adversely influence the society as a whole. Consequently successful 170 integration policies and practices preventing isolation of certain groups are a way to enhance the fulfilment of respect for common European and national values. 3. COMMON BASIC PRINCIPLES FOR IMMIGRANT INTEGRATION POLICY IN THE EUROPEAN UNION: Employment is a key part of the integration process and is central to the participation of immigrants, to the contributions immigrants make to the host society, and to making such contributions visible. Employment is an important way for immigrants to make a visible contribution to Member State societies and to participate in the host society. At the workplace integration of immigrants can be promoted by the recognition of qualifications acquired in another country, by training opportunities that provide skills demanded at the workplace and policies and programmes that facilitate access to jobs and the transition to work. It is also important that there are sufficient incentives and opportunities for immigrants, in particular for those with the prospect of remaining, to seek and obtain employment. The targeting of measures to support immigrants in the European Employment Strategy is an indication of the important influence of employment on the integration process. It is important to make greater use of the European Employment Strategy and the European Social Inclusion Process, backed up by the European Social Fund (ESF), including the lessons learnt from the Equal Community Initiative to reach the Lisbon targets and to promote the combat against all forms of discrimination at the workplace. It is important that Member States, in cooperation with the social partners, pay particular attention to and undertake effective action against discrimination in the recruitment policies of employers on the grounds of ethnic origin of the candidates. 4. Basic knowledge of the host society's language, history, and institutions is indispensable to integration; enabling immigrants to acquire this basic knowledge is essential to successful integration. The importance of basic linguistic, historical, and civic knowledge is reflected in the increasing emphasis placed by several Member States on introductory programmes that focus on putting together the most appropriate toolkit to start the integration process. Pursuing such programmes will allow immigrants to quickly find a place in the key domains of work, housing, education, and health, and help start the longer-term process of normative adaptation to the new society. At the same time, such programmes become strategic investments in the economic and social well-being of society as a whole. Acquiring the language and culture of the host society should be an important focus. Full respect for the immigrants' and their descendants' own language and culture should be also an important element of integration policy. 171 5. COMMON BASIC PRINCIPLES FOR IMMIGRANT INTEGRATION POLICY IN THE EUROPEAN UNION: Efforts in education are critical to preparing immigrants, and particularly their descendants, to be more successful and more active participants in society. Education is an important way to prepare people to participate in society, especially for newcomers. However, lifelong learning and employability are not the only benefits of education. Transferring knowledge about the role and working of societal institutions and regulations and transmitting the norms and values that form the binding element in the functioning of society are also a crucial goal of the educational system. Education prepares people to participate better in all areas of daily life and to interact with others. Consequently, education not only has positive effects for the individual, but also for the society as a whole. Educational arrears are easily transmitted from one generation to the next. Therefore, it is essential that special attention is given to the educational achievement of those who face difficulties within the school system. Given the critical role played by education in the integration of those who are new in a society – and especially for women and children – scholastic underachievement, early school-leaving and of all forms of migrant youth delinquency should be avoided and made priority areas for policy intervention. 6. Access for immigrants to institutions, as well as to public and private goods and services, on a basis equal to national citizens and in a non-discriminatory way is a critical foundation for better integration. If immigrants are to be allowed to participate fully within the host society, they must be treated equally and fairly and be protected from discrimination. EU law prohibits discrimination on the grounds of racial or ethnic origin in employment, education, social security, healthcare, access to goods and services, and housing. Consequently, transparent rules, clearly articulated expectations and predictable benefits for law-abiding immigrants are prerequisites to better immigration and integration policies. Any legal exceptions to this accessibility must be legitimate and transparent. Access also implies taking active steps to ensure that public institutions, policies, housing, and services, wherever possible, are open to immigrants. These steps need to be in accordance with the implementation of the Council Directive concerning the status of third-country nationals who are long-term residents. It is important to monitor and evaluate the success of public institutions in serving immigrants, and that adjustments are being made on an ongoing basis. COMMON BASIC PRINCIPLES FOR IMMIGRANT INTEGRATION POLICY IN THE EUROPEAN UNION: 172 Conversely, uncertainty and unequal treatment breed disrespect for the rules and can marginalise immigrants and their families, socially and economically. The adverse implications of such marginalisation continue to be seen across generations. Restrictions on the rights and privileges of non-nationals should be transparent and be made only after consideration of the integration consequences, particularly on the descendants of immigrants. Finally, the prospect of acquiring Member State citizenship can be an important incentive for integration. 7. Frequent interaction between immigrants and Member State citizens is a fundamental mechanism for integration. Shared forums, inter-cultural dialogue, education about immigrants and immigrant cultures, and stimulating living conditions in urban environments enhance the interactions between immigrants and Member State citizens. Integration is a process that takes place primarily at the local level. The frequency and quality of private interactions and exchanges between immigrants and other residents are key elements of greater integration. There are many ways to encourage interaction. An important aspect is a greater focus on promoting the use of common forums, intercultural dialogue, spaces, and activities in which immigrants interact with other people in the host society, and on the sustained education of the host society about immigrants and immigrant cultures. Good cooperation among the different involved actors is necessary in order to stimulate these processes. Furthermore, implementation of active anti-discrimination policies, anti- racism policies, and awareness-raising activities to promote the positive aspects of a diverse society are important in this regard. The level of economic welfare in neighbourhoods, the feeling of safety, the condition of public spaces, and the existence of stimulating havens for immigrant children and youngsters and other living conditions are all aspects that affect the image of the people who live in these areas. In many Member States, immigrant population groups are often concentrated in poor urban areas. This does not contribute to a positive integration process. Positive interaction between immigrants and the host society and the stimulation of this interaction contribute to successful integration and are therefore needed. Therefore, improving the living environment in terms of decent housing, good health care, neighbourhood safety, and the availability of opportunities for education, voluntary work and job training is also necessary. 173 8. The practice of diverse cultures and religions is guaranteed under the Charter of Fundamental Rights and must be safeguarded, unless practices conflict with other inviolable European rights or with national law. COMMON BASIC PRINCIPLES FOR IMMIGRANT INTEGRATION POLICY IN THE EUROPEAN UNION: In this context particularly consideration needs to be given to the impact of immigration on public services like education, social services and others, especially at the level of regional and local administrations, in order to avoid a decrease in the quality standards of these services. S The integration of immigrants is deeply influenced by a broad array of policies that cut across institutional competencies and levels of government. In this context particularly consideration needs to be given to the impact of immigration on public services like education, social services and others, especially at the level of regional and local administrations, in order to avoid a decrease in the quality standards of these services. Accordingly, not only within Member States but also at the European level, steps are needed to ensure that the focus on integration is a mainstream consideration in policy formulation and implementation while at the same time specifically targeted policies for administrations, in order to avoid a decrease in the quality standards of these services. Accordingly, not only within Member States but also at the European level, steps are needed to ensure that the focus on integration is a mainstream consideration in policy formulation and implementation, while at the same time specifically targeted policies for integrating migrants are being developed. Although Governments and public institutions at all levels are important actors, they are not the only ones. Integration occurs in all spheres of public and private life. Numerous non-governmental actors influence the integration process of immigrants and can have an additional value. Examples in this respect are, trade unions, businesses, employer organisations, political parties, the media, sports clubs and cultural, social and religious organisations. Cooperation, coordination and communication between all of these actors are important for effective integration policy. The involvement of both immigrant and the other people in the host society is also necessary. 11. Developing clear goals, indicators and evaluation mechanisms are necessary to adjust policy, evaluate progress on integration and to make the exchange of information more effective. Irrespective of the level of integration policy efforts, it is important to know whether these efforts are effective and make progress. Although it is a process rather than an outcome, integration can be measured and policies evaluated. Sets of integration indicators, goals, evaluation mechanisms and benchmarking can assist measuring and comparing progress, monitor trends and developments. COMMON BASIC PRINCIPLES FOR IMMIGRANT INTEGRATION POLICY IN THE EUROPEAN UNION: The cultures and religions that immigrants bring with them can facilitate greater understanding among people, ease the transition of immigrants into the new society and can enrich societies. Furthermore, the freedom to practice one's religion and culture is guaranteed under the Charter of Fundamental Rights. Member States have an obligation to safeguard these rights. Furthermore, EU law prohibits discrimination in employment or occupation on the grounds of religion or belief. However, Member States also have a responsibility to ensure that cultural and religious practices do not prevent individual migrants from exercising other fundamental rights or from participating in the host society. This is particularly important as it pertains to the rights and equality of women, the rights and interests of children, and the freedom to practice or not to practice a particular religion. Constructive social, inter-cultural and inter- religious dialogue, education, thoughtful public discourse, support for cultural and religious expressions that respect national and European values, rights and laws (as opposed to expressions that violate both the letter and spirit of such values and rights), and other non-coercive measures are the preferred way of addressing issues relating to unacceptable cultural and religious practices that clash with fundamental rights. However if necessary according to the law legal coercive measures can also be needed. 9. The participation of immigrants in the democratic process and in the formulation of integration policies and measures, especially at the local level, supports their integration. Allowing immigrants a voice in the formulation of policies that directly affect them may result in policy that better serves immigrants and enhances their sense of belonging. Wherever possible, immigrants should become involved in all facets of the democratic process. Ways of stimulating this participation and generating mutual understanding could be reached by structured dialogue between immigrant groups and governments. Wherever possible, immigrants could even be involved in elections, the right to vote and joining political parties. When unequal forms of membership and levels of engagement persist for longer than is either reasonable or necessary, divisions or differences can become deeply rooted. This requires urgent attention by all Member States. 174 10. Mainstreaming integration policies and measures in all relevant policy portfolios and levels of government and public services is an important consideration in public- policy formation and implementation. The integration of immigrants is deeply influenced by a broad array of policies that cut across institutional competencies and levels of government. COMMON BASIC PRINCIPLES FOR IMMIGRANT INTEGRATION POLICY IN THE EUROPEAN UNION: The purpose of such evaluation is to learn from experience, a way to avoid possible failures of the past, adjust policy accordingly and showing interest for each others efforts. When Member States share information about their evaluative tools at European level and, where appropriate, develop European criteria (indicators, benchmarks) and gauges for the purposes of comparative learning, the process of knowledge-sharing will be made more effective. The exchange of information has already proven to be useful within the National Contact Points on integration. Exchanging information provides for 175 taking into account the different phases in which Member States find themselves in the development of their own integration policies and strategies. COMMON BASIC PRINCIPLES FOR IMMIGRANT INTEGRATION POLICY IN THE EUROPEAN UNION: (Council of The European Union, 2004) (Council of The European Union, 2004) 176 Appendix 2 Example course content for ESOL community course Course Outline: 177 Appendix 2 Example course content for ESOL community course Course Outline: Classroom: Community Centre: SEPTEMBER 2018 Monday Tuesday Wednesday Thursday Friday 24 – Classroom 10am-12:30pm Course introduction 25 26 27 – Community centre 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 28 OCTOBER 2018- Black History Month Monday Tuesday Wednesday Thursday Friday 1 – Classroom 10am-12:30pm ESOL class 1-2:30pm Language Lab 2 3 4 – Community centre 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 5 8 – Classroom 10am-12:30pm ESOL class 1-2:30pm Community leadership training 9 10 11 – Community centre 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 12 15 – Classroom World Food Day 10am-12:30pm ESOL class 1-2:30pm Language Lab or workshop 16 17 18 – Community centre British Summer Time 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 19 HALF TERM HOLIDAYS 22-26 October NOVEMBER 2018 Monday Tuesday Wednesday Thursday Friday 29/10 – Classroom Halloween 10am-12:30pm ESOL class 1-2:30pm Language Lab or workshop 30/10 31/10 1 – Community centre 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 2 177 178 5 – Classroom 10am-12:30pm ESOL class 1-2:30pm Community leadership training 6 7 8 – Community centre Diwali 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 9 12 – Classroom Remembrance 10am-12:30pm ESOL class 1-2:30pm Language Lab or workshop 13 14 15 – Community centre 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 16 19 – Classroom 10am-12:30pm ESOL class 1-2:30pm Language Lab or workshop 20 21 22 – Community centre 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 23 26 – Classroom International Day for the Elimination of Violence against Women 10am-12:30pm ESOL class 1-2:30pm Community leadership training 27 28 29 – Community centre 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 30 DECEMBER 2018- Universal Month for Human Rights Monday Tuesday Wednesday Thursday Friday 3 – Classroom 10am-12:30pm ESOL class 1-2:30pm Language Lab or workshop 4 5 6 – Community centre 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 7 10 – Classroom 10am-12:30pm ESOL class 1-2:30pm Language Lab or workshop 11 12 13 – Community centre 9:30am-1:30pm Skills Sharing Workshop / Celebration Lunch 14 17 – Classroom 18 19 20 21 178 International Migrants Day 10am-12:30pm ESOL class 1-2:30pm Community leadership training NO CLASS/LUNCH JANUARY 2019 Monday Tuesday Wednesday Thursday Friday 7 – Classroom 10am-12:30pm ESOL class 1-2:30pm Community leadership training 8 9 10 – Community centre 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 11 14 – Classroom 10am-12:30pm ESOL class 1-2:30pm Language Lab or workshop 15 16 17 – Community centre 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 18 21 – Classroom World Religion Day 10am-12:30pm ESOL class 1-2:30pm Language Lab or workshop 22 23 24 – Community centre 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 25 28 – Classroom Holocaust Memorial 27/01/19 10am-12:30pm ESOL class 1-2:30pm Community leadership training 29 30 31 – Community centre 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 1 Feb FEBRUARY 2019- LGBT History Month Monday Tuesday Wednesday Thursday Friday 4 – Classroom 10am-12:30pm ESOL class 1-2:30pm Community 5 6 7 – Community centre 9:30am-11:30am Volunteering in conversation club 8 179 Action Forum event 12:30-1:30pm Community lunch 11 – Classroom 10am-12:30pm ESOL class 1-2:30pm Language Lab or workshop 12 13 14 – Community centre Valentine’s Day 9:30am-11:30am Volunteering in conversation club 12:30-1:30pm Community lunch 15 END OF COURSE 180 Appendix 3 ESOL Functional Skills Descriptors Appendix 3 ESOL Functional Skills Descriptors Entry 3 Level 1 At this level, adults can At this level, adults can listen and respond to spoken language, including straightforward information and narratives, and follow straightforward explanations and instructions, both faceto- face and on the telephone listen and respond to spoken language, including information and narratives, and follow explanations and instructions of varying length, adapting response to speaker, medium and context speak to communicate information, feelings and opinions on familiar topics, using appropriate formality, both face-to-face and on the telephone speak to communicate information, ideas and opinions, adapting speech and content to take account of the listener(s) and medium engage in discussion with one or more people in a familiar situation, making relevant points and responding to what others say to reach a shared understanding about familiar topics engage in discussion with one or more people in familiar and unfamiliar situations, making clear and relevant contributions that respond to what others say and produce a shared understanding about different topics An adult will be expected to: An adult will be expected to: listen for and follow the gist of explanations, instructions and narratives in different contexts listen for and understand explanations, instructions and narratives on different topics in a range of contexts listen for detail in explanations, instructions and narratives in different contexts listen for and identify relevant information from explanations and presentations on a range of straightforward topics listen for and identify relevant information and new information from discussions, explanations and presentations use strategies to clarify and confirm understanding, e.g. COMMON BASIC PRINCIPLES FOR IMMIGRANT INTEGRATION POLICY IN THE EUROPEAN UNION: facial expressions, body language and verbal prompts use strategies to clarify and confirm understanding, e.g. facial expressions or gestures provide feedback and confirmation when listening to others listen to and respond appropriately to other points of view Department for Education and Skills, 2001, p. 18 181 Appendix 4 English Language Levels ESOL and CEFR Appendix 4 English Language Levels ESOL and CEFR Appendix 4 English Language Levels ESOL and CEFR ESOL and CEFR Levels ESOL CEFR C1 Level 2 B2 Level 1 B1 Entry-level 3 A2 Entry-level 2 A1 Entry-level 1 Appendix 4 English Language Levels ESOL and CEFR ESOL and CEFR Levels ESOL CEFR C1 Level 2 B2 Level 1 B1 Entry-level 3 A2 Entry-level 2 A1 Entry-level 1 ESOL and CEFR Levels ESOL and CEFR Levels Taken from (Mallows, 2013) 182 Appendix 5 Participant difficulties pp p Participant Examples of the difficult personal situations they were experiencing Elma Homeless and in temporary accommodation in a different borough from the one that her older child was in school, and younger child was in nursery. Threatened with eviction. Later threatened with deportation. Her daughter was awaiting surgery and then had a playground accident. In the final few months of the research her son had an accident and was in hospital and she too was hospitalised for illness not long after. Asma Experienced controlling behaviour from her in-laws and as a result was experiencing stress and depression. She returned to Pakistan to give her time from her husband’s family but later managed to return to live with her husband. Flora Homeless and in temporary accommodation with her 4 year old child after the sudden death of her husband. She had regular issues with the temporary accommodation including violence and threats from other service users. Suffering from depression. Lucy Experienced bullying from her stepdaughter and husband for wanting to work and be independent. Experienced being taken advantage of by a two employers and people who promised her work. Samira Carer for her husband, a refugee who suffers from PTSD, and three children. Was placed in temporary and unsuitable accommodation by the council and had to fight to be relocated. Brief table of personal circumstances of participants 183 Appendix 6 Participant Profiles Name Basic information (e.g. country of origin, ethnic background) Family background Socio- economic background/sit uation Work experience Home country Education Level Prior language-learning experience Elma Somalia via Egypt, Somali, lived in UK for Large family. Father died when she was young. At 13, family fled to Egypt. She had to look after younger siblings. Destitute in the refugee camps, relied on relief. Had to work as a teenager. Yes 2 yrs in Egypt and a year in the UK. Went to school in Egypt. Did not finish secondary school as was ill in hospital. Learnt Arabic at the refugee camp in Egypt. Learnt a little English when working as a tour guide. Flora India, Bengali, lived in UK 6 years Small family. Very close to brothers. Good socio- economic situation. Were able to live comfortably. No, but worked in the UK for less than a year. Completed secondary school. ESOL and CEFR Levels Had learnt English in school and college in India. She had an intermediate level of English when she came to the UK. Lucy China, Chinese, lived in UK for five years. Small family; had a grown up daughter. Had elderly parents in China. Financially independent having worked in many high level jobs and achieved high level qualifications. Yes 35+ yrs, worked for all her adult life, straight from university. Highly qualified and has studied to MA level, a professor in Mandarin, a mandarin test teacher, Confucius school scholar and has a teaching certificate. Had been learning English for more than 20 years, not fluent yet and had a lower level of speaking and listening (lower intermediate level) than in reading and writing. Samira Afghanistan, Afghan, lived in the UK for four years. Lived with a large family. Father killed in the war. Mother was disabled and Very low income and Samira was working from a young age. Yes 3+ yrs as a nursery assistant and tailor at home. Mid secondary- had to look after her mother and family. Fluent in three languages from Afghanistan. Learnt basic English from television and others. Fluent in three languages from Afghanistan. Learnt basic English from television and others. 184 Samira had to look after her mother and younger siblings. Asma Pakistan, Pakistani, one year in UK. Large and extended family. Family had good socio-economic situation; could afford to live comfortably, owned a shop. Yes 2 yrs as a boutique dress- maker Completed high school. Learnt English at school and from family members. Had an intermediate level of English when she moved to the UK. Noor The Netherlands, Moroccan Average family size for Netherlands. Close to sister and family. Family in good socio-economic situation. Moved to UK with husband. Yes 3+ yrs in office jobs. College educated. Had learnt Dutch as a young child. Learnt English at school but as a subject and also through watching English language shows on television. ESOL and CEFR Levels 185 185 186 Appendix 8 Codes: Places of social interactio a Priori Codes of areas identified by IFS Community Centre Playgroup Work place Doctor's Surgery Hospital Child's school Nursery Travel- underground Travel- bus At a SELT English test centre At a LIUK English test centre At a Driving test centre At the bank Talking to estate agents At a local corner shop At a retail store On social media/ online/ websites On Whatsapp Places Codes after Inductive analysis 1 Community Centre 2 Playgroup 3 Work place 4 Doctor's Surgery 5 Hospital 6 Child's school with teachers 6a PTA 6b School gates 7 Nursery 7a Nursery teachers 7b Nursery parents 8 Travel- underground 8a Station 9 Travel- bus Appendix 8 Codes: Places of social interactions a Priori Codes of areas identified by IFS Community Centre Playgroup Work place Doctor's Surgery Hospital Child's school Nursery Travel- underground Travel- bus At a SELT English test centre At a LIUK English test centre At a Driving test centre At the bank Talking to estate agents At a local corner shop At a retail store On social media/ online/ websites On Whatsapp Places Codes after Inductive analysis 1 Community Centre 2 Playgroup 3 Work place Appendix 8 Codes: Places of social interactions Places Codes after Inductive analysis 1 Community Centre 2 Playgroup 3 Work place 4 Doctor's Surgery 5 Hospital 6 Child's school with teachers 6a PTA 6b School gates 7 Nursery 7a Nursery teachers 7b Nursery parents 8 Travel- underground 8a Station 9 Travel- bus 187 10 At a SELT English test centre 11 At a LIUK English test centre 12 At a Driving test centre 13 At the bank 14 Talking to estate agents 15 At a local corner shop 16 At a retail store 17 On social media/ online/ websites 18 On Whatsapp 19 Home- in-laws/husband 20 Home- own 20a Home- neighbours 21 Public- street/open places 22 ESOL class 23 College 24 Home Office 25 Home- other families 26 Social Services 27 Housing support services 28 Travel-trains 29 Child Maintennance agency 30 Library 31 Family friends home 32 Home Community 33 Children's Centre 34 Mosque 35 Home- temp/hostel 36 Volunteer placement 36a Prospective employer 37 Friend's houses/other people 38 Jobcentre 39 Council 40 CAB 41 Child's friend's home 42 Opticians 43 Counselling services 43b Physio 44 Temple 45 Café/restaurant 46 Park 47 Workshop 188 48 Conversation Club 49 Driving lesson 50 Places of interest 51 Utilities/services 189 Appendix 9 Diagrams of places of social interactions Elma The data shows that Elma’s main places of participation are with her home community, in her ESOL class, at the hospital, home, and in the homes of her child’s friends. The other places that she participates in most are at the school and in the PTA, with and at the council. The data shows that Elma’s main places of participation are with her home community, in her ESOL class, at the hospital, home, and in the homes of her child’s friends. The other places that she participates in most are at the school and in the PTA, with and at the council. 190 Flora The data shows that Flora’s main spheres of participation are at her child’s nursery, with people in her hostel accommodation, in her ESOL class, her counselling sessions and with the council. Other areas that she participates most in are with parents at the school gates of her nursery, the jobcentre, at the homes of family friends, the GP, at the temple and in the park. The data shows that Flora’s main spheres of participation are at her child’s nursery, with people in her hostel accommodation, in her ESOL class, her counselling sessions and with the council. Other areas that she participates most in are with parents at the school gates of her nursery, the jobcentre, at the homes of family friends, the GP, at the temple and in the park. 191 Asma Asma The data shows that Asma has very limited participation in places. The main places she participates in are in the home of her in-laws, in her class and with classmates out of class via phones and WhatsApp, and other online communications, and in her workplace. The data shows that Asma has very limited participation in places. The main places she participates in are in the home of her in-laws, in her class and with classmates out of class via phones and WhatsApp, and other online communications, and in her workplace. 192 Lucy The data shows that Lucy participates in these places the most: in her home with her husband and family, but also with friends who visit and have a language exchange arrangement, in her ESOL class and vocational qualification class, with her home community, and with prospective employers as she is constantly applying for jobs (mainly in schools to teach Chinese). Other places are in her own business set-up, which involved partners, the library, in work and volunteer work placement. The data shows that Lucy participates in these places the most: in her home with her husband and family, but also with friends who visit and have a language exchange arrangement, in her ESOL class and vocational qualification class, with her home community, and with prospective employers as she is constantly applying for jobs (mainly in schools to teach Chinese). Other places are in her own business set-up, which involved partners, the library, in work and volunteer work placement. The data shows that Lucy participates in these places the most: in her home with her husband and family, but also with friends who visit and have a language exchange arrangement, in her ESOL class and vocational qualification class, with her home community, and with prospective employers as she is constantly applying for jobs (mainly in schools to teach Chinese). Other places are in her own business set-up, which involved partners, the library, in work and volunteer work placement. 193 Samira Samira Samira The data shows that Samira participates in these spheres the most: in her class, at the college she attends for childcare, at the council, at home with her neighbours, and at the GP and health visitor. She also participates in these spheres: the nursery, her child’s school, her childcare work placement and a conversation club. The data shows that Samira participates in these spheres the most: in her class, at the college she attends for childcare, at the council, at home with her neighbours, and at the GP and health visitor. She also participates in these spheres: the nursery, her child’s school, her childcare work placement and a conversation club. The data shows that Samira participates in these spheres the most: in her class, at the college she attends for childcare, at the council, at home with her neighbours, and at the GP and health visitor. She also participates in these spheres: the nursery, her child’s school, her childcare work placement and a conversation club. 194 Noor Noor The data shows that Noor had smaller number of participation in places and they were concentrated to two main places- the mosque and home. The data shows that Noor had smaller number of participation in places and they were concentrated to two main places- the mosque and home. 195 Appendix 10- Types of Social Interactions 196 197 Category Types of interactions Formal/In Asking for information Questions about diagnosis Formal Asking for information Requesting support for understanding complex medical terminology Formal Asking for information Requests for information Formal Asking for information Requests for services. Formal Asking for support- from social group Requests for help Informal Asking for support- from social group Requests for support Informal Asking for support- from social group Requests to other classmates Informal Asking support- from public services Requests for help to ambulance operators, reception/admission staff, triage nurses and waiting for information Formal Asking support- from public services Requests for support Formal Building relations- business Making business plans with other people. Formal Building relationships Asking each other questions about children and family Informal Building relationships Asking for information about each other Informal Building relationships Giving information to others Informal Building relationships Greetings Informal Building relationships Other parents to be friendly- mainly asking how they are some in English some in Bengali Informal Building relationships Requesting and giving information to other mosque-goers. Informal Building relationships Saying hello and good morning to hostel caretaker/manager; Informal Building relationships Taking part in the PTA Informal Building relationships Talking about children to her friends Informal Building relationships Talking about emotions and personal mental health situation Informal Building relationships Talking about food- they always cooked for each other. Informal Building relationships Talking about real llife situations. Informal Building relationships Talking to child on phone/video call- reassuring them that Elma will be home soon. Informal Building relationships Talking to daughter about school, playing with daughter, looking after daughter. Informal Building relationships Talking to other children's parents Informal Building relationships Talking to teacher at the school Formal Business proposals Presenting ideas/pitches to people about the value of Mandarin classes in libraries and community venues. Appendix 10- Types of Social Interactions Formal Complaining Expressing frustration to the staff Formal Getting advice and support- from social group Asking for advice Informal Getting advice and support- from social group Asking questions about advice and guidance received Informal Getting advice and support- from social group Consulting others on decisions Informal Giving information Communicating with school admin about absences, accidents etc Formal Giving information Giving information to others about her experience and qualifications Formal Giving information Making payments- giving information to staff to make payments Formal Giving information Requests for information and giving information Formal Giving information Responding to queries and requests for information Formal Giving information Responding to requests for information from administrators Formal Giving information Safeguarding process support from teachers and external organisations Formal Giving information Taking part in the PTA Formal Giving information Talking to child on phone/video call- reassuring them that Elma will be home. Informal Learning- from experts Listening to talks about the religion. Formal Learning- from experts Talking to teachers about her daughter's progress and issues/problems Formal Learning- from social group Discussing with other mosque-goers about topics in the talks or about the Informal Learning- from social group Discussions about legal position, rights and options Informal Learning- from social group Listening to others' ideas Informal Learning- from social group Talking about language. Informal Learning- from social group Talking about real llife situations. Informal Learning- from social group Talking to other children's parents Informal Receiving information Requests for information and giving information Formal Receiving information Talking to daughter about school, playing with daughter, looking after daughter. Appendix 10- Types of Social Interactions Informal Receiving information Talking to nursery nurses Formal Receiving information and instructions Being told the rules by hostel manager/caretaker Formal Receiving information and instructions Getting messages from the school about tasks for children to complete (homework) Formal Receiving information and instructions Listening for understanding of complex medical terminology Formal Receiving information and instructions Receiving advice for recovery (physio for son's fingers) Formal Receiving information and instructions Requests/instructions to do thing you need to eat this medicine; you need to take this scan Formal Receiving information and instructions Taking instructions Formal Receiving information and instructions Taking part in the PTA Formal Receiving information and instructions Talking to teachers about her daughter's progress and issues/problems Formal Receiving information and instructions Talking to teachers at the school Formal Self advocacy Appealing a decision Formal Self advocacy Making demands of staff based on rights not being met Formal Serving customers Asking customers what they wanted- personal consultation about the service Formal Serving customers Asking for and receiving payment. Formal Serving customers Greeting customers Formal Serving customers Making conversation/small talk with customers having treatment Formal Supporting each other Advice and guidance for each other Informal Supporting each other Other patients- sharing, supporting each other Informal Supporting each other Updating family members about their condition and talking about other things (taking mind off the situation) Informal
https://openalex.org/W2906319866
https://sciforum.net/paper/download/6075/manuscript
English
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&lt;span&gt;Experimental and Computational Studies Addressed to 1,3-Dipolar Cycloadditions of D-Erythrose 1,3-Dioxane 1,5-Lactone with &lt;/span&gt;&lt;em&gt;Regio&lt;/em&gt;&lt;span&gt;&lt;em&gt;- and&lt;/em&gt;&lt;br /&gt;&lt;/span&gt;&lt;em&gt;Stereo-selectivity&lt;/em&gt;
Proceedings of MOL2NET 2018, International Conference on Multidisciplinary Sciences, 4th edition
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MOL2NET, 2018, 4, ISSN: 2624-5078, ISBN: 978-3-03842-820-6 http://sciforum.net/conference/mol2net-04 MOL2NET, 2018, 4, ISSN: 2624-5078, ISBN: 978-3-03842-820-6 http://sciforum.net/conference/mol2net-04 1 MDPI MOL2NET, International Conference Series on Multidisciplinary Sciences Experimental and Computational Studies Addressed to 1,3- Dipolar Cycloadditions of D-Erythrose 1,3-Dioxane 1,5-Lactone with Regio- and Stereo-selectivity Cristina E. A. Sousaa, António M. P. Ribeiroa, António Gil Fortesa, Nuno M. F. S. A. Cerqueirab, and Maria J. Alvesa a Departamento de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal b UCIBIO@REQUIMTE, Departamento de Biomedicina da Faculdade de Medicina da Universidade do Porto, 4200-319 Porto, Portugal a Departamento de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal b UCIBIO@REQUIMTE, Departamento de Biomedicina da Faculdade de Medicina da Universidade do Porto, 4200-319 Porto, Portugal . Graphical Abstract Graphical Abstract Graphical Abstract Abstract A new D-erythrose 1,3-dioxane 1,5-lactone derivative was synthetized and found to be a highly stereo-selective template as dipolarophile in 1,3-dipolar cycloadditions. Different regio- selectivities were obtained depending on the nature of the 1,3-dipole: complete, with alkyl azides and diazomethylbenzene, inexistent, with nitrile oxides. To understand the mechanisms of cycloadditions with the three types of 1,3- dipoles, computational studies were performed, giving full agreement with the experimental data. The computational results showed that all the studied cycloadditions are concerted processes, involving exoenergonic, and small free activation energies. The stereoselectivity of MOL2NET, 2018, 4, ISSN: 2624-5078, ISBN: 978-3-03842-820-6 http://sciforum.net/conference/mol2net-04 2 http://sciforum.net/conference/mol2net-04 the reactions is due to a combination of the steric effect endorsed by hydrogen H-8 and the hyper conjugative effect of the incoming 1,3- dipole with the lactone. The regioselectivity observed in alkyl azides and phenyldiazomethane is mostly dependent on the distortion effect during the cycloaddition process. This distortion effect is however higher in the alkyl azide compounds than in phenyldiazomethane. This distortion effect is absent from nitrile oxides. This study provides a specific example where apparent similar chemistry was found to proceed via different mechanisms, leading to different output results. p the reactions is due to a combination of the steric effect endorsed by hydrogen H-8 and the hyper conjugative effect of the incoming 1,3- dipole with the lactone. The regioselectivity observed in alkyl azides and phenyldiazomethane is mostly dependent on the distortion effect during the cycloaddition process. This distortion effect is however higher in the alkyl azide compounds than in phenyldiazomethane. This distortion effect is absent from nitrile oxides. This study provides a specific example where apparent similar chemistry was found to proceed via different mechanisms, leading to different output results. the reactions is due to a combination of the steric effect endorsed by hydrogen H-8 and the hyper conjugative effect of the incoming 1,3- dipole with the lactone. The regioselectivity observed in alkyl azides and phenyldiazomethane is mostly dependent on the distortion effect during the cycloaddition process. This distortion effect is however higher in the alkyl azide compounds than in phenyldiazomethane. This distortion effect is absent from nitrile oxides. This study provides a specific example where apparent similar chemistry was found to proceed via different mechanisms, leading to different output results. Sousa, C.E.A.; Ribeiro, A. M. P.; Gil Fortes, A.; N. M. F. S. A. Cerqueira; Alves, M. J. J. Org. Chem., 2017, 82 (2), pp 982–991. Ribeiro, A. M. P.; Gil Fortes, A.; N. M. F. S. A. Cerqueira; Alves, M. J. J. Org. Chem., 2017, 82 References Sousa, C.E.A.; Ribeiro, A. M. P.; Gil Fortes, A.; N. M. F. S. A. Cerqueira; Alves, M. J. J. Org. Chem., 2017, 82 (2), pp 982–991.
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https://www.researchsquare.com/article/rs-2097064/latest.pdf
English
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An Efficient Method to Prepare Barcoded cDNA Libraries from Plant Callus for Long-Read Sequencing
Methods and protocols
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Method Article Keywords: amplification-free protocol, direct cDNA sequencing, high-throughput RNA sequencing, long- reads, MinION, Oxford Nanopore Technologies®, poly(A) RNA, tamarillo Posted Date: September 28th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2097064/v1 Conclusions An efficient method for preparing barcoded cDNA libraries from plant callus is presented. An efficient method for preparing barcoded cDNA libraries from plant callus is presented. Long-read sequencing using these libraries allows a comprehensive analysis of plant transcriptomes. cient method for preparing barcoded cDNA libraries from plant callus is pre Results A detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies® was developed. Through this method, 8 barcoded cDNA libraries from two tamarillo (Solanum betaceum Cav.) types of callus (friable and compact) and with distinct embryogenic competencies (embryogenic non-embryogenic) have been successfully prepared and sequenced. DOI: https://doi.org/10.21203/rs.3.rs-2097064/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Methods and Protocols on March 15th, 2023. See the published version at https://doi.org/10.3390/mps6020031. Page 1/17 Page 1/17 Background Long-read sequencing methods allow a comprehensive analysis of transcriptomes in identifying full- length transcripts. This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies. However, some plant tissues are recalcitrant to the successful preparation of cDNA libraries thus impairing further analyses. Background In recent years, transcriptome sequencing has been an invaluable tool in gene expression profiling. By unravelling gene regulatory mechanisms, high-throughput sequencing approaches have shown widespread applications in many fields of biology, including plant physiology and developmental analysis [1–3]. Nanopore sequencing from Oxford Nanopore Technologies® (ONT) is a high-throughput sequencing technology that detects ionic current changes as DNA or RNA molecules pass through nanopores [4]. Compared to other next-generation sequencing methods, Nanopore offers native and long-read sequencing (> 4Mb in a single read), which, together with the template-switching method, allows the detection of complete full-length transcripts [5]. Moreover, barcoding kits are available allowing cDNA tagging. Multiple barcoded libraries can be pooled and sequenced in a single run optimising the assay for high-throughput. With Nanopore, it is also possible to sequence cDNA directly using a PCR-free protocol, thus avoiding amplification bias and preserving samples' quantitative information [6]. Despite the rapid advances in the development and optimization of sequencing technologies, some methods are not so straightforward applicable to particular organisms. Due to the richness of secondary metabolites and high ribonucleases (RNases) content, plant tissues have proven to be a complicated challenge for the application of cutting-edge sequencing technologies and to obtain high-quality RNA Page 2/17 Page 2/17 yields for subsequent construction of good-quality cDNA libraries [7]. Although several kits are available for total RNA extraction and poly(A) RNA isolation, they have proved to be somewhat inefficient for some plant tissues, making it difficult to reproduce results between different laboratories, which is still a limitation to gene expression studies, particularly in non-model species [8]. Furthermore, RNA extraction yields widely vary according to species, genotypes, sample type, age and oxidative state [9–12]. For instance, in vitro growing callus, rich in starch, pectins and other polysaccharides (e.g., arabinoxylan, β- glucan) [13, 14], has shown to be a particularly difficult tissue to achieve high yield and quality RNA extractions for further processing methods [9]. Finally, the polyadenylated transcripts (such as mRNAs and some long non-coding (lncRNAs)), the most targeted in transcriptome analysis studies, comprise only a small part of the total RNA in eukaryotes [15, 16]. Because of its great morphogenic plasticity, callus samples are valuable experimental models for fundamental research as well as for industrial and bioengineering applications [17]. However, straightforward optimized procedures for high-throughput analysis are still often required. Plant material Two tamarillo (Solanum betaceum) callus lines were used: a compact embryogenic callus (EC, Fig. 1a) and a friable non-embryogenic (NEC, Fig. 1b). Both were induced from leaf explants of in vitro growing shoots and had been maintained in Murashige and Skoog medium [18] supplemented with 20 μM Picloram plus 9% (w/v) sucrose, solidified with 0.25% (w/v) PhytagelTM (Sigma-Aldrich) at pH 5.7 and incubated at 24 ± 1 ºC under dark conditions, following a procedure previously described [19]. For each callus line, EC and NEC, four biological replicates were used. Background Here, a detailed methodology that combines poly(A) RNA isolation and barcoded cDNA library preparation is described for plant callus samples. To develop this protocol Dynabeads™ mRNA DIRECT™ Kit from Invitrogen™ and Direct cDNA Sequencing Kit (SQK-DCS109) with Native Barcoding Expansion 1–12 (EXP-NBD104) from ONT were used to prepare barcoded cDNA libraries from two tamarillo (solanaceous tree) callus lines with different cytological (compact and friable) and morphogenic competencies (embryogenic and non- embryogenic). Page 4/17 ( ) LongAmp® Taq 2X Master Mix (NEB, Cat. No. M0287) Maxima H Minus Reverse Transcriptase (Thermo Scientific™, Cat. No. EP0751) Native Barcoding Expansion 1-12 (ONT, Cat. No. EXP-NBD104) NEBNext® Quick Ligation Module (NEB, Cat. No. E6056) NEBNext® Ultra™ II End Repair/dA-Tailing Module (NEB, Cat. No. E7546) Nuclease-free water (PanReac AppliChem ITW Reagents, Cat. No. A7398) NZYRibonuclease Inhibitor (NZY, Cat. No. MB084) QubitTM dsDNA HS Assay Kit (InvitrogenTM, Cat. No. Q32851) QubitTM RNA High Sensitivity (HS) Assay Kit (InvitrogenTM, Cat. No. Q32852) RNase Cleaner (NZY, Cat. No. MB16001) RNase CocktailTM Enzyme Mix (InvitrogenTM, Cat. No. AM2286) Supplies  0.2 mL PCR tubes 1.5 mL microcentrifuge tubes 21-gauge needles, sterile Liquid nitrogen Low-binding P10 barrier tips, sterile Low-binding P1000 barrier tips, sterile Low-binding P200 barrier tips, sterile Mortars Pestles QubitTM Assay Tubes (InvitrogenTM, Cat. No. Q32856) LongAmp® Taq 2X Master Mix (NEB, Cat. No. M0287) NEBNext® Ultra™ II End Repair/dA-Tailing Module (NEB, Cat. No. E7546) Nuclease-free water (PanReac AppliChem ITW Reagents, Cat. No. A7398) Nuclease-free water (PanReac AppliChem ITW Reagents, Cat. No. A7398) NZYRibonuclease Inhibitor (NZY, Cat. No. MB084) Page 3/17 Agencourt AMPure XP beads (Beckman Coulter, Inc, Cat. No. A63880) Blunt/TA Ligase Master Mix (New England Biolabs (NEB), Cat. No. M0367) Direct cDNA Sequencing Kit (ONT, Cat. No. SQK-DCS109) dNTPs NZYMix (NZYTech, Lda. - Genes and Enzymes(NZY), Cat. No. MB086) DynabeadsTM mRNA DIRECTTM Kit (InvitrogenTM, Cat. No. 61011) Page 3/17 Agencourt AMPure XP beads (Beckman Coulter, Inc, Cat. No. A63880) Blunt/TA Ligase Master Mix (New England Biolabs (NEB), Cat. No. M0367) Direct cDNA Sequencing Kit (ONT, Cat. No. SQK-DCS109) dNTPs NZYMix (NZYTech, Lda. - Genes and Enzymes(NZY), Cat. No. MB086) DynabeadsTM mRNA DIRECTTM Kit (InvitrogenTM, Cat. No. 61011) Page 3/17 Agencourt AMPure XP beads (Beckman Coulter, Inc, Cat. No. A63880) Blunt/TA Ligase Master Mix (New England Biolabs (NEB), Cat. No. M0367) Direct cDNA Sequencing Kit (ONT, Cat. No. SQK-DCS109) dNTPs NZYMix (NZYTech, Lda. - Genes and Enzymes(NZY), Cat. No. MB086) DynabeadsTM mRNA DIRECTTM Kit (InvitrogenTM, Cat. No. 61011) Page 3/17 DynabeadsTM mRNA DIRECTTM Kit (InvitrogenTM, Cat. No. 61011) Flow Cell R9.4.1 (ONT, Cat. No. FLO-MIN106) Spatulas Spatulas Sterilized syringues Equipment DynaMagTM-2 magnet (InvitrogenTM, Cat. No. 12321D) Heating block Software MinKnow (ONT, version 21.10.8) Supplies QubitTM Assay Tubes (InvitrogenTM, Cat. No. Q32856) Page 4/17 Page 4/17 1. Sample preparation a. Weight 100 mg of fresh callus samples and flash-freeze in liquid nitrogen. a. Weight 100 mg of fresh callus samples and flash-freeze in liquid nitrogen b. Grind the frozen material in liquid nitrogen, using mortar and pestle, and transfer the fine powder to a microcentrifuge tube with a spatula. Maintain tubes in liquid nitrogen while all samples are processed. c. Add 1250 µL of lysis/binding buffer and vortex for 2 min. Don’t let the samples heat up, place tubes on ice. c. Add 1250 µL of lysis/binding buffer and vortex for 2 min. Don’t let the samples heat up, place tubes on ice. d. Force the lysate through a 21-gauge needle 3–5 times using a 1 mL syringe to shear the DNA. Spin down briefly. 2. Dynabeads preparation 2. Dynabeads preparation 2. Dynabeads preparation a. Bring lysis/binding buffer and washing buffers A and B to room temperature before use. Keep 10 mM Tris-HCl at 4 °C. b. Resuspend DynabeadsTM Oligo(dT)25 thoroughly. b. Resuspend DynabeadsTM Oligo(dT)25 thoroughly. c. Transfer 250 μL of beads to a 1.5 mL microcentrifuge tube and place the tube on the DynaMag™-2 magnet. d. When the suspension is clear discard the supernatant. d. When the suspension is clear discard the supernatant. e. Remove the tube from the magnet and wash the beads by resuspending in 250 µL of fresh lysis/binding buffer. Setup Autoclave twice all no nuclease-free materials. Autoclave twice all no nuclease-free materials. Autoclave twice all no nuclease-free materials. Clean mortars, pestles and spatulas with ethanol and RNase Cleaner before autoclaving. Work in RNase-free environment, use RNase Cleaner and wear gloves. Bring Agencourt AMPure XP beads to room temperature 30 min before use. Bring Agencourt AMPure XP beads to room temperature 30 min before use. Prepare fresh 70% (v/v) ethanol before each Agencourt AMPure XP beads purification step. Poly(A) RNA purification Prepare fresh 70% (v/v) ethanol before each Agencourt AMPure XP beads purification step. Poly(A) RNA purification Poly(A) RNA purification Page 5/17 Poly(A) RNA was purified directly from callus samples using the DynabeadsTM mRNA DIRECTTM Kit (InvitrogenTM) (Fig. 2). 1. Sample preparation 1. Sample preparation 1. Sample preparation 3. Poly(A) RNA isolation a. Place the washed beads on the magnet and discard the supernatant when the suspension is clear. b. Remove the tube from the magnet and add the sample lysate from step 1.d., pipetting to resuspend the beads completely. c. Incubate the mixture on a mixer for 15 min at room temperature. d. Place the tube on the magnet for 5 min. Rotate the tube slowly 90° to the right and then back to the starting position. Do the same for the left and hold for more 5 min. When the suspension is clear, discard the supernatant. d. Place the tube on the magnet for 5 min. Rotate the tube slowly 90° to the right and then back to the starting position. Do the same for the left and hold for more 5 min. When the suspension is clear, discard the supernatant. e. Remove the tube from the magnet and wash the RNA-beads complex twice with 1 mL of washing buffer A by pipetting until beads are thoroughly resuspended. Use the magnet to remove completely all traces between each washing step. Page 6/17 f. Wash the RNA-beads complex twice with 1 mL of washing buffer B by pipetting until beads are thoroughly resuspended. Use the magnet to remove completely all traces between each washing step. g. Elute the poly(A) RNA from the beads by adding 12 μL of 10 mM Tris–HCl (pH 7.5). Pipet until beads are thoroughly resuspended and keep the tube at 80 °C for 2 min. h. Immediately place the tube on the magnet and transfer the supernatant containing the poly(A) RNA to a new RNase-free tube. Place poly(A) RNA on ice. i. Analyse 1 μL of the poly(A) RNA using Qubit™ RNA High Sensitivity (HS) Assay Kit and 1 μL using a NanoDrop™ spectrophotometer. Use only samples with A260/A280 ratio between 1.8 and 2.1 and A260/A230 ratio higher than Use only samples with A260/A280 ratio between 1.8 and 2.1 and A260/A230 ratio higher than 1.8 for cDNA synthesis. 1.8 for cDNA synthesis. 1.8 for cDNA synthesis. Reverse transcription and strand-switching Barcoded libraries were prepared following the Direct cDNA Native Barcoding protocol from ONT with some modifications (Fig. 2). 1. Transfer 100 ng poly(A) RNA into a 0.2 mL PCR tube and adjust the volume to 7.5 μL with nuclease- free water. 2. Add 2.5 μL of VN primer and 1 μL of dNTPs (10mM). 3. Mix thoroughly by pipetting and spin down briefly. g y y p p g p y 4. Incubate at 65 °C for 5 min and then snap cool on a pre-chilled freezer block. Incubate at 65 °C for 5 min and then snap cool on a pre-chilled freezer bloc 5. Prepare a master mix with the following reagents: μL 5x RT Buffer μL NZYRibonuclease Inhibitor μL nuclease-free water μL Strand-Switching Primer 6. Mix thoroughly by pipetting and spin down briefly. μL Strand-Switching Primer 6. Mix thoroughly by pipetting and spin down briefly. μL Strand-Switching Primer 6. Mix thoroughly by pipetting and spin down briefly. 7. Add 8 μL of the master mix to the snap-cooled RNA. Mix by flicking the tube and spin down briefly. 8. Incubate at 50 ºC for 2 min. 9. Add 1 μL of Maxima H Minus Reverse Transcriptase. Mix gently by flicking the tube and spin down briefly. 9. Add 1 μL of Maxima H Minus Reverse Transcriptase. Mix gently by flicking the tube and spin down briefly. 10. Incubate at 50 ºC for 120 min and at 85 ºC for 5 min. Then hold at 4 ºC. RNA degradation 1. Add 1 μL RNase Cocktail™ Enzyme Mix to the reverse transcription reaction. 2. Incubate the reaction at 37 °C for 10 min. 3. Resuspend the beads by vortexing and transfer 36 μL to a new 1.5 mL microcentrif 1. Add 1 μL RNase Cocktail™ Enzyme Mix to the reverse transcription reaction. 3. Resuspend the beads by vortexing and transfer 36 μL to a new 1.5 mL microcentrifuge tube. 3. Resuspend the beads by vortexing and transfer 36 μL to a new 1.5 mL microcentrifuge tube. Page 7/17 Page 7/17 4. Add the reaction to the beads and mix thoroughly by pipetting. 4. Add the reaction to the beads and mix thoroughly by pipetting. 4. Add the reaction to the beads and mix thoroughly by pipetting. 5. Incubate on a mixer for 15 min at room temperature. 5. Incubate on a mixer for 15 min at room temperature. 6. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when the suspension is clear. 6. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when the suspension is clear. 7. Keep the tube on the magnet and wash the beads twice with 200 μL of freshly prepared 70% ethanol without disturbing the pellet. After 1 min remove the ethanol using a pipette and discard. 8. Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking. 8. Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking. 9. Remove the tube from the magnet and resuspend the pellet in 40 μL nuclease-free water. 10. Incubate on a mixer for 15 min at room temperature. 10. Incubate on a mixer for 15 min at room temperature. 11. Pellet beads on magnet until the eluate is clear and colourless. 11. Pellet beads on magnet until the eluate is clear and colourless. 12. Remove and retain 40 μL of eluate into a new 1.5 mL microcentrifuge tu 12. Remove and retain 40 μL of eluate into a new 1.5 mL microcentrifuge tube. 13. Let the tubes cold down on ice and take them open to a vacuum concentrator to reduce the sample volume up to 23 μL. 13. Let the tubes cold down on ice and take them open to a vacuum concentrator to reduce the sample volume up to 23 μL. Second strand synthesis 1. Transfer the samples to a 0.2 mL PCR tube and adjust the volume to 23 μL with nuclease-free water. 2. Add 25 μL of LongAmp® Taq 2X Master Mix and 2 μL of PR2 primer. 1. Transfer the samples to a 0.2 mL PCR tube and adjust the volume to 23 p j μ 2. Add 25 μL of LongAmp® Taq 2X Master Mix and 2 μL of PR2 primer. 2. Add 25 μL of LongAmp® Taq 2X Master Mix and 2 μL of PR2 primer. 3. Mix thoroughly by pipetting and spin down briefly. 3. Mix thoroughly by pipetting and spin down briefly. 4. Incubate at 94 ºC for 1 min, 50 ºC for 1 min and 65 ºC for 20 min. Then hold at 4 °C. 5 Resuspend the beads by vortexing and transfer 90 μL to a new 1 5 mL microcentrifuge tube at 94 ºC for 1 min, 50 ºC for 1 min and 65 ºC for 20 min. Then hold at 4 °C. 4. Incubate at 94 ºC for 1 min, 50 ºC for 1 min and 65 ºC for 20 min. Then h 5. Resuspend the beads by vortexing and transfer 90 μL to a new 1.5 mL microcentrifuge tube. 6. Repeat steps 4-12 from “RNA degradation”. 7. Analyse 1 μL of the strand-switched DNA using QubitTM dsDNA HS Assay Kit from InvitrogenTM. 8. If necessary, pool cDNA samples to reach 70-200 ng in a maximum volume of 50 μL. Use a vacuum concentrator to reduce the sample volume. End-prep 1. Transfer the samples to a 0.2 mL PCR tube and adjust the volume to 50 μL with nuclease-free water. 2 Add 7 μL of NEBNext Ultra II End Prep Reaction Buffer and 3 μL of NEBNext Ultra II End Prep Enzyme 1. Transfer the samples to a 0.2 mL PCR tube and adjust the volume to 50 μL with nuclease-free water. 1. Transfer the samples to a 0.2 mL PCR tube and adjust the volume to 50 μL with nuclease-free water. 2. Add 7 μL of NEBNext Ultra II End Prep Reaction Buffer and 3 μL of NEBNext Ultra II End Prep Enzyme Mix. 1. Transfer the samples to a 0.2 mL PCR tube and adjust the volume to 50 μL with nuclease-free water. 2. Add 7 μL of NEBNext Ultra II End Prep Reaction Buffer and 3 μL of NEBNext Ultra II End Prep Enzyme Mix. 2. Add 7 μL of NEBNext Ultra II End Prep Reaction Buffer and 3 μL of NEBNext Ultra II End Prep Enzyme Mix. 3. Mix thoroughly by pipetting and spin down briefly. 4. Incubate at 20 ºC for 30 min and 65 ºC for 30 min. Page 8/17 Page 8/17 5. Resuspend the beads by vortexing and transfer 108 μL to a new 1.5 mL microcentrifuge tube. 6. Repeat steps 4-12 from “RNA degradation”. 6. Repeat steps 4-12 from “RNA degradation”. 7. Let the tubes cold down on ice and take them open to a vacuum concentrator to reduce the sample volume up to 22.5 μL. Barcode ligation 1. Transfer Blunt/TA Ligase Master Mix to ice before reaction set up. Mix tube by finger flicking before use. 1. Transfer Blunt/TA Ligase Master Mix to ice before reaction set up. Mix tube by finger flicking before use. 2. Adjust the volume sample to 22.5 μL with nuclease-free water. 2. Adjust the volume sample to 22.5 μL with nuclease-free water. 3. Add 2.5 μL of a native barcode to each sample and 25 μL of Blunt/TA Li 3. Add 2.5 μL of a native barcode to each sample and 25 μL of Blunt/TA Ligase Master Mix. 4. Mix thoroughly by pipetting and spin down briefly. 5. Incubate at 25 °C for 15 min. 6. Resuspend the beads by vortexing and transfer 90 μL to the reaction. Mix thoroughly by pipetting. 7. Repeat steps 5-12 from “RNA degradation”. 6. Resuspend the beads by vortexing and transfer 90 μL to the reaction. Mi 7 R t t 5 12 f “RNA d d ti ” 6. Resuspend the beads by vortexing and transfer 90 μL to the reaction. Mix thoroughly by pipetting. 7. Repeat steps 5-12 from “RNA degradation”. 8. Analyse 1 μL of the eluted sample using QubitTM dsDNA HS Assay Kit from InvitrogenTM. 8. Analyse 1 μL of the eluted sample using QubitTM dsDNA HS Assay Kit 9. Estimate cDNA sequences’ average length by electropherogram analysis (e.g., Agilent’s Bioanalyzer) or from previously obtained transcriptomic data analysis. Adapter ligation Return the tube to the magnet, allow beads to pellet and pipette off the supernatant. 13. Remove the tube from the magnet and resuspend the pellet in 13 μL of Elution Buffer (EB). 14. Incubate on a mixer for 15 min at room temperature. 15. Pellet beads on magnet until the eluate is clear and colourless. 16. Remove and retain 13 μL of eluate into a clean 1.5 mL microcentrifuge tube. 17. Quantify 1 μL of the eluted sample using QubitTM dsDNA HS Assay Kit from InvitrogenTM. 17. Quantify 1 μL of the eluted sample using QubitTM dsDNA HS Assay Kit from InvitrogenTM. Quality assessment of cDNA libraries and ONT sequencing 1. Fragment size and purity of cDNA libraries can be assessed using an Agilent Bioanalyzer High Sensitivity DNA chip. 1. Fragment size and purity of cDNA libraries can be assessed using an Agilent Bioanalyzer High Sensitivity DNA chip. 2. High throughput ONT sequencing. 2. High throughput ONT sequencing. 2. High throughput ONT sequencing. Adapter ligation 1. Based on the predicted average length of sequences and not exceeding 200 fmol, calculate the maximum cDNA concentration to use. For example, if the average length of sequences is 1000 bp, use a maximum of 130 ng. 2. Calculate the volume to be used from each barcoded sample to do a pool with the same concentration for all samples. 3. Pool barcoded samples to get the maximum cDNA concentration in a 1.5 mL microcentrifuge tube. Adjust the volume to 65 μL by adding nuclease-free water or using a Vacuum Concentrator to reduce the sample volume. 4. Thaw Wash Buffer (WSB), Elution Buffer (EB) and NEBNext Quick Ligation Reaction Buffer (5x) at room temperature. Mix by vortexing, spin down and place on ice. Check if the contents of each tube are clear of any precipitate. Spin down the T4 Ligase and the Adapter Mix II (AMII) and place them on ice. 5. Add 5 μL of Adapter Mix II (AMII), 20 μL of NEBNext Quick Ligation Reaction Buffer (5X) and 10 μL of Quick T4 DNA Ligase to the pooled and barcoded DNA, mixing by flicking the tube between each sequential addition. 5. Add 5 μL of Adapter Mix II (AMII), 20 μL of NEBNext Quick Ligation Reaction Buffer (5X) and 10 μL of Quick T4 DNA Ligase to the pooled and barcoded DNA, mixing by flicking the tube between each sequential addition. 6. Spin down and incubate at 20 °C for 15 min. 6. Spin down and incubate at 20 °C for 15 min. Page 9/17 Page 9/17 7. Resuspend the beads by vortexing and transfer 50 μL to the reaction. 7. Resuspend the beads by vortexing and transfer 50 μL to the reaction. 8. Mix thoroughly by pipetting. 9. Incubate on a mixer for 15 min at room temperature. 9. Incubate on a mixer for 15 min at room temperature. 10. Pellet beads on magnet and pipette off the supernatant when the suspension is clear. 11. Wash beads-DNA complex twice with 140 μL of the Wash Buffer (WSB). Close the tube lid and resuspend the beads by flicking the tube. Return the tube to the magnet, allow beads to pellet and pipette off the supernatant. 11. Wash beads-DNA complex twice with 140 μL of the Wash Buffer (WSB). Close the tube lid and resuspend the beads by flicking the tube. Results And Discussion Aiming to perform ONT sequencing in difficult plant tissues, an optimized protocol for preparing barcoded cDNA libraries from callus samples with very distinct histological and morphogenic features was developed. The protocol combines high yield and quality poly(A) RNA isolation and barcoded cDNA library preparation. Poly(A) RNA isolation was performed from total RNA using the Dynabeads™ mRNA Purification Kit (Invitrogen™), at the beginning of this assay. Total RNA from tamarillo EC and NEC was extracted using NZYol (NZY, Cat. No. MB18501) and the Direct-zol™ RNA MicroPrep from Zymo© Research Corporation. As recommended by the manufacturer’s protocol, sample pools with 75 µg of total RNA were used as starting material. Nevertheless, even starting with 85–100 µg of total RNA and following the manufacturer’s protocol, it was only possible to obtain the required 13 ng/µL of poly(A) RNA for the Direct cDNA Native Barcoding protocol in less than 50% of the NEC samples used (Table 1). This approach thus proved ineffective to reach the required quantity of total RNA (75–100 µg), entailing significant losses of tissue samples and reagents due to the need to pool total RNA extractions from three replicates of ~ 100 mg fresh callus samples. Therefore, another kit for poly(A) RNA isolation was used, Dynabeads™ mRNA Page 10/17 Page 10/17 DIRECT™ Kit (Invitrogen™). With this kit, poly(A) RNA was isolated directly from callus samples, which enabled to get 100 ng of poly(A) RNA (Table 2) with much less tissue, extractions and time. DIRECT™ Kit (Invitrogen™). With this kit, poly(A) RNA was isolated directly from callus samples, which enabled to get 100 ng of poly(A) RNA (Table 2) with much less tissue, extractions and time. g g p y( ) ( ) , Table 1 Poly(A) RNA yield from embryogenic (EC) and non-embryogenic (NEC) callus using Dynabeads™ mRNA Purification Kit. EC NEC Total RNA (µg) Poly(A) RNA (ng/µL) Total RNA (µg) Poly(A) RNA (ng µL) 75 17.3 61 4.0 75 14.7 86 6.2 75 10.5 88 15.0 75 26.8 91 14.2 88 4.2 93 24.8 97 4.4 107 0 98 18.9 100 0 Table 2 Poly(A) RNA yield from embryogenic (EC) and non-embryogenic (NEC) callus using Dynabeads™ mRNA DIRECT™ Kit. EC NEC Poly(A) RNA (ng) Poly(A) RNA (ng) 40.4 11.6 58.0 14.1 72.2 21.8 94.4 27.0 l b h d f l fi For the cDNA libraries preparation, the Direct cDNA Native Barcoding from ONT protocol was first followed. Results And Discussion However, DNA losses were verified. In fact, several hard-to-remove contaminants inherent to plant tissues (e.g., phenolic compounds and polysaccharides) inhibit enzymes in library preparation processes for nucleic acid sequencing [10, 12, 20]. Therefore, optimizations were also needed. First, the incubation time of all reactions was prolonged. Also, the incubation temperature of the Maxima H Minus Reverse Transcriptase (Thermo Scientific™) was 50 ºC (and not 42 ºC, as suggested by ONT protocol), since this was the optimal reaction temperature described for this enzyme by the manufacturer. Finally, to guarantee that all DNA was recovered in the Agencourt AMPure XP beads purification steps, adequate beads and elution volume were used. As recommended by the manufacturer’s protocol, 1.8 µL AMPure XP were added per 1.0 µL of sample (almost twice the beads volume suggested by ONT protocol). Withal, Page 11/17 Page 11/17 after ethanol wash, beads were always eluted in 40 µL, since, as indicated by the manufacturer, recovery decreases with a decrease in elution volume. In several cases, to overcome the problem of too much volume a Vacuum Concentrator was used. With this protocol, we have prepared and sequenced 8 barcoded cDNA libraries from two tamarillo cell lines with different histological and embryogenic competencies. Libraries were pooled and sequenced using one flow cell. Direct cDNA Sequencing was carried out using MinION equipment. In a single run, 4.07 M reads with N50 ~ 1.2 kb were generated. Per base sequence quality of reads from each barcoded cDNA library was verified using the standard Phred-scaled quality score (Fig. 3). Reads showed an average quality score across all bases over 15 and all above 7 as recommended by Nanopore. This indicates an accuracy higher than 96.8% [21], which is considered as assigning confidence for long reads with little effect on downstream analysis [22, 23]. Therefore, barcoded libraries prepared using this protocol are suitable for further analysis. Conclusion An optimized and detailed protocol that combines poly(A) RNA isolation using Dynabeads™ mRNA DIRECT™ Kit (Invitrogen™) and cDNA library preparation using Direct cDNA Sequencing Kit (ONT, Cat. No. SQK-DCS109) with Native Barcoding Expansion 1–12 (ONT, Cat. No. EXP-NBD104) was developed. Barcoded cDNA libraries prepared following this method can be used for long-read sequencing methods. Moreover, this protocol can be applied to other plant species, overcoming the recalcitrance of some plant tissues to these molecular biology methods that remain a limitation of gene expression studies. Ethics approval and consent to participate Not applicable. Availability of data and materials Not applicable. Consent for publication Not applicable. Declarations Ethics approval and consent to participate Authors' contributions DC, SC and CS conceived the idea and designed the protocol described. DC and AP isolated total RNA. DC and SC performed mRNA purification. DC prepared cDNA. DC, AC and CS performed end-prep, barcode ligation, adaptor ligation and sequencing. SC, CS, JC managed resources and funding acquisition. DC wrote the manuscript and the other authors reviewed and edited the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Funding Page 12/17 Foundation for Science and Technology (Portugal) supported Daniela Cordeiro PhD fellowship (SFRH/BD/136925/2018). This work was carried out at the R&D Unit Center for Functional Ecology— Science for People and the Planet (CFE), with reference UIDB/04004/2020, financed by FCT/MCTES through national funds (PIDDAC), and at CATAA, this work was made in the scope of CULTIVAR project (CENTRO-01-0145-FEDER-000020), co-financed by the Regional Operational Programme Centro 2020, Portugal 2020, and the European Union, through the European Fund for Regional Development (ERDF), Collaborative Project. Acknowledgements The authors are grateful to the Centre for Neuroscience and Cell Biology (CNC) for providing the vacuum concentrator. The authors also acknowledge CD Genomics, for helping with the bioinformatic assessment of sequencing quality parameters. References Zeng Y, Yang T. RNA isolation from highly viscous samples rich in polyphenols and polysaccharides. Plant Mol Biol Report. 2012;20:417. 11. Zeng Y, Yang T. RNA isolation from highly viscous samples rich in polyphenols and polysaccharides. Plant Mol Biol Report. 2012;20:417. 12. Inglis PW, Marilia de Castro RP, Resende L V., Grattapaglia D. Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications. PLoS One. 2018;13:e0206085. 13. Ikeuchi M, Sugimoto K, Iwase A. Plant Callus: Mechanisms of induction and repression. Plant Cell. 2013;25:3159–73. 14. Endress V, Barriuso J, Ruperez P, Martin JP, Blazquez A, Villalobos N, et al. Differences in cell wall polysaccharide composition between embryogenic and non-embryogenic calli of Medicago arborea L. Plant Cell Tissue Organ Cult. 2009;97:323–9. 15. Livyatan I, Harikumar A, Nissim-Rafinia M, Duttagupta R, Gingeras TR, Meshorer E. Non- polyadenylated transcription in embryonic stem cells reveals novel non-coding RNA related to pluripotency and differentiation. Nucleic Acids Res. 2013;41:6300–15. 16. Chekanova JA. Long non-coding RNAs and their functions in plants. Curr Opin Plant Biol. 2015;27:207–16. 16. Chekanova JA. Long non-coding RNAs and their functions in plants. Curr Opin Plant Biol. 2015;27:207–16. 17. Fehér A. Callus, dedifferentiation, totipotency, somatic embryogenesis: What these terms mean in the era of molecular plant biology? Front Plant Sci. 2019;10:536. 17. Fehér A. Callus, dedifferentiation, totipotency, somatic embryogenesis: What these terms mean in the era of molecular plant biology? Front Plant Sci. 2019;10:536. 18. Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962;15:473–97. 18. Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962;15:473–97. 19. Correia S, Canhoto JM. Somatic embryogenesis of tamarillo (Solanum betaceum Cav.). In: Jain SM, Gupta PK, editors. Step Wise Protoc Somat Embryog Important Woody Plants. Springer, Cham; 2018. p. 171–9. 20. Nath O, Fletcher SJ, Hayward A, Shaw LM, Agarwal R, Furtado A, et al. A comprehensive high-quality DNA and RNA extraction protocol for a range of cultivars and tissue types of the woody crop avocado. Plants. 2022;11:242. 21. Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998; 8:175-85. 21. Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998; 8:175-85. 22. References 1. Jiao Y, Tausta SL, Gandotra N, Sun N, Liu T, Clay NK, et al. A transcriptome atlas of rice cell types uncovers cellular, functional and developmental hierarchies. Nat Genet. 2009;41:258–63. 1. Jiao Y, Tausta SL, Gandotra N, Sun N, Liu T, Clay NK, et al. A transcriptome atlas of rice cell types uncovers cellular, functional and developmental hierarchies. Nat Genet. 2009;41:258–63. 1. Jiao Y, Tausta SL, Gandotra N, Sun N, Liu T, Clay NK, et al. A transcriptome atlas of rice cell types uncovers cellular, functional and developmental hierarchies. Nat Genet. 2009;41:258–63. 2. Tian C, Wang Y, Yu H, He J, Wang J, Shi B, et al. A gene expression map of shoot domains reveals regulatory mechanisms. Nat Commun. 2019;10:141. 2. Tian C, Wang Y, Yu H, He J, Wang J, Shi B, et al. A gene expression map of shoot domains reveals regulatory mechanisms. Nat Commun. 2019;10:141. 2. Tian C, Wang Y, Yu H, He J, Wang J, Shi B, et al. A gene expression map of shoot domains reveals regulatory mechanisms. Nat Commun. 2019;10:141. 3. Zhao L, Zhang H, Kohnen M V., Prasad KVSK, Gu L, Reddy ASN. Analysis of transcriptome and epitranscriptome in plants using pacbio iso-seq and nanopore-based direct RNA sequencing. Front Genet. 2019;10:253. 4. Wang Y, Zhao Y, Bollas A, Wang Y, Au KF. Nanopore sequencing technology, bioinformatics and applications. Nat Biotechnol. 2021;39:1348–65. 5. Zhu YY, Machleder EM, Chenchik A, Li R, Siebert PD. Reverse transcriptase template switching: A SMARTTM approach for full-length cDNA library construction. Biotechniques. 2001;30:892–7. 6. Quantitative RNA-seq: PCR-cDNA, PCR-free Direct cDNA and Direct RNA sequencing. In: Oxford Nanopore Technologies. 2019. https://nanoporetech.com/resource-centre/quantitative-rna-seq-pcr- cdna-pcr-free-direct-cdna-and-direct-rna-sequencing#image1&. Accessed 24 Feb 2022. 7. MacRae E. Extraction of Plant RNA. Methods Mol Biol. 2007;15–24. 7. MacRae E. Extraction of Plant RNA. Methods Mol Biol. 2007;15–24. Page 13/17 Page 13/17 8. Smart M, Roden LC. A small-scale RNA isolation protocol useful for high-throughput extractions from recalcitrant plants. South African J Bot. 2010;76:375–9. 9. Vennapusa AR, Somayanda IM, Doherty CJ, Jagadish SVK. A universal method for high-quality RNA extraction from plant tissues rich in starch, proteins and fiber. Sci Rep. 2020;10:16887. 10. Rezadoost MH, Kordrostami M, Kumleh HH. An efficient protocol for isolation of inhibitor-free nucleic acids even from recalcitrant plants. 3 Biotech. 2016;6:61. 10. Rezadoost MH, Kordrostami M, Kumleh HH. An efficient protocol for isolation of inhibitor-free nucleic acids even from recalcitrant plants. 3 Biotech. 2016;6:61. 11. a - Tamarillo embryogenic (EC) and b - non-embryogenic callus (NEC). 23. Delahaye C, Nicolas J. Sequencing DNA with nanopores: troubles and biases. PLoS ONE. 2021; 16:e0257521. References Rivara-Espasandín M, Balestrazzi L, Álvarez GD, Ochoa I, Seroussi G, Smircich P, et al. Nanopore quality score resolution can be reduced with little effect on downstream analysis. Bioinf. Adv. 2022; 2:vbac054. Page 14/17 Figures Figures Figure 1 Figure 1 Figure 1 a - Tamarillo embryogenic (EC) and b - non-embryogenic callus (NEC) Page 15/17 Page 15/17 Figure 2 Schematic workflow for preparing barcoded cDNA libraries from plant callus for using DynabeadsTMmRNA DIRECTTM Kit (InvitrogenTM) and Direct cDNA Seq SQK-DCS109) with Native Barcoding Expansion 1-12 (ONT, Cat. No. EXP-NBD10 Figure 2 Schematic workflow for preparing barcoded cDNA libraries from plant callus for long-read sequencing, using DynabeadsTMmRNA DIRECTTM Kit (InvitrogenTM) and Direct cDNA Sequencing Kit (ONT, Cat. No. SQK-DCS109) with Native Barcoding Expansion 1-12 (ONT, Cat. No. EXP-NBD104). Figure 2 Schematic workflow for preparing barcoded cDNA libraries from plant callus for long-read sequencing, using DynabeadsTMmRNA DIRECTTM Kit (InvitrogenTM) and Direct cDNA Sequencing Kit (ONT, Cat. No. SQK-DCS109) with Native Barcoding Expansion 1-12 (ONT, Cat. No. EXP-NBD104). Page 16/17 Page 16/17 Figure 3 Mean quality scores of long reads for eight barcoded cDNA libraries. Libraries were prepared from t b i (EC) d f i bl b i (NEC) S b t ) ll i Figure 3 Mean quality scores of long reads for eight barcoded cDNA libraries. Libraries were prepared from compact embryogenic (EC) and friable non-embryogenic (NEC) S. betaceum) callus, using DynabeadsTMmRNA DIRECTTM Kit (InvitrogenTM) and Direct cDNA Sequencing Kit (ONT, Cat. No. SQK- DCS109) with Native Barcoding Expansion 1-12 (ONT, Cat. No. EXP-NBD104). Figure 3 Mean quality scores of long reads for eight barcoded cDNA libraries. Libraries were prepared from compact embryogenic (EC) and friable non-embryogenic (NEC) S. betaceum) callus, using DynabeadsTMmRNA DIRECTTM Kit (InvitrogenTM) and Direct cDNA Sequencing Kit (ONT, Cat. No. SQK- DCS109) with Native Barcoding Expansion 1-12 (ONT, Cat. No. EXP-NBD104). Page 17/17
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Retracted: Immunization Coverage: Role of Sociodemographic Variables
Advances in preventive medicine
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Hindawi Publishing Corporation Advances in Preventive Medicine Volume 2014, Article ID 890248, 1 page http://dx.doi.org/10.1155/2014/890248 Hindawi Publishing Corporation Advances in Preventive Medicine Volume 2014, Article ID 890248, 1 page http://dx.doi.org/10.1155/2014/890248 Hindawi Publishing Corporation Advances in Preventive Medicine Volume 2014, Article ID 890248, 1 page http://dx.doi.org/10.1155/2014/890248 Advances in Preventive Medicine Advances in Preventive Medicine Received 16 December 2014; Accepted 16 December 2014; Published 29 December 2014 Copyright © 2014 Advances in Preventive Medicine. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The paper titled “Immunization Coverage: Role of Sociode- mographic Variables” [1], published in Advances in Preven- tive Medicine, has been retracted upon the authors’ request due to a flaw in data acquisition. [1] B. Sharma, H. Mahajan, and G. D. Velhal, “Immunization cover- age: role of sociodemographic variables,” Advances in Preventive Medicine, vol. 2013, Article ID 607935, 5 pages, 2013. References [1] B. Sharma, H. Mahajan, and G. D. Velhal, “Immunization cover- age: role of sociodemographic variables,” Advances in Preventive Medicine, vol. 2013, Article ID 607935, 5 pages, 2013.
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Reward Activates Stimulus-Specific and Task-Dependent Representations in Visual Association Cortices
˜The œjournal of neuroscience/˜The œJournal of neuroscience
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To cite this version: Anne-Marike Schiffer, T Muller, Nick Yeung, Florian Waszak. Reward Activates Stimulus-Specific and Task-Dependent Representations in Visual Association Cortices. Journal of Neuroscience, 2014, 34 (47), pp.15610-15620. ￿10.1523/JNEUROSCI.1640-14.2014￿. ￿hal-02385067￿ Reward Activates Stimulus-Specific and Task-Dependent Representations in Visual Association Cortices Anne-Marike Schiffer, T Muller, Nick Yeung, Florian Waszak To cite this version: Anne-Marike Schiffer, T Muller, Nick Yeung, Florian Waszak. Reward Activates Stimulus-Specific and Task-Dependent Representations in Visual Association Cortices. Journal of Neuroscience, 2014, 34 (47), pp.15610-15620. ￿10.1523/JNEUROSCI.1640-14.2014￿. ￿hal-02385067￿ Anne-Marike Schiffer, T Muller, Nick Yeung, Florian Waszak. Reward Activates Stimulus-Specific and Task-Dependent Representations in Visual Association Cortices. Journal of Neuroscience, 2014, 34 (47), pp.15610-15620. ￿10.1523/JNEUROSCI.1640-14.2014￿. ￿hal-02385067￿ Reward Activates Stimulus-Specific and Task-Dependent Representations in Visual Association Cortices Anne-Marike Schiffer,1 Timothy Muller,1 Nick Yeung,1 and Florian Waszak2,3 1Department of Experimental Psychology, University of Oxford, Oxford OX1 3UD, United Kingdom, 2Universite´ Paris Descartes, Sorbonne Paris Cite´, 75270 Paris, France, and 3CNRS Laboratoire Psychologie de la Perception, Unite´ Mixte de Recherche 8242, 75006 Paris, France Anne-Marike Schiffer,1 Timothy Muller,1 Nick Yeung,1 and Florian Waszak2,3 1Department of Experimental Psychology, University of Oxford, Oxford OX1 3UD, United Kingdom, 2Universite´ Paris Descartes, Sorbonne Paris Cite´, 75270 Paris, France, and 3CNRS Laboratoire Psychologie de la Perception, Unite´ Mixte de Recherche 8242, 75006 Paris, France Humans reliably learn which actions lead to rewards. One prominent question is how credit is assigned to environmental stimuli that are acted upon. Recent functional magnetic resonance imaging (fMRI) studies have provided evidence that representations of rewarded stimuli are activated upon reward delivery, providing possible eligibility traces for credit assignment. Our study sought evidence of postreward activation in sensory cortices satisfying two conditions of instrumental learning: postreward activity should reflect the stimulus category that preceded reward (stimulus specificity), and should occur only if the stimulus was acted on to obtain reward (task dependency). Our experiment implemented two tasks in the fMRI scanner. The first was a perceptual decision-making task on degraded face and house stimuli. Stimulus specificity was evident as rewards activated the sensory cortices associated with face versus house perception more strongly after face versus house decisions, respectively, particularly in the fusiform face area. Stimulus specificity was further evident in a psychophysiological interaction analysis wherein face-sensitive areas correlated with nucleus accumbens activity afterface-decisionrewards,whereashouse-sensitiveareascorrelatedwithnucleusaccumbensactivityafterhouse-decisionrewards.The second task required participants to make an instructed response. The criterion of task dependency was fulfilled as rewards after face versus house responses activated the respective association cortices to a larger degree when faces and houses were relevant to the performed task. Our study is the first to show that postreward sensory cortex activity meets these two key criteria of credit assignment, and does so independently from bottom-up perceptual processing. Key words: credit assignment; fMRI; reward-related learning; stimulus-specific postreward activation Key words: credit assignment; fMRI; reward-related learning; stimulus-specific postreward activation ral signatures of potential eligibility traces, i.e., stimulus repre- sentations that have two crucial properties. To guarantee precision of ensuing reward predictions, activated representa- tions should be stimulus specific and task dependent. Stimulus specificity ensures that the precise environmental conditions that preceded reward will trigger its prediction. Received April 23, 2014; revised Aug. 12, 2014; accepted Aug. 17, 2014. Author contributions: A.-M.S., N.Y., and F.W. designed research; A.-M.S. and T.M. performed research; A.-M.S., T.M., N.Y., and F.W. analyzed data; A.-M.S., N.Y., and F.W. wrote the paper. ThisworkissupportedbyBiotechnologyandBiologicalSciencesResearchCouncilGrantBB/I019847/1,awarded to N.Y. and FW. The authors declare no competing financial interests. This article is freely available online through the J Neurosci Author Open Choice option. Correspondence should be addressed to Anne-Marike Schiffer at the above address. E-mail: anne-marike.schiffer@psy.ox.ac.uk. DOI:10.1523/JNEUROSCI.1640-14.2014 Copyright © 2014 Schiffer et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. Reward Activates Stimulus-Specific and Task-Dependent Representations in Visual Association Cortices Task dependency warrants that environmental conditions are only associated with reward if they were used to perform the rewarded action. HAL Id: hal-02385067 https://hal.science/hal-02385067v1 Submitted on 28 Nov 2019 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. 15610 • The Journal of Neuroscience, November 19, 2014 • 34(47):15610–15620 Introduction Humans learn how to act on stimuli to gain reward. Substantial effort has been devoted to understanding how reward delivery fosters associative learning (Rescorla and Wagner, 1972; Schultz, 2007). This research has revealed that reward-driven learning depends on midbrain dopamine neurons, which display a firing pattern resembling reward prediction error signals in models of reinforcement learning (Schultz et al., 1997; Waelti et al., 2001). However, whereas computational approaches provide solutions to the critical problem of credit assignment—determining which features are predictive of positive outcomes—little is known about how such eligibility traces (Sutton and Barto, 1990) are represented in the brain. In this study, we aimed to identify neu- Some recent studies have investigated related questions, fo- cusing on the hypothesis that learning should depend on activa- tion of stimulus representations at the time of reward delivery (Pleger et al., 2008, 2009; Weil et al., 2010; FitzGerald et al., 2012; Arsenault et al., 2013). However, whereas functional magnetic resonance imaging (fMRI) in animals has demonstrated stimulus-specific reward-related activity (Arsenault et al., 2013), corresponding evidence in human studies has not been consis- tently observed (Weil et al., 2010; FitzGerald et al., 2012). It there- fore remains unclear whether reward-based activity in human sensory cortex is stimulus specific. Received April 23, 2014; revised Aug. 12, 2014; accepted Aug. 17, 2014. Author contributions: A.-M.S., N.Y., and F.W. designed research; A.-M.S. and T.M. performed research; A.-M.S., T.M., N.Y., and F.W. analyzed data; A.-M.S., N.Y., and F.W. wrote the paper. ThisworkissupportedbyBiotechnologyandBiologicalSciencesResearchCouncilGrantBB/I019847/1,awarded to N.Y. and FW. The authors declare no competing financial interests. This article is freely available online through the J Neurosci Author Open Choice option. Correspondence should be addressed to Anne-Marike Schiffer at the above address. E-mail: anne-marike.schiffer@psy.ox.ac.uk. DOI:10.1523/JNEUROSCI.1640-14.2014 Copyright © 2014 Schiffer et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. To investigate this question, we conceived a novel paradigm in which subjects performed a perceptual discrimination task, de- ciding whether degraded stimuli contained images of faces or houses. The analysis focused on trials in which, unbeknownst to participants, the stimulus was pure noise. This renders activation by reward independent of initial bottom-up activation as well as of potential category-specific reward expectations, while mini- Correspondence should be addressed to Anne-Marike Schiffer at the above address. This article is freely available online through the J Neurosci Author Open Choice option. Received April 23, 2014; revised Aug. 12, 2014; accepted Aug. 17, 2014. DOI:10.1523/JNEUROSCI.1640-14.2014 J. Neurosci., November 19, 2014 • 34(47):15610–15620 • 15611 J. Neurosci., November 19, 2014 • 34(47):15610–15620 • 15611 J. Neurosci., November 19, 2014 • 34(47):15610–15620 • 15611 Schiffer et al. • Stimulus-Specific Task-Dependent Reward Activation mizing the possible effects of neural adaptation (Grill-Spector et al., 2006; FitzGerald et al., 2012). mizing the possible effects of neural adaptation (Grill-Spector et al., 2006; FitzGerald et al., 2012). Rewards consisted of images showing either one or two moneybags, indicating gains of 10 or 20 points, respectively. Penalties were shown as one or two bombs, indicating loss of either 10 or 20 points. Participants were told that rewards and penalties were contingent on the correctness of their previous response, but were in fact randomly assigned in noise trials. Participants received feedback on their accumulated score every 50 trials. Their final score was converted into a monetary bonus of £5 after the scan, which was added to their usual remuneration of £20. The ex- perimental sequence in the scanner consisted of 275 trials, 138 of which were noise trials. Thirty-five noise trials and 33 signal trials were instruc- tion trials; 24–26 noise question trials were followed by a neutral out- come, the remaining noise trials were followed in equal numbers by large rewards, small rewards, small penalties, and large penalties. Outcomes in the signal trials were contingent on performance, but outcome size was randomly determined. Just before the scanning session, participants per- formed 16 practice trials of the experimental paradigm. We sought evidence of reward-dependent, stimulus-specific cortical activity; that is, activity in our regions of interest (ROIs), the fusiform face area (FFA) and the parahippocampal place area (PPA), at the time of reward delivery. This was our first criterion for a neural signature that could serve as an eligibility trace. Our second criterion was task dependency. Postreward activation should be stronger for stimuli that were used to gain reward. We therefore compared postreward activation in the perceptual de- cision task with activation in a second, instructed response task, hypothesizing that stimulus activity for reward outcomes would be restricted to trials in which outcomes were experienced as a consequence of a perceptual decision. In summary, we predicted that activity in the ROIs would show a positive correlation with reward size for associated deci- sions (stimulus specificity), and would be more influenced by reward following a perceptual decision than following an in- structed response (task dependency). J. Neurosci., November 19, 2014 • 34(47):15610–15620 • 15611 The experimental task was followed by a functional localizer task to determine the ROIs for all planned contrasts. Participants performed a one-back task while they were presented with two sequences of six blocks of 18 images that appeared on the screen for 150 ms each, followed by an interstimulus interval of 400 ms. Participants had a short break after the first six blocks. They had to switch from making responses with one hand to the other after the break. Each block contained images of only one category; these categories were as follows: unscrambled face images; un- scrambled house images; easy, medium, and hard face images; easy, me- dium, and hard house images; pure noise images; and unscrambled object images. Stimulus material l h Grayscale photographs of faces and front views of houses (Fig. 1) served as stimulus material. In a first step, all images were adjusted in luminance and spatial frequency to the mean of the stimulus pool using the SHINE (Spectrum, Histology and Intensity Normalization and Equalization) Matlab tool (Willenbockel et al., 2010). This measure was taken to pre- vent categorization based on surface similarities (Schyns and Oliva, 1994; Rajimehr et al., 2011). Images were Fourier transformed and a variable percentage of all phases in each Fourier transformed image was scram- bled. Images were then back-transformed into native space. Three de- grees of phase scrambling were applied per category to yield stimuli producing easy, medium, or hard levels of difficulty in perceptual dis- crimination. Face images were phase scrambled to 70, 75, and 85%. House images were phase scrambled to 50, 65, or 75%. These degrees of scrambling were chosen based on pilot testing to produce comparable performance for house and face stimuli across the three levels of degra- dation. In addition to the three difficulty levels, half of the images were pure noise images with 100% of all phases scrambled. Materials and Methods Eighteen right-handed, healthy participants (10 women; ages 20–32 years mean age 24 years) took part in the study. The participants reported no psychiatric or neurological past or present condition. All procedures were approved by the local ethics committee of the University of Oxford and all participants gave written informed consent. Behavioral analysis h h k Behavioral analysis Behavior in the task was recorded to establish that participants showed the expected performance modulation by the degree of phase scrambling of the signal stimuli, as well as to assess changes in performance over time and win–stay/lose–shift behavior as markers of learning. We also tested whether participants made both face and house judgments in trials with pure noise stimuli. fMRI procedure Th f ti l i f p The functional imaging session took place in a 3T Siemens Magnetom Trio scanner (Siemens). During the scan, participants lay supine on the scanner bed with their left and right index fingers resting on two buttons of a centrally placed response box. Participants wore sound-attenuating headphones that allowed communication with the experimenter. They viewed the stimuli on the screen via a mirror built into the head coil. Stimuli were displayed at 5° of visual angle to prevent head and eye movements. The functional session engaged a single-shot gradient echo- planar imaging (EPI) sequence sensitive to blood oxygen level- dependent (BOLD) contrast (32 slices, 192 mm field of view, 4 mm slice thickness, 3  3  4 in-plane resolution, orientation parallel to the bicommisural plane, 30 ms TE, 90° flip angle, 2000 ms TR, interleaved, descending recording). After the functional session was completed, high- resolution 3D T1-weighted whole-brain modified driven equilibrium Fourier transformation (MDEFT) sequences were recorded for every participant (128 slices, 256 mm field of view, 256  256 pixel matrix, 1 mm slice thickness, 0.25 mm spacing). Introduction E-mail: anne-marike.schiffer@psy.ox.ac.uk. y This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. Task When the questio peared,participantsindicatedtheirdecisionwithaleftorrightbuttonpress.Theythenreceivedpositive(asshown),negative,orneutralfeedback.Thesecondtaskmadeup25%ofalltria e initial stimulus was followed by an exclamation mark on top of two boxes, one of which was darkened; participants had to press the button corresponding to the side of the darken edbackwasagaindeliveredinthesameformatasfortheperceptualdecisiontaskandcontingentonresponseaccuracy.B,Luminance-adjustedgrayscaleimagesoffacesandhouseswere nsformed and phase scrambled for use as stimuli. Within the experiment, three levels of degradation and noise trials were included, yielding graded performance levels (the displayed l ase scrambling for signal stimuli are for illustrative purposes only and deviate from the actual levels in the main experiment; see Materials and Methods). Noise stimuli were 100% ambled.Participantswereunawareoftheexistenceofnoisestimuli.C,Participantsexperiencedfivelevelsofoutcomevalence,twolevelsofreward,twolevelsofpenalty,andaneutralou rgeandsmallrewardsorpenaltiesresultedinthegainorlossof20or10points,respectively.Outcomevalence(rewardorpenalty)wasperformancecontingentonsignaltrialsandintheins sponse task, but randomly assigned for perceptual decisions in noise trials. A quarter of all trials were followed by neutral outcomes instead independent of performance. + x ! x ! x ? x ? Decision Response Outcometypes Stimuli Stimulus Outcome Instructed Response C B 2s 1.5 s 2 - 6 s 2s 1.5 s 2 - 6 s 1 s 2 - 6 s phase scrambling easy medium hard noise Outcometypes Reward Penalty Neutral Stimuli Response C B phase scrambling easy medium hard noise x x x x x x A, The main task was a perceptual decision task in which participants were first presented with a stimulus that they had to classify as either face or house. When the ques participantsindicatedtheirdecisionwithaleftorrightbuttonpress.Theythenreceivedpositive(asshown),negative,orneutralfeedback.Thesecondtaskmadeup25%ofalltr stimulus was followed by an exclamation mark on top of two boxes, one of which was darkened; participants had to press the button corresponding to the side of the dark wasagaindeliveredinthesameformatasfortheperceptualdecisiontaskandcontingentonresponseaccuracy.B,Luminance-adjustedgrayscaleimagesoffacesandhouseswe ed and phase scrambled for use as stimuli. Within the experiment, three levels of degradation and noise trials were included, yielding graded performance levels (the displaye mbling for signal stimuli are for illustrative purposes only and deviate from the actual levels in the main experiment; see Materials and Methods). Noise stimuli were 100 P ti i t fth i t f i ti li C P ti i t i dfi l l f t l t l l f d t l l f lt d t l Stimuli Response B phase scrambling easy medium hard noise Figure 1. A, The main task was a perceptual decision task in which participants were first presented with a stimulus that they had to classify as either face or house. Task In each trial, participants were first presented with a stimulus image for 2 s. Stimulus presentation was followed by a task cue, displayed on the screen for 1.5 s; the task cue was either a question mark or an exclamation mark. Question marks instructed participants to press the left or right button to indicate whether they had seen a face or a house (perceptual decision task). Participants were unaware that half of the images were noise images and instructed to always decide and respond. Images of exclamation marks contained a darkened box on the left or right side underneath the exclamation mark (Fig. 1A). In these trials, participants had to press the button on the side corresponding to the darkened box (instructed response task). Importantly, since participants did not know the trial type at the time of stimulus presentation, they had to make a perceptual decision in all trials. Participants had 1.5 s to respond, after which the task image stayed on the screen for the remaining response– stimulus interval (RSI). A cross in a box to the left or right of the task image was displayed during this interval, indicating their previous re- sponse. The length of this RSI was randomly drawn from a Poisson distribution with lambda 4 s, minimum 2 s, maximum 6 s, and jittered in steps of 500 ms. This interval was followed by feedback, which could be rewarding, neutral, or penalizing. fMRI data analysis fMRI data analysis was conducted with the LIPSIA (Leipzig Image Pro- cessing and Statistical Inference Algorithms) processing tool (Lohmann et al., 2001). For spatial registration, EPI data and 3D MDEFT data were first oriented along the ac–pc axis. The matching parameters (six degrees of freedom, three rotational, three translational) of the functional data onto the individual 3D MDEFT reference set were used to calculate the transformation matrices for linear registration. These matrices were sub- sequently normalized to Talairach brain size (x  135 mm, y  175 mm, z  120 mm; Talairach and Tournoux, 1988) by linear scaling. The normalized transformation matrices were then applied to the functional slices, to transform them using trilinear interpolation and align them with the 3D reference set in the stereotactic coordinate system. The gen- erated output had a spatial resolution of 3  3  3 mm. Cubic-spline interpolation was used to correct for the temporal offset between the 15612 • J. Task Neurosci., November 19, 2014 • 34(47):15610–15620 Schiffer et al. • Stimulus-Specific Task-Dependent Reward Activation cquired in one scan. To remove low-frequency signal changes and dom (Worsley and Friston, 1995). A spatial Gaussian filter of 5. + x ! x ! + x ? x ? Decision Response Outcometypes Reward Penalty Neutral Stimuli Tasks Stimulus Outcome Instructed Response A C B 2s 1.5 s 2 - 6 s 1 s 2 - 6 s 2s 1.5 s 2 - 6 s 1 s 2 - 6 s phase scrambling easy medium hard noise x x x x x x . A, The main task was a perceptual decision task in which participants were first presented with a stimulus that they had to classify as either face or house. When the ques d,participantsindicatedtheirdecisionwithaleftorrightbuttonpress.Theythenreceivedpositive(asshown),negative,orneutralfeedback.Thesecondtaskmadeup25%ofalltr al stimulus was followed by an exclamation mark on top of two boxes, one of which was darkened; participants had to press the button corresponding to the side of the dark kwasagaindeliveredinthesameformatasfortheperceptualdecisiontaskandcontingentonresponseaccuracy.B,Luminance-adjustedgrayscaleimagesoffacesandhouseswe med and phase scrambled for use as stimuli. Within the experiment, three levels of degradation and noise trials were included, yielding graded performance levels (the displaye rambling for signal stimuli are for illustrative purposes only and deviate from the actual levels in the main experiment; see Materials and Methods). Noise stimuli were 10 ed.Participantswereunawareoftheexistenceofnoisestimuli.C,Participantsexperiencedfivelevelsofoutcomevalence,twolevelsofreward,twolevelsofpenalty,andaneutral dsmallrewardsorpenaltiesresultedinthegainorlossof20or10points,respectively.Outcomevalence(rewardorpenalty)wasperformancecontingentonsignaltrialsandinthe task, but randomly assigned for perceptual decisions in noise trials. A quarter of all trials were followed by neutral outcomes instead independent of performance. J. Neurosci., November 19, 2014 34(47):15610 15620 Schiffer et al. Stimulus Specific Task Dependent Reward + x ! x ! + x ? x ? Decision Response Tasks Stimulus Outcome Instructed Response A 2s 1.5 s 2 - 6 s 1 s 2 - 6 s 2s 1.5 s 2 - 6 s 1 s 2 - 6 s 2 - 6 s + x ! x ! + x ? x ? Decision Response Outcometypes Reward Penalty Neutral Stimuli Stimulus Outcome Instructed Response C B 2s 1.5 s 2 - 6 s 1 s 2s 1.5 s 2 - 6 s 1 s 2 - 6 s phase scrambling easy medium hard noise x x x x x x gure 1. A, The main task was a perceptual decision task in which participants were first presented with a stimulus that they had to classify as either face or house. Task These four PPI analyses (two hemispheres  two contrasts) were then combined in two conjunc- tion analyses. The first conjunction combined the left nucleus accumbens-seed PPI in house reward after noise trials versus face reward after noise trials with the corresponding right nucleus accumbens-seed PPI (house-vs-face PPI, hereafter). The second conjunction analogously combined the face reward after noise trials versus house reward after noise trials PPIs from both hemispheres (face-vs-house PPI, hereafter). LIPSIA controls for the inflated -error in conjunction analyses (Lohm- ann et al., 2001). The second step was to determine areas within the vicinity of the PPA ROI that were significantly activated in the house-vs- face PPI analysis and determining areas within the vicinity of the FFA ROI that were significantly activated in the face-vs-house PPI analysis. Selection of these ROIs was performed on not cluster-corrected data, as the search radius was limited based on previous ROIs and anatomy. Last, the areas identified in step two were used as ROIs (masks of a 2  2  2 cube in each hemisphere) in the localizer contrast of face blocks versus house blocks and house blocks versus face blocks (GLM1). This ensured that these ROIs not only correlated with nucleus accumbens activity after rewards corresponding to the perceptual decision in favor of one specific type of stimulus, but were also globally more sensitive to one stimulus category than the other. For example, areas that correlated with the nu- cleus accumbens more after face decisions on noise trials than house decisions on noise trials were required also to be more active during blocks of face images than blocks of house images in the functional localizer, thus supporting the argument that reward-related activity is stimulus specific. Decision-specific activation at noise stimulus presentation. To establish whether noise stimuli were treated as if they contained signal, a first contrast tested whether the ROIs would show significant activation in line with the perceptual decision on noise trials. Two regressors were entered into one GLM (GLM3), one accounting for the presentation of noise stimuli that were followed by a house decision and the correspond- ing regressor for noise stimuli that were followed by a face decision. Events were time-locked to noise stimulus presentation, modeled with an event length of 1 s and event amplitude of 1. Task The combi- nation of cluster size and cluster value decreases the risk of neglecting true activations in small structures. Thus, all reported activations were significant at p  0.05, corrected for multiple comparisons at the cluster level. Events were modeled with a 1 s duration, time-locked to reward presen- tation. The main parametric contrasts to estimate stimulus specificity were FaceDecisionReward and HouseDecisionReward. The estimated  values for postreward activity scaling with reward size in the FFA and PPA for face versus house decisions respectively were entered into a 2  2 repeated measures ANOVA to test for stimulus-specific postreward activation. Because these analyses were calculated on noise trials, with pseudorandomized response–reward contingencies, there was no signif- icant correlation between reward size and stimulus-specific regressors related to the earlier perceptual decision (r  0.034). Events were modeled with a 1 s duration, time-locked to reward presen- tation. The main parametric contrasts to estimate stimulus specificity were FaceDecisionReward and HouseDecisionReward. The estimated  values for postreward activity scaling with reward size in the FFA and PPA for face versus house decisions respectively were entered into a 2  2 repeated measures ANOVA to test for stimulus-specific postreward activation. Because these analyses were calculated on noise trials, with pseudorandomized response–reward contingencies, there was no signif- icant correlation between reward size and stimulus-specific regressors related to the earlier perceptual decision (r  0.034). level. ROI definition. Functional ROIs were determined in a two-step ap- proach. As a first step, all blocks from the functional localizer that con- tained house images, all blocks that contained face images, and the object image blocks were entered separately as regressors into a GLM (GLM1). Events were modeled with a box-car function and event length set to block length. House blocks were contrasted with face blocks (Houselo- calizerBlock  FacelocalizerBlock) and vice versa (FacelocalizerBlock  HouselocalizerBlock) on the single-subject level and averaged into t map contrast images. In a separate analysis, the face and house signal trials from the main experiment were entered separately into another GLM (GLM2). A parametric increase for event amplitudes was determined by signal strength, with amplitude increasing from 1 to 3 for hard to easy trials. The regressor accounting for house trials was then contrasted with the face-trial regressor (HouseSignalStrength  FaceSignalStrength) and vice versa (FaceSignalStrength  HouseSignalStrength). Task The resulting contrast images were masked with the contrast images generated based on the functional localizer task. The masked images then entered second- level random-effects analysis. One-sample t tests were used for the group analyses across the contrast images of all subjects to test whether ob- served differences between conditions were significantly different from zero. The bilateral peak voxels of activity in the parahippocampal gyri were used as centers for the PPA ROI. ROIs were established as 2  2  2 voxel cubes centered on the bilateral peak coordinates. Peak voxels for the FFA ROI were generated in a parallel approach, locating peak voxels in the fusiform gyrus; the bilateral ROI was set as a cube of 2  2  2 voxels around these centers. Task-dependent activation at reward outcome. The second parametric contrast calculated from this GLM (GLM5) tested for the assumption that reactivation should be task dependent, i.e., depend on a perceptual decision, as opposed to an instructed response. To test this hypothesis, we performed a repeated-measures ANOVA on the  values from the four regressors of interest: FaceDecisionReward, FaceInstructionRe- ward, HouseDecisionReward, and HouseInstructionReward. Further, we directly contrasted FaceDecisionReward  FaceInstructionReward in the FFA ROI. Events were modeled time-locked to reward presentation with an event duration of 1 s. Reward size for instructed responses was modeled parametrically with the same amplitude vector as in the stimulus-specificity analysis. p y y Reward-driven activation of stimulus-specific areas: psychophysiological interaction analysis. To investigate whether proposed effects of stimulus- specific BOLD increase with reward size were linked to activity in regions associated with reward processing, we conducted a psychophysiological interaction analysis (PPI) to complement the interaction analysis out- lined above. The PPI procedure aimed to establish whether activity in areas coding for reward magnitude would correlate with activity in areas showing stimulus-specific activation. This analysis was implemented in three steps. First, we established the peak voxel of activity in the left and right nucleus accumbens in the contrast measuring the reward-network response (above). We then conducted two PPI analyses for each hemi- sphere. One of these correlated the seed voxel with whole-brain activa- tion in a contrast of house reward after noise trials versus face reward after noise trials, while the second correlated the same seed voxel with the reverse contrast. PPI analyses used an unconvolved regressor and mod- eled event lengths of 3 TRs (O’Reilly et al., 2012). Task When the question mark appeared,participantsindicatedtheirdecisionwithaleftorrightbuttonpress.Theythenreceivedpositive(asshown),negative,orneutralfeedback.Thesecondtaskmadeup25%ofalltrials.Here, the initial stimulus was followed by an exclamation mark on top of two boxes, one of which was darkened; participants had to press the button corresponding to the side of the darkened box. Feedbackwasagaindeliveredinthesameformatasfortheperceptualdecisiontaskandcontingentonresponseaccuracy.B,Luminance-adjustedgrayscaleimagesoffacesandhouseswereFourier transformed and phase scrambled for use as stimuli. Within the experiment, three levels of degradation and noise trials were included, yielding graded performance levels (the displayed levels of phase scrambling for signal stimuli are for illustrative purposes only and deviate from the actual levels in the main experiment; see Materials and Methods). Noise stimuli were 100% phase scrambled.Participantswereunawareoftheexistenceofnoisestimuli.C,Participantsexperiencedfivelevelsofoutcomevalence,twolevelsofreward,twolevelsofpenalty,andaneutraloutcome. Largeandsmallrewardsorpenaltiesresultedinthegainorlossof20or10points,respectively.Outcomevalence(rewardorpenalty)wasperformancecontingentonsignaltrialsandintheinstructed response task, but randomly assigned for perceptual decisions in noise trials. A quarter of all trials were followed by neutral outcomes instead independent of performance. dom (Worsley and Friston, 1995). A spatial Gaussian filter of 5.65 mm FWHM was applied. Unless otherwise stated, the design matrix was gen- erated by hemodynamic modeling using a -function in all contrasts. The onset vectors were modeled with a duration of 1 s in a time-locked event-related fashion unless otherwise stated. No derivatives were in- cluded in the models. slices acquired in one scan. To remove low-frequency signal changes and baseline drifts, a high-pass filter of 1/75 Hz was applied for event-related analysis and a high-pass filter of 1/125 Hz was applied to the analysis of the localizer blocks. Filter lengths were chosen based on the optimal filter length for a specific design file as suggested by LIPSIA. Statistical evalu- ation was based on a least-square estimation using the general linear model (GLM) for serially autocorrelated observations (Worsley and Fris- ton, 1995). Temporal Gaussian smoothing (4 s FWHM) was applied to deal with temporal autocorrelation and determine the degrees of free- For group analyses, t tests and repeated-measures ANOVAs were per- formed on  values from the contrast of regressors in the respective GLMs. Acquired t values were transformed to z scores. For whole-brain J. Neurosci., November 19, 2014 • 34(47):15610–15620 • 15613 Schiffer et al. • Stimulus-Specific Task-Dependent Reward Activation analyses, an initial z threshold of 2.56 (p  0.01, one tailed) was then applied to the activation map. All voxels showing a positive activation above this threshold entered the second step of the correction. Here, a Monte Carlo simulation was used to define thresholds for cluster size and cluster value at a significance level of p  0.05 (one tailed). Behavioral analysis Analysis of participants’ performance in- dicated that they engaged with the task, and confirmed that the paradigm effec- tively created three different levels of dif- ficulty, with performance in the hardest level of difficulty being close to the fixed chance performance in noise trials. Partic- ipants made on average 75.8% correct re- sponses (SD, 12.9%) on signal trials. One dataset was excluded from the analysis be- cause performance was 2 SDs from the mean. The remaining 17 participants achieved on average 88.9, 79.9, and 63.3% correct on easy, medium, and hard face signal trials, respectively, and 92.1, 79.4, and 58.4% correct on the cor- responding house signal trials. In instructed response trials, par- ticipants reached on average 89.27% correct responses (SD, 9.67%). According to postexperiment reports, errors on these instructed trials typically occurred when participants failed to suppress a prepared response to the stimulus. To test that signal trials created a plausible context for noise trials, but were at the same time not clearly distinguishable from noise trials, we as- sessed how many levels of degradation participants thought they had encountered. Of 17 participants, 9 indicated that the ex- periment implemented three levels of difficulties, while 4 par- ticipants believed that there had been “3–4” levels of difficulty. Only 2 participants correctly estimated that there had been four levels of difficulty, while the remaining partic- ipants indicated five, and 50 levels of degradation, respec- tively. It thus appears as if for most subjects, noise trials were not clearly distinguishable from signal trials. Incidentally, only one participant reported noticing that a few trials did not contain any signal, but nevertheless did not realize that fully half of all trials were noise trials. 1st half Easy 00 Figure2. A,Feedbackinteg bars)oftheexperimenttothes evidentinwin–stay/lose–shif when the response was rewar Figure2. A,Feedbackintegrationforsignalstimuliwasapparentintheperformanceimprovementfromthefirsthalf(darkgray bars)oftheexperimenttothesecondhalf(lightgraybars)onperceptualdecisiontrials.B,Feedbackintegrationfornoisetrialswas evidentinwin–stay/lose–shiftbehavior.Participantsweresignificantlymorelikelytostaywitharesponsethantoshiftresponse when the response was rewarded and more likely to shift than to stay when a response was penalized. away from a penalized one. Such a win–stay/lose–shift behavior would indicate pseudolearning from positive feedback. A one-sample t test revealed significant difference in stay probabil- ities for rewarded compared with penalized noise trials (t(16)  2.99, p  0.004), with participants being more likely to repeat a rewarded response. While this trial-by-trial analysis was primarily aimed at uncovering correlates of feedback integration in noise trials, which do not allow assessment of true learning, we also performed the same analysis on signal trials. Task for house responses in noise decision trials, face responses in noise decision trials, in- structed “house” responses in noise trials, and instructed “face” responses in noise trials. To allow comparison, the GLM further included the corresponding regressors for signal trials. Mean s from all parametric contrasts were entered into a 2  2  2  2 repeated- measures ANOVA for further analysis. Task We estimated the main effect of each regressor separately and contrasted face noise trials with house noise trials (FaceDecision  HouseDecision). The mean  scores ex- tracted from the FFA and PPA ROIs entered a repeated-measures ANOVA to estimate main effects and interactions of decision (face or house) and ROI (FFA or PPA). Reward network response. The second contrast aimed to show that reward after noise trials would result in the network response associated with learning from rewards. We parametrically modeled BOLD increase from neutral to large reward trials after both face and house decisions to noise stimuli. Events were time-locked to reward presentation and mod- eled with an event length of 1 s and an event amplitude ranging from 1 (neutral) to 3 (large reward). Stimulus-specific activation at reward outcome. The most critical fea- tures for postreward activation to be qualified as a signature of reward- driven learning were stimulus specificity and task dependency. Analyses for both effects were conducted in the same GLM (GLM5), which con- tained the following eight regressors. The four main regressors of interest implemented a parametric modulation of BOLD by reward size: (1) after house responses in noise decision trials, (2) after face responses in noise decision trials, (3) after instructed “house” responses, and (4) after in- structed “face” responses. Large rewards were modeled with an ampli- tude of 2 and small rewards were modeled with an amplitude of 1. Further, this GLM contained separate regressors of no interest for pen- alty outcomes modeled by size after face decisions and house decisions and separate regressors for neutral outcomes after each type of decision. Trial-type sensitivity. The last contrast tested whether noise stimuli would deliver a more sensitive context for post-reward activation effects than signal stimuli. We therefore defined a final GLM (GLM6), which included the following four main regressors to assess stimulus specificity and task dependency. These modeled reward magnitude parametrically Schiffer et al. • Stimulus-Specific Task-Dependent Reward Activation 15614 • J. Neurosci., November 19, 2014 • 34(47):15610–15620 Learning in signal trials A win stay lose shift 1st half 2nd half Easy Medium Hard Proportion correct Probability 0.6 0.5 0.4 Win stay - lose shift B 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 0.8 Figure2. A,Feedbackintegrationforsignalstimuliwasapparentintheperformanceimprovementfromthefirsthalf(darkgray bars)oftheexperimenttothesecondhalf(lightgraybars)onperceptualdecisiontrials.B,Feedbackintegrationfornoisetrialswas evidentinwin–stay/lose–shiftbehavior.Participantsweresignificantlymorelikelytostaywitharesponsethantoshiftresponse when the response was rewarded and more likely to shift than to stay when a response was penalized. Behavioral analysis We expected the effect in signal trials to be weaker, because use of feedback to guide behavior seems more plausible if the rewarded or penalized trial is visually similar to the current trial. Seven of 17 subjects made no errors on easy, or easy and medium trials, preventing us from comparing effects of noise trials separately for each level of degradation. We therefore averaged win–stay probability, win– shift probability, lose–stay probability, and lose–shift probability across all levels of degradation, including noise trials. To test whether win–stay/lose–shift behavior was influenced by signal strength, we calculated a repeated-measures ANOVA with the two-level factor DEGRADATION on trial N  1 (signal/noise), VALENCE on trial N  1 (reward/penalty), and BEHAVIOR on trial N (stay/shift). This analysis revealed no reliable main effects, a marginally significant interaction VALENCE on N  1  BEHAVIOR (F(1,16)  4.251, p  0.056) and no three-way interac- tion. Thus, although win–tay/lose–shift behavior was numerically smaller after signal trials than noise trials, the difference was not significant. In a next step, we assessed whether participants made use of feedback to adapt their behavior. We therefore assessed par- ticipants’ performance changes over the course of the experi- ment. As expected, performance improved, as revealed in a 2  3 repeated-measures ANOVA with the factors TIME (level: first half of experiment/second half of experiment) and DEGRADATION (level: easy/medium/hard). This analysis re- vealed a marginally significant main effect of time (F(1,15)  4.41, p  0.051), a significant main effect of degradation (F(1,16)  42.85, p  0.000), and a significant interaction between the two main factors (F(1,16)  4.23, p  0.032). Descriptively, partici- pants’ performance improved particularly on hard trials (Fig. 2A). It thus seemed that participants learned from feedback inte- grating this information to modify their behavior. As a final assessment of the credibility of the manipulation, and also to establish the comparability of BOLD effects across face versus house decisions, we compared the distribution of per- ceptual judgments on noise trials. Participants showed balanced judgments, with no strong preferences on the group level. Face decisions were on average made on 49% of all trials (SD, 7.8 standardized percentage; range, 31–61%). Stimulus-specific activation at reward outcome Thus, visually identical reward images (pictures of money bags) activated stim- ulus representations differentially in a decision-contingent man- ner, an effect mostly carried by an increase in FFA activity following reward stimuli to face decisions compared with reward stimuli following house decisions (Fig. 4). ( , ) with the hypothesis of stimulus-specific postreward ROI activity (Fig. 4). In a follow-up analysis, we investigated the degree to which both areas contributed to this overall effect. To this end, we assessed  weights within both ROIs separately for the following two parametric contrasts: (1) parametric increase in BOLD re- sponse with reward size after face decisions (FaceDecisionRe- ward) and (2) parametric increase in BOLD response with reward size after house decisions (HouseDecisionReward). The FaceDe- cisionReward parameter yielded a significant result in the FFA ROI (t(16)  2.45, p  0.013), but no significant result in the PPA ROI. No significant parametric BOLD increase could be estab- lished in either ROI for HouseDecisionReward. Thus, visually identical reward images (pictures of money bags) activated stim- ulus representations differentially in a decision-contingent man- ner, an effect mostly carried by an increase in FFA activity following reward stimuli to face decisions compared with reward stimuli following house decisions (Fig. 4). To determine whether participants treated noise stimuli as if they contained signal, we estimated the BOLD activity in the ROIs at the time when noise stimuli were presented in relation to the subsequent perceptual judgment. Activity in these stimulus- specific ROIs provided clear evidence that noise stimuli were treated as if they contained some (albeit weak) signal. Partici- pants’ individual  values for the two conditions—viewing noise stimuli that were then judged to be faces (FaceDecision) and viewing noise stimuli that were judged to be houses (HouseDeci- sion)—were estimated in the two ROIs (Fig. 3A). These individual  values were then entered into a repeated- measures ANOVA with the factors DECISION (face/house) and ROI (FFA/PPA). This yielded no significant effect of decision, but a significant main effect of ROI (F(1,16)  5.93, p  0.027) and a significant interaction between DECISION and ROI (F(1,16)  38.33, p  0.001). The significant interaction is further illustrated by the direct contrasts of conditions within the ROIs. fMRI analysis Th A O f The FFA ROI for all analyses except the PPI was derived by mask- ing the FaceSignalStrength  HouseSignalStrength contrast with the FacelocalizerBlock  HouselocalizerBlock contrast, and was centered on the peak coordinates x  38, y  51, z  15, and x  34, y  60, z  15. The PPA ROI for all analyses except the PPI was derived by masking the HouseSignal- Because feedback was assigned randomly in noise trials (for which learning was impossible), performance modification by feedback for these trials was assessed on a trial-by-trial basis in- stead. To this end, we assessed for successive noise trials how likely participants were to repeat a rewarded response or switch J. Neurosci., November 19, 2014 • 34(47):15610–15620 • 15615 Schiffer et al. • Stimulus-Specific Task-Dependent Reward Activation B PPA FFA A x = -38, y = -51, z = -15 x = 34, y = -60, z = -15 x = -26 , y = -41 , z = -6 x = 25, y = -44 , z = -6 Functional ROIS * * * * * PPA FFA A FaceDecision HouseDecision Decision-specific activation 0.1 0.075 0.05 0.025 0 -0.05 -0.025 ? x ? x Figure 3. A, Functional ROIs were created by masking the contrast FacelocalizerBlock  HouselocalizerBlock and Houseloc izerBlock  FacelocalizerBlock, respectively, with the corresponding parametric contrast for BOLD increase with signal streng fromthemainexperiment.B,ContrastingBOLDresponseforstimuliprecedingfacedecisions(darkgray)onsignaltrialswiththo preceding house decisions (light gray) revealed significantly stronger activity for face decisions in the FFA (left) and for hou decisions in the PPA (right) compared with the respective alternative decision. B PPA FFA A x = -38, y = -51, z = -15 x = 34, y = -60, z = -15 x = -26 , y = -41 , z = -6 x = 25, y = -44 , z = -6 Functional ROIS A D i i ifi ti ti level, a positive correlation of BOLD sig- nal with reward magnitude was estab- lished in the hypothesized network of areas, classically associated with reward processing, including the right nucleus ac- cumbens and right subgenual anterior cin- gulate gyrus/ventromedial PFC (vmPFC). The network further included bilateral hip- pocampal activation (see Fig. 5A). Stimulus-specific activation at reward outcome The primary aim of the present study was to identify neural correlates of reward- driven learning, reflected in postreward activation in ROIs that represent stimulus categories. This stimulus specificity was defined as the first criterion to make postreward activation a plausible corre- late of credit assignment. The stimulus- specificity effect was assessed in two separate contrasts that modeled the para- metric effect of reward size. The two parameters modeled BOLD activity in- crease in the ROIs separately for reward after face and house decisions, respec- tively. A repeated-measures ANOVA on the mean  values from the parametric contrasts with the factors ROI (PPA/FFA) and RESPONSE (house/face) yielded a significant main effect of ROI (F(1,16)  5.104, p  0.038), no significant main ef- fect of RESPONSE (F(1,16)  1.59, p  0.22), and a statistically significant inter- action (F(1,16)  8.98, p  0.009), in line f ti l ifi t d ROI ti it * * * * * PPA FFA FaceDecision HouseDecision 0.1 0.075 0.05 0.025 0 -0.05 -0.025 ? x ? x Figure 3. A, Functional ROIs were created by masking the contrast FacelocalizerBlock  HouselocalizerBlock and Houselocal- izerBlock  FacelocalizerBlock, respectively, with the corresponding parametric contrast for BOLD increase with signal strength fromthemainexperiment.B,ContrastingBOLDresponseforstimuliprecedingfacedecisions(darkgray)onsignaltrialswiththose preceding house decisions (light gray) revealed significantly stronger activity for face decisions in the FFA (left) and for house decisions in the PPA (right) compared with the respective alternative decision. Strength  FaceSignalStrength contrast with the Houselocalizer- Block  FacelocalizerBlock contrast, and was centered on the peak coordinates in x  26, y  41, z  6, and x  25, y  44, z  6 (Fig. 3). action (F(1,16) 8.98, p 0.009), in line with the hypothesis of stimulus-specific postreward ROI activity (Fig. 4). In a follow-up analysis, we investigated the degree to which both areas contributed to this overall effect. To this end, we assessed  weights within both ROIs separately for the following two parametric contrasts: (1) parametric increase in BOLD re- sponse with reward size after face decisions (FaceDecisionRe- ward) and (2) parametric increase in BOLD response with reward size after house decisions (HouseDecisionReward). The FaceDe- cisionReward parameter yielded a significant result in the FFA ROI (t(16)  2.45, p  0.013), but no significant result in the PPA ROI. No significant parametric BOLD increase could be estab- lished in either ROI for HouseDecisionReward. Stimulus-specific activation at reward outcome These con- trasts showed significantly more activity in the FFA for pending face versus house judgments (t(16)  2.25, p  0.019) and signif- icantly more activity in the PPA preceding house versus face judg- ments (t(16)  3.39, p  0.002; Fig. 3B). Task dependent activation at reward outcome Our second criterion for postreward activation to be a marker of credit assignment was task dependency. Task dependency re- quires postreward activation to be specific to perceptual decision tasks. We modeled four separate parametric contrasts: reward size for face decisions, house decisions, instructed “face” re- sponses, and instructed “house” responses. Entering the  values from the RESPONSE (face/house), ROI (FFA/PPA), and TASK (decision/instructed) conditions of the parametric analysis into a 2  2  2 repeated-measures ANOVA revealed no significant main effects, but a marginally significant interaction for RE- We further established that positive outcomes to noise trials would activate the network of brain regions associated with learning from reward (O’Doherty et al., 2003; O’Doherty, 2004). After correction for multiple comparisons at the whole-brain NSE  TASK (F(1,16)  3.125, p  0.096) and the predicted i t i t ti f ROI  TASK  RESPONSE (F dependency criterion. Because activity in the FFA ROI wa l t d t hi h d b ti l ifi it ? x ! x ? x ! x > FFA PPA > mean ß decision - instruction (a.u.) A Stimulus specificity mean ß (a.u.) FFA PPA FaceDecRewParam HouseDecRewParam * B Task dependency FaceDecRewParam HouseDecRewParam FaceInstructionRewParam HouseInstructionRewParam Parametric increase Task dependency face response > > Task dependency house response 0.2 0.1 0 -0.1 0.2 0.1 0 -0.05 0.05 0.15 * Contrast of parametric increase 2 1 amp 2 1 amp 2 1 amp 2 1 amp 2 1 amp 2 1 amp x x x x x x x x x x x x 4. A,Stimulus-specificactivityinnoisetrialswasmeasuredasanincreaseinBOLDactivitywithrewardsize,whichwassignificantlystrongerforthedecisionassociatedwitha houseforPPA)thanfortheoppositedecision.Darkgraybarsshowmeansforparametricincreasewithrewardmagnitudeforfacedecisions;lightgraybarsshowmeansforp withrewardmagnitudeforhousedecisions.Left,ActivityintheFFAROI.Right,ActivityinthePPAROI.B,Taskdependencywasreflectedinalargerparameterestimate(mean with reward size in the associated ROIs (FFA left, PPA right) in the perceptual decision, compared with the instructed response tasks. Markers refer to the difference of mean ual decision and instructed response tasks; the difference for face responses are shown in dark gray, house responses in light gray. J. Neurosci., November 19, 2014 • 34(47):15610–15620 Schiffer et al. • Stimulus-Specific Task-Dependent Reward 15616 • J. Neurosci., November 19, 2014 • 34(47):15610–15620 Schiffer et al. J. Neurosci., November 19, 2014 • 34(47):15610–15620 • 15617 J. Neurosci., November 19, 2014 • 34(47):15610–15620 • 15617 Schiffer et al. • Stimulus-Specific Task-Dependent Reward Activation To compare the two types of trials, we modeled reward pa- rameters for noise as well as signal trials in each task, implement- ing eight separate regressors for the factorial combination of TRIAL-TYPE (noise/signal), RESPONSE (house/face), and TASK (decision/instructed). As a first pass, we established whether the documented stimulus-specificity and task- dependency effects were again reliable in noise trials in a 2  2  2 (ROI  RESPONSE  TASK) repeated-measures ANOVA. This yielded a marginally significant interaction of ROI and TASK (F(1,16)  3.09, p  0.098) and a significant three-way interaction between ROI, RESPONSE, and TASK (F(1,16)  10.19, p  0.006), with ROI activity being greater for the associ- ated response in decision tasks. Thus, we find support for the result from the original stimulus-specificity and task-dependency analyses in this alternative GLM. tionReward) yielded a significant result in the FFA ROI (t(16)  2.02 p  0.03), showing that the significant effect of stimulus specificity in the FFA was task dependent (Fig. 4b). tionReward) yielded a significant result in the FFA ROI (t(16)  2.02 p  0.03), showing that the significant effect of stimulus specificity in the FFA was task dependent (Fig. 4b). Reward-driven activation of stimulus-specific areas: PPI analyses As a parallel to the preceding ROI analysis, which assessed whether stimulus-selective regions in sensory cortex would show activity at the time of reward presentation, our PPI analyses aimed to identify whether regions showing reward-related mod- ulation would simultaneously exhibit evidence of stimulus spec- ificity. Consistent with this notion, the face-vs-house PPI analysis yielded significant activation (z  2.3, p  0.01) in an area of the posterior fusiform gyrus centered on x  34, y  78, z  15 (Talairach coordinates). As a complementary left-hemisphere ROI, we chose the closest peak of activation to the mirrored co- ordinates in the left hemisphere (x  34), which identified a peak voxel at x  38, y  78, z  6 that was marginally significant (z  1.63; p  0.053). A mask consisting of two cubes of 2  2  2 voxels around these centers was then applied to the face block versus house block functional localizer and yielded a significant result (z  2.09, p  0.018). J. Neurosci., November 19, 2014 • 34(47):15610–15620 • 15617 Thus, these bilateral ROIs in the fusiform gyrus (1) correlated more positively with the nu- cleus accumbens after reward delivery for face than house deci- sions on noise trials, and (2) were more involved in face than house processing in a separate task. These ROIs were somewhat posterior to the FFA ROIs discussed above. PPI activation peaks were evident close to these FFA ROIs in the right hemisphere (x  31, y  60, z  15) and left hemisphere (x  35, y  48, z  12), but these activations were only marginally reliable in the PPI analysis (right hemisphere: z  1.39, p  0.082; left hemi- sphere: z  1.506, p  0.066), while of course exhibiting evidence of stimulus specificity (z  1.66, p  0.047). y Given this confirmation, we next compared postreward BOLD responses for the two different trial types in a 2  2  2  2 repeated-measures ANOVA, including the factor ROI (FFA/ PPA) crossed with RESPONSE, TASK, and TRIAL-TYPE. This analysis replicated the stimulus-specificity effect in a marginally significant interaction of ROI  RESPONSE (F(1,16)  3.47 p  0.081). Further, it showed a significant four-way interaction (F(1,16)  18.11, p  0.001). The significant four-way interaction indicates trial-type sensitivity of the established effects, expressed as a difference between noise and signal trials with regard to task dependency. Because the differential activation of the ROIs was repeatedly shown to be decision dependent, and not pronounced for instruction trials, we focused our further comparison of noise and signal trials on decision trials only. We therefore investigated the effect of trial-type sensitivity for decision trials in a 2  2  2 repeated-measures ANOVA with the factors ROI (FFA/PPA), RESPONSE (face/house), and TRIAL-TYPE (noise/signal) and found a significant interaction of ROI and RESPONSE (F(1,16)  7.32, p  0.016), indicating stimulus specificity in decision tasks, a significant interaction of ROI and TRIAL-TYPE (F(1,16)  10.13, p  0.006), and a significant three-way interaction be- tween ROI, RESPONSE, and TRIAL-TYPE (F(1,16)  7.45, p  0.015), with more stimulus-specific, reward-modulated ROI ac- tivity after perceptual decisions on noise trials than on signal trials. Thus, we find confirmation of the stimulus-specificity and task-dependency effect, but a difference between noise and signal trials. J. Neurosci., November 19, 2014 • 34(47):15610–15620 • 15617 p y p The corresponding house-vs-face PPI analysis yielded signif- icant activation (z  2.14, p  0.016) in an area in the hippocam- pal gyrus centered on x  16, y  48, z  3 (Talairach coordinates). As a complementary left-hemisphere ROI, we chose the closest peak of activation to the mirrored coordinates in the left hemisphere (x  16), yielding a peak voxel at x  17, y  45, z  9 that was significantly activated (z  1.72; p  0.041). A mask consisting of two cubes of 2  2  2 voxels around these centers was then applied to the house block ver- sus face block functional localizer and yielded a significant result (z  2.507, p  0.006). Thus, we identified two ROIs in the parahippocampal region which correlated more positively with the nucleus accumbens after reward delivery for house than face decisions on noise trials, and these ROIs were more involved in house than face processing in a separate (localizer) task (Fig. 5). Discussion Humans learn how to acquire rewarding outcomes and the neu- ral basis of reward processing has been studied extensively. How- ever, we know very little about how reward-yielding tasks are represented in the brain. Machine learning describes how reward-driven learning depends on eligibility traces that signal which events are predictors of reward (Weil et al., 2010; cf. credit assignment, FitzGerald et al., 2012). It has been proposed that these learning processes are evident at the neural level as activa- tion of the sensory cortices that represent components of the rewarded task. We investigated the neural correlates of postreward task representations in visual association cortices in a perceptual decision task. We defined two criteria for potential neural correlates of eligibility traces: postreward activation in a sensory association cortex should be stimulus specific (i.e., reflect the stimulus category of the rewarded response) and task depen- dent (i.e., should only occur if the stimulus was relevant to the task). We indeed found the representation of a stimulus to be activated postreward, especially if it was relevant for the correct response. This effect was established in a significant interaction of Stimulus-specific activation at reward outcome • Stimulus-Specific Task-Dependent Reward Activation A Stimulus specificity mean ß (a.u.) FFA PPA FaceDecRewParam HouseDecRewParam * Parametric increase 0.2 0.1 0 -0.1 * 2 1 amp 2 1 amp x x x x Parametric increase 2 mean ß (a.u.) HouseDecRewParam ? x ! x ? x ! x > FFA PPA > mean ß decision - instruction (a.u.) B Task dependency FaceDecRewParam HouseDecRewParam FaceInstructionRewParam HouseInstructionRewParam Task dependency face response > > Task dependency house response 0.2 0.1 0 -0.05 0.05 0.15 Contrast of parametric increase 2 1 amp 2 1 amp 2 1 amp 2 1 amp x x x x x x x x A,Stimulus-specificactivityinnoisetrialswasmeasuredasanincreaseinBOLDactivitywithrewardsize,whichwassignificantlystrongerforthedecisionassociatedwitha useforPPA)thanfortheoppositedecision.Darkgraybarsshowmeansforparametricincreasewithrewardmagnitudeforfacedecisions;lightgraybarsshowmeansfor ithrewardmagnitudeforhousedecisions.Left,ActivityintheFFAROI.Right,ActivityinthePPAROI.B,Taskdependencywasreflectedinalargerparameterestimate(mean ith reward size in the associated ROIs (FFA left PPA right) in the perceptual decision compared with the instructed response tasks Markers refer to the difference of mean ? x ! x > n ß decision - instruction (a.u.) B Task dependency FaceDecRewParam FaceInstructionRewParam Task dependency face response > Task dependency house response 0.2 0.1 0 0.05 0.15 Contrast of parametric increase 2 1 amp 2 1 amp x x x x Contrast of parametric increase ? x ! x > HouseDecRewParam HouseInstructionRewParam > Task dependency house response 2 1 amp 2 1 amp x x x x Figure4. A,Stimulus-specificactivityinnoisetrialswasmeasuredasanincreaseinBOLDactivitywithrewardsize,whichwassignificantlystrongerforthedecisionassociatedwithanROI(face forFFA,houseforPPA)thanfortheoppositedecision.Darkgraybarsshowmeansforparametricincreasewithrewardmagnitudeforfacedecisions;lightgraybarsshowmeansforparametric increasewithrewardmagnitudeforhousedecisions.Left,ActivityintheFFAROI.Right,ActivityinthePPAROI.B,Taskdependencywasreflectedinalargerparameterestimate(mean)forBOLD increase with reward size in the associated ROIs (FFA left, PPA right) in the perceptual decision, compared with the instructed response tasks. Markers refer to the difference of mean s in the perceptual decision and instructed response tasks; the difference for face responses are shown in dark gray, house responses in light gray. dependency criterion. Because activity in the FFA ROI was mod- ulated to a higher degree by stimulus specificity, we assessed within the FFA whether this stimulus-specific activity was also task dependent. Therefore, face decisions were contrasted with trials in which participants made an instructed response with the same key. This contrast (FaceDecisionReward  FaceInstruc- SPONSE  TASK (F(1,16)  3.125, p  0.096) and the predicted significant interaction of ROI  TASK  RESPONSE (F(1,16)  10.84, p  0.005). This significant three-way interaction is indic- ative of a stronger positive relationship between reward size and response-specific ROI activity in the perceptual decision task than in the instructed response task (Fig. 4), satisfying the task- Trial-type sensitivity Th di The present paradigm aimed to establish stimulus specificity and task dependency by focusing on trials with noise stimuli, using stimuli with true (house or face) signal primarily to create a cred- ible context for those critical noise trials within our perceptual judgment task. It is nevertheless instructive to analyze reward- induced activity following the signal trials for comparison with other recent studies of reward-related activation in sensory cor- tex (Pleger et al., 2008; Weil et al., 2010; FitzGerald et al., 2012). Signal trials differ from noise trials for several notable reasons. They differ, for example, because these perceptual decisions would involve more reliance on bottom-up features and because reward probability and neural adaptation effects are confounded with signal strength. Schiffer et al. • Stimulus-Specific Task-Dependent Reward Activation 15618 • J. Neurosci., November 19, 2014 • 34(47):15610–15620 Reward activation Hippocampus vmPFC N. Acc. N. Acc. vmPFC PPI analysis FaceDecRew HouseDecRew > HouseDecRew FaceDecRew > N. Acc. N. Acc. seed voxel 2 PPIs per seed conjunction yields ROIs applied to localiser ROI comparison [(face blocks > house blocks) I face signal] [(house blocks > face blocks) I house signal] Face PPI conjunction ROI* House PPI conjunction ROI* A B C i ii iii iv x x x 2 1 amp 3 alternative Face PPI conjunction ROI † Block Localizer Contrasts Functional ROIs for Main Analysis PPI-derived ROIs BlockLocalizer: Face > House mean (a.u.) PPI-derived ROIs 0.1 0.2 0 -0.1 -0.2 Face House (face blocks > house blocks) contrast †conjunction significant at p < 0.1 (house blocks > face blocks) contrast *conjunction significant at p < 0.05 A,Rewardingoutcomesafterdecisionsonnoisetrialsactivatedanetworkclassicallyassociatedwithrewarddelivery,includingtheventromedialPFC(vmPFC)andnucleus as well as the anterior hippocampus. B, Each N.Acc. peak voxel (i) was used as a seed voxel in two PPI analyses (ii). The first PPI analysis implemented a contrast of the incre ft f d i i t i t i l BOLDi ft h d i i t i t i l Th th PPIi l t dth t t A j ti fth PPI d i df Neurosci., November 19, 2014 • 34(47):15610–15620 Schiffer et al. • Stimulus-Specific Task-Dependent Rewa Reward activation Hippocampus vmPFC N. Acc. N. Acc. vmPFC A x x x 2 1 amp 3 Reward activation A vmPFC N. Acc. N. Acc. Hippocampus PPI analysis B N. Acc. N. Acc. N. Acc. N. Acc. Trial-type sensitivity Th di ROI comparison [(face blocks > house blocks) I face signal] [(house blocks > face blocks) I house signal] Face PPI conjunction ROI* House PPI conjunction ROI* C alternative Face PPI conjunction ROI † Block Localizer Contrasts Functional ROIs for Main Analysis PPI-derived ROIs (face blocks > house blocks) contrast †conjunction significant at p < 0.1 (house blocks > face blocks) contrast *conjunction significant at p < 0.05 Figure5. A,Rewardingoutcomesafterdecisionsonnoisetrialsactivatedanetworkclassicallyassociatedwithrewarddelivery,includingtheventromedialPFC(vmPFC)andnucleusaccumbens (N. Acc.), as well as the anterior hippocampus. B, Each N.Acc. peak voxel (i) was used as a seed voxel in two PPI analyses (ii). The first PPI analysis implemented a contrast of the increase in BOLD responseafterfacedecisionstonoisetrialsversusBOLDincreaseafterhousedecisionstonoisetrials.TheotherPPIimplementedthereversecontrast.AconjunctionofthePPIsderivedfromdifferent hemispheres for FaceDecisionReward  HouseDecisionReward contrasts yielded ROIs in the posterior fusiform gyrus. The conjunction of the bilateral PPIs implementing the HouseDecisionRe- wardFaceDecisionRewardcontrastsyieldedROIsintheparahippocampalregion(iii).ApplyingtheseROIstothefunctionallocalizerblockshowedthatareasthatcorrelatedwiththeN.Acc.inthe FaceDecisionReward  HouseDecisionReward contrast were also selectively activated by blocks of face images (iv). Conversely, areas that correlated with the N.Acc. in the reverse contrast were moreselectivelyactivatedbyblocksofhouseimagesthanfaceimages.C,WhiletheROIsderivedfromthePPIapproach(right)divergefromtheROIsthatwereselectedonthecriteria:responsiveness to a specific category in the functional localizer and correlation with signal strength (middle), both sets of areas lie clearly within the large parts of cortex that show either preferential response to faceblocksorpreferentialresponsetohouseblocks,respectively.Themoststronglyactivatedface-selectiveareaintheFaceDecisionRewardHouseDecisionRewardPPIwaslocatedposteriorto classicalFFAcoordinates.Anareaclosertothesecoordinates(denotedasalternativeFacePPIconjunctionROI)showedmarginallysignificantactivityintheFaceDecisionRewardHouseDecision- Reward PPI and also displayed face selectivity in the block localizer. J. Neurosci., November 19, 2014 • 34(47):15610–15620 • 15619 Schiffer et al. • Stimulus-Specific Task-Dependent Reward Activation top-down-driven postreward activation rely both on feedback projections, which differ from feedforward projections convey- ing sensory input. They might therefore activate the same level in the cortical hierarchy of stimulus representation (Markov et al., 2013). This could increase the overlap of the locus of BOLD response measured for decisions under noise and top-down- driven activation, increasing positive correlation between decision-specific activity and postreward stimulus-specific activ- ity. Third, in studies with true “signal” stimuli, strong anticipa- tion of reward may modulate activity in the sensory cortices before reward delivery (Serences, 2008; Brosch et al., 2011). Thus, reward delivery may have had little effect on activity given that it was delivered in a performance-dependent manner in tasks where participants performed above chance (Pleger et al., 2008; Weil et al., 2010; FitzGerald et al., 2012). Here, however, rewards in noise trials could not be anticipated, as feedback was assigned randomly, limiting a positive correlation between signal strength, reward anticipation, and reward. Collectively, these features of noise stimuli in our design may have made our study very sensi- tive to stimulus-specific postreward effects. Trial-type sensitivity Th di response category (face or house decision) and BOLD activity in the FFA or PPA. Moreover, the effect was specific to trials in which the stimulus was task relevant, fulfilling the criterion of task dependency. The effects of stimulus-specific postreward ac- tivation received further support from a PPI analysis. This anal- ysis identified an area in the parahippocampal region that correlated with nucleus accumbens activity after rewarded house responses and that was sensitive to house processing in the sepa- rate localizer task, whereas an area in the fusiform gyrus that correlated with nucleus accumbens activity after rewarded face responses was sensitive to face processing in the separate localizer task. Task dependency M bj i Most objects require a specific manipulation to yield desired out- comes. However, at any given moment, many objects are present in our environment, and any given object may afford different actions, depending on the task. Hence, reward needs to activate the specific representations of only those objects that were in- volved in the current task. Global postreward activation, includ- ing irrelevant stimulus representations, would yield a new credit assignment problem (Roelfsema and van Ooyen, 2005). In fact, stimulus specificity and task dependency relate to the difference between Pavlovian and instrumental conditioning. Pavlovian conditioning on the one hand requires a stimulus-specific asso- ciation between the conditional stimulus and the unconditional stimulus to be formed; this association needs to be stimulus spe- cific, but is not necessarily related to a specific action. On the other hand, instrumental conditioning requires the association of a stimulus, an appropriate response, and the eventual reinforcer. Thus, instrumental conditioning should crucially incorporate task dependency. Credit assignment f l Reinforcement learning theory explains how reward-predicting events are assigned a higher value and become targets of behavior (Sutton, 1988; Sutton and Barto, 1990; Daw and Doya, 2006; Dayan and Niv, 2008). However, the neural underpinnings of this credit assignment mechanism are unclear. In particular, it is known that reward prediction and prediction errors elicit neural activity in the basal ganglia and vmPFC and result in increased firing of dopamine neurons in the midbrain, but it is yet to be established how this reward response fosters the representations of rewarded tasks. One proposal is that reward signals increase synaptic plasticity in sensory areas (Jay, 2003; Lisman et al., 2011; Pennartz et al., 2011). Supporting evidence comes from studies showing modulation of neural activity by anticipated reward (Serences, 2008; Brosch et al., 2011). These findings indicate that pairing with reward changes the neuronal representation of an event’s value in sensory cortices; this may explain why associa- tions between stimuli and reward can prime behavior (Hickey et al., 2010; Hickey and van Zoest, 2012; Wimmer and Shohamy, 2012). However, while these studies show that credit assignment takes place, and may be linked to dopamine release, they do not reveal how neural activity representing relevant stimuli is linked to neural correlates of reward during learning. The present study sheds light on this question by demonstrating stimulus specificity and task dependency of postreward activation. The stimulus-specific effects we identified in terms of interac- tions between decisions and ROIs were reliable only within the FFA. A possible explanation for this finding is that we chose the ROIs based on a localizer that correlated BOLD increase with the increase in signal strength in the stimulus. We thus biased our analysis toward ROIs that responded more strongly if more category-specific bottom-up input was present. However, whether, for example, subregions of the PPA are differentially involved in top-down-driven versus bottom-up-driven scene processing has yet to be empirically tested (cf. Park et al., 2010). Conclusion Th y y y difference models and reward-related learnin ron 38:329–337. CrossRef Medline The present study has established stimulus-specific and task- dependent activation following reward delivery in a perceptual decision task. The established features of stimulus specificity and task dependency suggest that postreward activation may be a cortical signature of eligibility traces for credit assignment. This finding is a substantial step toward closing the gap between well defined computational concepts in reward-based learning and their neural implementation. The next important step will be to clarify how information is maintained during the intervals be- tween the stimulus, the response, and the ensuing feedback. Rep- resentations could be maintained as persistent activation in sensory cortices, which would be modulated by the eventual feed- back. Alternatively, increased activation of representations might only persist in PFC during the delay and be reinstated in sensory cortices by reward. Further, an important task for future research will be to test the suggested functional relationship between postreward activation and the dopaminergic modulation of syn- aptic plasticity directly. Establishing such a link would open the field to further exciting questions regarding the mechanisms of maintaining relevant representations until reward delivery, the nature of dopaminergic circuits that mediate this form of learn- ing, and the degree to which this learning depends on the avail- ability of dopamine, or the interplay between dopamine and other neurotransmitters that influence behavior. g ron 38:329–337. CrossRef Medline ron 38:329–337. CrossRef Medline O’Reilly JX, Woolrich MW, Behrens TE, Smith SM, Johansen-Berg H (2012) Tools of the trade: psychophysiological interactions and functional con- nectivity. Soc Cogn Affect Neurosci 7:604–609. CrossRef Medline Park S, Chun MM, Johnson MK (2010) Refreshing and integrating visual scenes in scene-selective cortex. J Cogn Neurosci 22:2813–2822. CrossRef Medline Pennartz CM, Ito R, Verschure PF, Battaglia FP, Robbins TW (2011) The hippocampalstriatal axis in learning, prediction and goal-directed behav- ior. Trends Neurosci 34:548–559. CrossRef Medline Pleger B, Blankenburg F, Ruff CC, Driver J, Dolan RJ (2008) Reward facilitates tactilejudgmentsandmodulateshemodynamicresponsesinhumanprimary somatosensory cortex. J Neurosci 28:8161–8168. CrossRef Medline Pleger B, Ruff CC, Blankenburg F, Klo¨ppel S, Driver J, Dolan RJ (2009) Influence of dopaminergically mediated reward on somatosensory decision-making. PLoS Biol 7:e1000164. CrossRef Medline Rajimehr R, Devaney KJ, Bilenko NY, Young JC, Tootell RB (2011) The parahippocampal place area responds preferentially to high spatial fre- quencies in humans and monkeys. PLoS Biol 9:e1000608. Stimulus specificity f d i i If postreward activation of the task underlies learning, an exact representation of the previous stimulus should be traceable after reward delivery. Although tested in a number of studies (Pleger et al., 2008, 2009; Weil et al., 2010; FitzGerald et al., 2012), results of paradigms studying reward-related activation in visual associa- tion areas have been ambiguous. Using fMRI in monkeys, Ar- senault and colleagues (2013) successfully showed postreward activation in sensory cortices, but only in trials that did not entail the visual stimulus itself. Conversely, several human fMRI studies have found no evidence for stimulus-specific activity in sensory cortex following reward delivery (Weil et al., 2010; FitzGerald et al., 2012). This reasoning implies that if postreward activation observed in the current study was a marker of a credit assignment, we would expect it to reflect whether the preceding stimulus was relevant to the rewarded task. Previously, postreward activation has been tested using designs in which stimuli that preceded re- ward were always task relevant, rendering it difficult to interpret the established effects as correlates of either credit assignment or less specific reward-driven activation. In the present study, only one of two tasks required a response that was stimulus specific. We showed that stimulus-specific postreward activation was de- pendent on the relevance of the stimulus for the reward-yielding task. Thus, our study shows that reward selectively increases ac- tivity in sensory areas representing objects that have been used to In the present study, we measured effects following decisions on pure noise stimuli. This may have rendered the design espe- cially sensitive to stimulus-specific activation for a number of reasons. First, adaptation to individual stimuli has been sug- gested to explain the observation of nonspecific activation effects (FitzGerald et al., 2012). In contrast, we analyzed postreward activation following perceptual decisions on noise. Activity in the respective ROIs was thus dependent on the judged category rep- resentations, not on low-level features of the individual stimuli. Category representations might be less prone to sensory adapta- tion than low-level features. Second, activation in noise trials and 15620 • J. Neurosci., November 19, 2014 • 34(47):15610–15620 Schiffer et al. • Stimulus-Specific Task-Dependent Reward Activation perform a task, and not globally in sensory areas representing any object present in the current context. perform a task, and not globally in sensory areas representing any object present in the current context. Stimulus specificity f d i i O’Doherty JP (2004) Reward representations and reward-related learning in the human brain: insights from neuroimaging. Curr Opin Neurobiol 14:769–776. CrossRef Medline O’Doherty JP, Dayan P, Friston K, Critchley H, Dolan RJ (2003) Temporal difference models and reward-related learning in the human brain. Neu- O’Doherty JP, Dayan P, Friston K, Critchley H, Dolan RJ (2003) Temporal difference models and reward-related learning in the human brain. Neu- ron 38:329–337. CrossRef Medline Conclusion Th Medline Rescorla RA, Wagner AR (1972) A theory of Pavlovian conditioning: varia- tions in the effectiveness of reinforcement and nonreinforcement. In: Classical conditioning II: Current research and theory (Black AH, Prokasy WF, eds), pp 64–69. New York: Appleton Century Crofts. WF, eds), pp 64–69. New York: Appleton Century Crofts. Roelfsema PR, van Ooyen A (2005) Attention-gated reinforcement learning of internal representations for classification. Neural Comput 17:2176– 2214. CrossRef Medline Schultz W (2007) Behavioral dopamine signals. Trends Neurosci 30:203– 210. CrossRef Medline References A l JT N Schultz W, Dayan P, Montague PR (1997) A neural substrate of prediction and reward. Science 275:1593–1599. CrossRef Medline Arsenault JT, Nelissen K, Jarraya B, Vanduffel W (2013) Dopaminergic re- ward signals selectively decrease fMRI activity in primate visual cortex. Neuron 77:1174–1186. CrossRef Medline Schyns PG, Oliva A (1994) From blobs to boundary edges: Evidence for time- and spatialscale-dependent scene recognition. Psychol Sci 5:195– 200. CrossRef Brosch M, Selezneva E, Scheich H (2011) Representation of reward feedback in primate auditory cortex. Front Syst Neurosci 5:5. CrossRef Medline Serences JT (2008) Value-based modulations in human visual cortex. Neu- ron 60:1169–1181. CrossRef Medline Daw ND, Doya K (2006) The computational neurobiology of learning and reward. Curr Opin Neurobiol 16:199–204. CrossRef Medline Sutton RS (1988) Learning to predict by the methods of temporal differ- ences. Machine Learning 3:9–44. CrossRef Sutton RS (1988) Learning to predict by the method ences. Machine Learning 3:9–44. CrossRef Dayan P, Niv Y (2008) Reinforcement learning: the good, the bad and the ugly. Curr Opin Neurobiol 18:185–196. CrossRef Medline Sutton RS, Barto AC (1990) Time-derivative models of Pavlovian reinforce- ment. In: Learning and Computational Neuroscience: Foundations of Adaptive Networks (Gabriel M, Moore J, eds), pp 497–537. Cambridge, MA: MIT. FitzGerald TH, Friston KJ, Dolan RJ (2012) Action-specific value signals in reward-related regions of the human brain. J Neurosci 32:16417–16423a. CrossRef Medline Grill-Spector K, Henson R, Martin A (2006) Repetition and the brain: neu- ral models of stimulus-specific effects. Trends Cogn Sci 10:14–23. CrossRef Medline Talairach J, Tournoux P (1988) Co-planar stereotaxic atlas of the human brain. 3-Dimensional proportional system: an approach to cerebral im- Talairach J, Tournoux P (1988) Co-planar stereotaxic atlas of the human brain. 3-Dimensional proportional system: an approach to cerebral im- aging. New York: Thieme Medical. aging. New York: Thieme Medical. Hickey C, van Zoest W (2012) Reward creates oculomotor salience. Curr Biol 22:R219–R220. CrossRef Medline Waelti P, Dickinson A, Schultz W (2001) Dopamine responses comply with basic assumptions of formal learning theory. Nature 412:43–48. CrossRef Medline HickeyC,ChelazziL,TheeuwesJ (2010) Rewardchangessalienceinhumanvision via the anterior cingulate. J Neurosci 30:11096–11103. CrossRef Medline Weil RS, Furl N, Ruff CC, Symmonds M, Flandin G, Dolan RJ, Driver J, Rees G (2010) Rewarding feedback after correct visual discriminations has both general and specific influences on visual cortex. J Neurophysiol 104: 1746–1757. CrossRef Medline Jay TM (2003) Dopamine: a potential substrate for synaptic plasticity and memory mechanisms. Prog Neurobiol 69:375–390. References A l JT N CrossRef Medline Jay TM (2003) Dopamine: a potential substrate for synaptic plasticity and memory mechanisms. Prog Neurobiol 69:375–390. CrossRef Medline Lisman J, Grace AA, Duzel E (2011) A neoHebbian framework for episodic memory; role of dopamine-dependent late LTP. Trends Neurosci 34: 536–547. CrossRef Medline Willenbockel V, Sadr J, Fiset D, Horne GO, Gosselin F, Tanaka JW (2010) Controlling low-level image properties: the SHINE toolbox. Behav Res Methods 42:671–684. CrossRef Medline Lohmann G, Mu¨ller K, Bosch V, Mentzel H, Hessler S, Chen L, Zysset S, von Cramon DY (2001) LIPSIA—a new software system for the evaluation of functional magnetic resonance images of the human brain. Comput Med Imaging Graph 25:449–457. CrossRef Medline Wimmer GE, Shohamy D (2012) Preference by association: how memory mechanisms in the hippocampus bias decisions. Science 338:270–273. CrossRef Medline Med Imaging Graph 25:449–457. CrossRef Medline Markov NT, Ercsey-Ravasz M, Van Essen DC, Knoblauch K, Toroczkai Z, Kennedy H (2013) Cortical high-density counterstream architectures. Science 342:1238406. CrossRef Medline Worsley KJ, Friston KJ (1995) Analysis of fMRI time series revisited—again. Neuroimage 2:173–181. CrossRef Medline
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Review of: "ProteinGLUE: A multi-task benchmark suite for self-supervised protein modeling."
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Qeios, CC-BY 4.0 · Review, December 24, 2021 Qeios ID: P5VEAL · https://doi.org/10.32388/P5VEAL Review of: "ProteinGLUE: A multi-task benchmark suite for self-supervised protein modeling." Jianfeng Gao1 1 Wuhan University Jianfeng Gao1 1 Wuhan University Potential competing interests: The author(s) declared that no potential competing interests exist. In the manuscript entitled “ProteinGLUE: A multi-task benchmark suite for self-supervised protein modeling”, the authors introduce the ProteinGLUE benchmark for the evaluation of protein representations: a set of seven tasks for evaluating learned protein representations. The authors also offer reference code, and they provide two baseline models with hyperparameters specifically trained for these benchmarks. Pre-training was done on two tasks, masked symbol prediction and next sentence prediction. The authors show that pre-training yields higher performance on a variety of downstream tasks such as secondary structure and protein interaction interface prediction, compared to no pre-training. As self-supervised language modeling is getting more attention, the work sounds interesting. However, the paper need to be revised carefully further for the following reasons. Firstly, the reliability, universality and stability of ProteinGLUE Benchmark Set need further confirmation. Secondly, the English must be improved in terms of grammar, formatting, sentence structure, Italicization, referencing and citation format. Together, I suggest that this paper is able to be accepted for publication with Minor Revision. 1/1
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Becoming obese in young adulthood: the role of career-family pathways in the transition to adulthood for men and women
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University of Groningen Becoming obese in young adulthood Mooyaart, Jarl E.; Liefbroer, Aart C.; Billari, Francesco C. Published in: BMC Public Health DOI: 10.1186/s12889-019-7797-7 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2019 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Mooyaart, J. E., Liefbroer, A. C., & Billari, F. C. (2019). Becoming obese in young adulthood: the role of career-family pathways in the transition to adulthood for men and women. BMC Public Health, 19(1), Article 1511. https://doi.org/10.1186/s12889-019-7797-7 University of Groningen Becoming obese in young adulthood Mooyaart, Jarl E.; Liefbroer, Aart C.; Billari, Francesco C. DOI: 10.1186/s12889-019-7797-7 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Mooyaart, J. E., Liefbroer, A. C., & Billari, F. C. (2019). Becoming obese in young adulthood: the role of career-family pathways in the transition to adulthood for men and women. BMC Public Health, 19(1), Article 1511. https://doi.org/10.1186/s12889-019-7797-7 Citation for published version (APA): Mooyaart, J. E., Liefbroer, A. C., & Billari, F. C. (2019). Becoming obese in young adulthood: the role of career-family pathways in the transition to adulthood for men and women. 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BMC Public Health (2019) 19:1511 https://doi.org/10.1186/s12889-019-7797-7 Open Access Becoming obese in young adulthood: the role of career-family pathways in the transition to adulthood for men and women Jarl E. Mooyaart1* , Aart C. Liefbroer2,3,4 and Francesco C. Billari5 Abstract Background: During the transition to adulthood many young adults become obese for the first time in their lives, yet relatively little research has examined why people in this life phase become obese. This study examines what career and family life-course pathways during the transition to adulthood are related to developing obesity in young adulthood. Methods: We use data from the NLSY97, a U.S. nationally representative panel survey conducted by the Bureau of Labor Statistics between 1997 to 2013 (N = 4688), and apply multichannel sequence analysis in order to identify clusters of typical career-family pathways during the transition to adulthood (age 17 to 27), and subsequently investigate whether these pathways are associated with becoming obese at the end of young adulthood (age 28), using logistic regression. We control for obesity at age 17 and family background factors (race, parental education, parental income, and family structure). To take into account the fact that the transition to adulthood has a different meaning for men and for women, we also interact career-family clusters with gender. Results: For women, pathways characterized by college education, early home leaving, and postponement of family formation decrease the probability of becoming obese. For men, pathways characterized by early marriage increase the probability of becoming obese. Conclusions: The results highlight the importance of gender differences in how career and family pathways are related to becoming obese in young adulthood. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background normal weight during their childhood, become obese for the first time during the transition to adulthood [21]. However, the explanation of why such a strong increase in obesity occurs during the transition to adulthood has received little attention [34]. The transition to adulthood is an eventful phase in the life-course. It is the time in which events such as leaving the parental home, entering the labor market, and/or postsecondary education, union formation, and parenthood take place in the lives of most individuals. There is evidence that specific events in the transition to adulthood are related to changes in weight, physical activity and dietary behavior. Events such as leaving the parental home and moving to college are associated with a less healthy diet and a drop in physical activity [57, 59]. Furthermore, getting married, becoming a parent, and starting a new paid job have The dramatic increase in obesity over the last few de- cades in the United States and other Western countries is a major public health concern [12, 32, 36]. Currently, about one in three adults are obese [35]. Because obesity has been linked to an increased risk of a number of dis- eases (See Kopelman [23] for an overview), it is crucial to identify risk factors for how obesity develops. While much research has focused on obesity during childhood and adolescence, a large increase in body mass index (BMI) occurs during the transition from ado- lescence to adulthood [22, 34, 48]. Many youths, having * Correspondence: jarl.mooyaart@gmail.com 1Department of Sociology, McGill University, Peterson Hall Building 3460 McTavish Street, Montréal, QC H3A 0E6, Canada Full list of author information is available at the end of the article * Correspondence: jarl.mooyaart@gmail.com 1Department of Sociology, McGill University, Peterson Hall Building 3460 McTavish Street, Montréal, QC H3A 0E6, Canada Full list of author information is available at the end of the article Mooyaart et al. BMC Public Health (2019) 19:1511 Page 2 of 12 Page 2 of 12 been related to more unhealthy food intake and a de- crease in physical activity [10, 17]. adulthood and obesity. Studies by Scharoun-Lee and col- leagues found that for women, being socio-economically disadvantaged throughout the transition to adulthood and foregoing post-secondary education increases the risk of obesity whereas this applies less for men [41–43]. Over the last decades, the transition to adulthood has become destandardized and diversified [46], meaning that there is no longer one typical way in which youths become adults, but rather there are diverse pathways marking the transition to adulthood. Marriage rates have decreased and cohabitation rates have increased [28]. Furthermore, the transition to adulthood has been pro- tracted since the second half of the twentieth century: union formation (be it marriage or cohabitation) and parenthood have been delayed, and youth have pro- longer their time spent in full-time education [19]. Given these important changes in the transition to adulthood, it is important to understand which of career and family pathways are nowadays associated with developing obes- ity in young adulthood. However, these studies do not fully take into account the ordering and timing of both career- and family- related events in the transition to adulthood. Events such as marriage and entering postsecondary education, ob- tain a specific meaning once the whole pathway of the transition to adulthood is taken into account [2, 6, 16]. While other studies link family and career sequences to health outcomes [11, 40], the present study is the first to link the transition to adulthood as a sequence of events to the development of obesity in young adulthood. Se- quences contain information on quantum (which events occur and how many times), ordering (what is the se- quencing of events), and timing (when events take place) of events [9]. This approach can provide more insight into what specific life-courses are linked to the risk of becoming obese. An important aspect of the transition to adulthood is the adoption of adult roles and responsibilities. The life- course approach acknowledges that individuals do not only move from one role to another; they can also adopt multiple roles at the same time in the career and family domains [17]. The interplay between career and family roles may have an impact on obesity, as the adoption of multiple roles may give rise to a conflict between career and family. Youth who experience family events before the completion of college are more likely to become obese [31]. Work-family conflict is related to more high- fat and high-sugar food consumption and less physical activity and therefore increase in BMI [5, 27, 39, 51]. For men and women both the meaning of career and family roles, and the strategies through which conflicts are managed are different [45]. We therefore can expect that the effect of the transition to adulthood on developing obesity differs by gender. Indeed, there is evidence for a different impact of life-course events and work-family conflict on BMI, diet and physical activity for men and women [5, 24, 39]. In this study, we focus in detail on the influence of life-course sequences in both career and family domains between ages 17 and 27. In order to deal with career and family sequences simultaneously, we use multichannel sequence analysis [20, 37], which enables us to obtain a measure of similarity between individuals’ career and family sequences. On the basis of these similarity mea- sures, individuals’ career-family sequences are grouped into clusters. In the final step we examine whether membership of a certain career-family sequence cluster is related to a higher or lower probability of developing obesity in young adulthood, with a specific focus on gen- der. Our research objective is to assess the influence of career-family trajectories on the risk of becoming obese towards the end of young adulthood and whether career-family trajectories are differently related to obes- ity for men and women. Our main research question is therefore: to what extent are career-family pathways dur- ing the transition to adulthood related to becoming obese for men and women? Research linking the transition to adulthood with the development of obesity is still limited. While some re- search focuses on single transitions such as college en- rollment [26, 33] and marriage [7, 49, 53], few studies examine the influence of multiple characteristics of the transition to adulthood on BMI. 2The NLSY97 reports weekly job status. We recoded this to monthly statuses using the conversion recommended by the NLS. If someone is employed for at least one week during that period, this person is considered employed. 1More information on the sampling design can be found on: https:// www.nlsinfo.org/content/cohorts/nlsy97/intro-to-the-sample/sample- design-screening-process Methods Data This study uses data from the National Longitudinal Survey of Youth from 1997 (hereafter referred to as NLSY97), a panel study conducted by the U.S. Bureau of Labor Statistics. Respondents were selected in 1997 at ages 12 to 17 (born 1980–1984), using a multi-stage area (housing units) stratified random sampling design,1 and were interviewed annually until 2013 (with the exception of 2012). The NLSY97 contains an oversample of re- spondents of Afro-American and Latino descent. When weighted, the NLSY97 provides a nationally representa- tive sample. The total sample consists of 8984 respon- dents. However, we only included those respondents who participated in all waves and for whom there is at least some information on body height and weight at (around) age 28. Most respondents are excluded, be- cause they do not provide full information on the timing of key events in the transition to adulthood, while only few respondents are excluded because of a missing or in- valid height and weight. In all, our analysis is based on N = 4688 cases (47% men, 53% women). We use sample weights designed specifically for the group of respon- dents that participated in all waves. Table 1 shows the proportions of all the categories of the family background variables in the sample and the percentage of obesity within these categories. Family background and control variables y g The first NLSY97 wave contains a “Parent Questionnaire”, from which we derived family background characteristics, such as parental income, education, and family structure. Parental education was coded as the highest education of the mother or father using five categories: lower than high school, high school, some college, 4-year college or higher, and missing. Parental income refers to the household in- come reported by one of the parents when the respondent was 12 to 16 years old and was coded in quartiles, also in- cluding a missing category. Family structure is the recorded family structure in 1997 and was coded in four categories: 1) Both biological parents, 2) 1 biological, 1 step-parent, 3) 1 biological parent, 4) other (no biological parents). For the main respondent, the gender variable (Female) was coded 0 for males and 1 for females, and Race was coded in four categories: 1) white (non-Hispanic), 2) black (non-His- panic), 3) Hispanic, 4) other (mixed). Finally, two controls were included. First, we control for obesity at the end of adolescence, so that we can examine how career-family se- quences during the transition to adulthood affect the prob- ability of becoming obese, rather than possibly viceversa obesity affecting career-family trajectories. We therefore in- cluded the variable Obesity age 17 as a dichotomous vari- able (0 = not obese, 1 = obese). We defined obesity at age 17 at a cut-off point of 28 rather than 30, as previous re- search has shown that a somewhat lower cut-off point more accurately captures obesity at younger ages [38]. Sec- ond, pregnant indicates whether the respondent was preg- nant (1) or not (0) at age 28. Macmillan and Fursten- berg [29] found that employed, married young adults with a 4-year college degree, having become parents after the transition to adulthood show a lower BMI in- crease than unemployed young adults with no college degree, who have not entered unions or parenthood. Scharoun-Lee et al. [41, 42] found that young adults who become residentially independent and enter the labor market and marriage early have an increased risk of obesity. There is also limited evidence for gender dif- ferences in the relationship between the transition to In assessing differences in the development obesity in young adulthood, also racial and family background dif- ferences may play an important role. Black and Hispanic youths are found to have a higher prevalence of obesity compared with whites [35]. Parental SES and family structure are also related to BMI for children from impoverished and broken families and lower-class households, who are more likely to develop obesity dur- ing their lifetimes [25, 41, 42, 44, 56, 58]. In the present study, we also take into account the influences of race, parental SES, and family structure, by examining whether these background factors continue to have an influence on becoming obese during young adulthood. Page 3 of 12 Mooyaart et al. BMC Public Health (2019) 19:1511 Mooyaart et al. BMC Public Health (2019) 19:1511 MacMillan and Furstenberg [29], all BMI scores below 12 or over 50 were considered invalid. MacMillan and Furstenberg [29], all BMI scores below 12 or over 50 were considered invalid. The advantages offered by protective factors may accu- mulate over the life-course, as in the “cumulative advan- tage” concept [14, 47, 55]. There is indeed evidence that cumulative advantage can also occur with respect to obesity risk [15, 41, 42]. Our research design allows us to test whether certain types of career-family sequences during the transition to adulthood increase the risk of becoming obese in early adulthood, and whether they have an effect independently and on top of disadvantage in childhood. Analytical strategy The NLSY97 contains measures of self-reported height in feet and inches and weight in pounds (lbs). BMI is calculated by (weight(lbs) × 703)/height2(inches). Our main dependent variable is a binary variable indicating whether or not the subject was obese at age 28, with this age chosen also because all respondents in the survey were at least 28 years old. If respondents did not report height and weight at age 28, their BMI at age 29 was used, and if this was also missing, their BMI at age 27 was used. In line with common practice [13], respon- dents were classified as obese when their BMI was 30 or higher. Furthermore, adopting the same approach as Multichannel analysis of career-family sequences In NLSY97, respondents reported the year and month in which specific life-course events occurred. In terms of education, in each wave they were asked whether they had entered or exited an educational institution in the previous year. Respondents were also asked to report the level of education in which they enrolled, i.e., secondary school, 2-year college, or 4-year college (including post- graduates). Regarding employment, respondents were asked to provide the start and end dates of each job they had in the previous year, including the number of Mooyaart et al. BMC Public Health (2019) 19:1511 Page 4 of 12 Page 4 of 12 Table 1 Descriptive statistics on family background variables (N = 4688) Proportion in sample (%) Obesity at Age 17 (%) Obesity at Age 28 (%) Gender Male 47.11 15.58 31.26 Female 52.89 14.12 34.27 Parental income Quartile 1 18.79 19.93 40.54 Quartile 2 18.91 17.55 35.66 Quartile 3 19.23 15.04 34.51 Quartile 4 19.77 8.29 23.90 Missing 23.30 13.79 30.59 Parental education Less than high school 15.32 20.00 40.42 High school diploma 31.06 16.10 36.03 Some college 23.79 16.19 33.36 4 year college or more 25.45 9.03 23.58 Missing 4.38 13.59 34.95 Family structure Both biological parents 52.15 12.28 30.27 1 biological, 1 step parent 12.26 14.06 30.21 Single parent 30.53 18.75 37.56 Other 5.06 18.91 37.39 Race White 52.49 11.19 27.20 Black 26.40 19.34 41.34 Hispanic 20.11 17.88 36.08 Other 1.00 23.40 40.43 create a sequence dataset it is necessary to define the ‘state space’, consisting of the different states individuals can occupy at each time-point. The career states cover educational enrollment and employment status. Analytical strategy Respon- dents are classified as being enrolled in high school, in a 2-year college education, a 4-year college education, or not enrolled. Where there are gaps between educational episodes, we consider someone as continuously enrolled if those gaps are shorter than 3 months. Regarding em- ployment, individuals are classified as employed 35 h per week or more, employed for less than 35 h per week, or not employed (the last category includes people who are not actively seeking employment, for instance stay-at- home mothers). Combining these educational and em- ployment statuses leads to 12 (4 × 3) possible different career states. Family states are defined in terms of living arrange- ments and parenthood status. Four living arrangements are distinguished: living with parents, living alone/inde- pendent, living with partner (cohabiting), and living with spouse (marriage). Within each of these options the re- spondent can either have had a child or not. Entering parenthood is considered irreversible. Once respondents have become parents, they are classified as parents for the rest of the sequence, independently of whether they co-reside with the child. This leads to 8 (4 × 2) possible family states. y Multichannel sequence analysis has been developed to compare life-course sequences on multiple dimensions [20, 37], such as career and family. In multichannel se- quence analysis, sequences are compared on both di- mensions simultaneously. The pathways of two different individuals are similar if the timing, occurrence, order- ing, and duration in states are similar to each other in both the career and family sequences. In order to de- velop a series of ideal-typical pathways in the transition to adulthood, we start from a dissimilarity, or distance, matrix and use cluster analysis. We use Optimal Match- ing Analysis to measure the level of dissimilarity of se- quences [1]. The measure is based on how many states would have to be substituted, deleted, or inserted in order to transform one sequence into another. The more of these operations are required, the less similar the se- quences are. However, some life-course transitions may occur more often than others. Therefore, in line with the literature we assign costs of substitutions based on the transition rates between different states [52]. When the transition rate from one state to another is low, the sub- stitution costs for these states is high, leading therefore to a larger distance between sequences. 2The NLSY97 reports weekly job status. We recoded this to monthly statuses using the conversion recommended by the NLS. If someone is employed for at least one week during that period, this person is considered employed. 3These questions were included from 2003 onwards, but in 2003 respondents also indicated the month and year of home return if this occurred in any of the previous years. 3These questions were included from 2003 onwards, but in 2003 respondents also indicated the month and year of home return if this occurred in any of the previous years. Analytical strategy working hours.2 With respect to family formation char- acteristics, respondents were asked whether they had started or ended a marriage or cohabiting relationship in the previous year, as well as the year and month of birth of each of their children. In each wave, respondents re- ported who was living in their household at that time. Furthermore, respondents were asked the month and year in which they first left and returned to the parental home (if they had done this).3 working hours.2 With respect to family formation char- acteristics, respondents were asked whether they had started or ended a marriage or cohabiting relationship in the previous year, as well as the year and month of birth of each of their children. In each wave, respondents re- ported who was living in their household at that time. Furthermore, respondents were asked the month and year in which they first left and returned to the parental home (if they had done this).3 We use NLSY97 information to construct a sequence- type life-course dataset, creating, for each individual, a sequence of 96 consecutive months between ages 17 and 27, along two dimensions: career and family. In order to Multichannel sequence analysis is performed using the TraMineR package in R. Based on the distance matrix resulting from the multichannel Optimal Matching pro- cedure, a weighted (using NLSY97 weights) hierarchical Mooyaart et al. BMC Public Health (2019) 19:1511 Page 5 of 12 Page 5 of 12 home (P), and lastly whether the majority of individuals has a child (CH). In the first cluster, the majority of young adults spend most of their time in the parental home. Re- garding career pathways, respondents in this cluster spend little time in college and most end up in full-time employ- ment, followed by part-time employment, and then in- activity. We therefore label this cluster UE-P. In the second cluster, the vast majority cohabit and have a child. Almost no one in this cluster attends college and employ- ment is relatively unstable, giving this cluster the UE-UC- CH code. The third cluster we label CO-E-M. Almost all respondents in this cluster are married, but relatively few have had children. Most spend time in either 2- or 4-year college education. The vast majority have stable full-time employment. Analyzing precursors of obesity We use binary logistic regression to identify the effects of career-family sequences on the risk of obesity at age 28. In addition to the family background and control variables, dummy variables for the set of career-family sequence clusters during the transition to adulthood are included, indicating whether someone is a member of a particular career-family cluster. The career-family cluster variables are interacted with gender in order to examine differences in the influence of each career-family type between men and women. Weights constructed by the NLSY were used to counter any potential selectivity of the sample. Analytical strategy The fourth cluster includes respondents who (previously) entered cohabitation or marriage, but by age 27 the majority have had a child and are not in a co- habiting relationship. Of all the clusters, respondents in this one spend most of their time in inactivity and least in employment and hardly anyone attends college. There- fore, we label this cluster UE-S-CH. In the fifth cluster, re- spondents marry and have children in quick succession. Most people in this cluster are in employment, either full- time or part-time at age 27, but there is also quite some time spent in inactivity, and few enter college, hence the label UE-M-CH. Entering cohabitation but not having children is the most salient feature of the sixth cluster. Most remain in cohabitation although some marry or be- come single again. Most enter college and have full-time employment when they reach 27. The label for this cluster is CO-E-UC. In the seventh cluster, almost all attend a 4- year college education. At age 27 most have finished their college education and have entered full-time employment. Regarding the family pathways of this group, most have left the parental home but experienced no other events, hence the label CO-E-S. In the final cluster, respondents spend very little to no time in college education. Most are full-time employed at age 27, but there is also time spent in part-time work and inactivity. They leave the parental home, but do not enter a union or have a child, thus the label for this cluster is UE-S. clustering procedure using Ward’s method was chosen to produce clusters of respondents with similar life se- quences. An advantage of the Ward algorithm is that it produces fairly equal-sized groups [3]. The choice of the optimal number of clusters is based on the best model fit in terms of the Akaike Information Criteria (AIC) [4]. We conduct multiple logistic regres- sions, in which each logistic regression differs in the number career-family clusters (based on the different cluster solutions) that are included as dummy variables, in order to test which set of career-family pathway vari- ables most adequately predict obesity at age 28. Table 2 shows that the 8-cluster solution provides the lowest AIC and therefore the best model fit, thus we opt for the 8-cluster solution. . Descriptive results on the transition to adulthood p In Fig. 1 we describe the eight career and family clusters. Some clusters have a similar career sequence, but differ in their family sequence and vice versa. To label the clusters we use a coding system that highlights whether most indi- viduals in the cluster attend college (CO), are continu- ously employed (E) or have more unstable employment (UE). For what concerns family behavior, our labels use the main relationship/residential status: married (M), un- married cohabitation (UC), single living (S) or in parental Table 3 shows the composition of the eight career- family clusters in terms of obesity at ages 17 and 28, gender, and pregnancy status at age 28. The UE-P clus- ter has the highest percentage of obesity at age 17 (20.56%), and the CO-E-M the lowest with 7.43%. At age 28, the cluster with the highest percentage of obesity is US-S-CH (38.22%), with the cluster CO-E-S having the lowest (21.25%). Pregnancy at age 28 is most prevalent in the CO-E-M cluster (18.47%), and least prevalent in the UE-S cluster (4.47%). Table 2 Model fit (AIC) of logistic regression for different number career-family clusters Number of clusters AIC 4 4887.19 5 4880.47 6 4883.93 7 4878.49 8 4876.91 9 4877.81 10 4881.52 Table 4 displays the distribution of career-family path- ways cluster membership for the different family back- ground variables. The respondents in the CO-E-S and Mooyaart et al. BMC Public Health (2019) 19:1511 Page 6 of 12 Fig. 1 Distribution of states for each of the multichannel career-family sequence clusters Mooyaart et al. The career-family clusters are coded using a the following scheme: CO college education, E (stable) employed, UE unemployed or u married, UC unmarried cohabitation, S single, P = living in the parental home, CH = having (had) a child(ren) are coded using a the following scheme: CO college education, E (stable) employed, UE unemployed or unstable employment, M The career-family clusters are coded using a the following scheme: CO college education, E (stable) employed, UE unemployed or unstable employment, M married UC unmarried cohabitation S single P = living in the parental home CH = having (had) a child(ren) g g g p y p y p y habitation, S single, P = living in the parental home, CH = having (had) a child(ren) 4Pregnant women at age 28 were excluded Descriptive results on the transition to adulthood However, even though there are dif- ferences between the clusters in terms of family back- ground, all clusters have a minimum of 5% per category (with the exception of “missing” or “other” categories, which have lower percentages among all different career- family pathway clusters), so that there is representation of all family backgrounds in each of the clusters. CO-E-UC clusters appear to be generally from more advantaged background as they predominantly white, have on average higher educated parent, grew up in relatively richer households, with a higher share of two-parent fam- ilies. Respondents in the UE-S-CH and UE-UC-CH clus- ters appear to be on average from a more disadvantaged background, with a higher share of racial minorities, with less affluent and educated parents, and a lower share of two-parent families. However, even though there are dif- ferences between the clusters in terms of family back- ground, all clusters have a minimum of 5% per category (with the exception of “missing” or “other” categories, which have lower percentages among all different career- family pathway clusters), so that there is representation of all family backgrounds in each of the clusters. Descriptive results on the transition to adulthood BMC Public Health (2019) 19:1511 Page 8 of 12 Page 8 of 12 Table 5 Log-odds estimates (and SE) from a logistic regressio model with obesity risk at age 28 as the dependent variable Coefficient Standard e Constant −1.020*** 0.191 Obesity age 17 2.798*** 0.126 Female 0.367* 0.181 Pregnant at 28 0.373** 0.135 Parental income Quartile 1 ref. Quartile 2 −0.105 0.130 Quartile 3 0.053 0.138 Quartile 4 −0.269 0.151 Missing −0.233 0.129 Parental education Less than high school ref. High school diploma −0.091 0.132 Some college −0.275 0.143 4 year college or more −0.436** 0.152 Missing 0.028 0.224 Family structure Both biological parents ref. 1 biological, 1 step-parent −0.126 0.131 Single parent 0.025 0.101 Other −0.237 0.204 Race White ref. Black 0.367*** 0.103 Hispanic 0.039 0.112 Other 0.326 0.333 Career-family clusters UE-P ref. UE-UC-CH −0.085 0.237 CO-E-M 0.470* 0.214 UE-S-CH −0.198 0.208 UE-M-CH 0.295 0.190 CO-E-UC −0.180 0.236 CO-E-S −0.176 0.177 UE-S −0.035 0.214 Interactions Career-family clusters*female UE-P*female ref. UE-UC-CH*female −0.008 0.347 CO-E-M*female −0.659* 0.300 UE-S-CH*female −0.031 0.277 UE-M-CH*female −0.532* 0.268 CO-E-UC*female −0.297 0.326 CO-E-S*female −0.772** 0.276 Table 5 Log-odds estimates (and SE) from a logistic regression model with obesity risk at age 28 as the dependent variable (Continued) Coefficient Standard error UE-S *female −0.220 0.379 Observations 4688 Obesity is the dependent variable and defined in the model as a dichotomous variable indicating 0 = not obese 1 = obese at age 28, this coding applies also for the independent variable obesity at age 17 The career-family clusters are coded using a the following scheme: CO college education, E (stable) employed, UE unemployed or unstable employment, M married, UC unmarried cohabitation, S single, P = living in the parental home, CH = having (had) a child(ren) *p < 0.05, **p < 0.01, ***p < 0.001 CO-E-UC clusters appear to be generally from more advantaged background as they predominantly white, have on average higher educated parent, grew up in relatively richer households, with a higher share of two-parent fam- ilies. Respondents in the UE-S-CH and UE-UC-CH clus- ters appear to be on average from a more disadvantaged background, with a higher share of racial minorities, with less affluent and educated parents, and a lower share of two-parent families. Descriptive results on the transition to adulthood BMC Public Health (2019) 19:1511 Page 7 of 12 Table 3 Obesity, gender, pregnancy and career-family sequence cluster membership (%) UE-P UE-UC-CH CO-E-M UE-S-CH UE-M-CH CO-E-UC CO-E-S UE-S Obesity at 17 No 79.44 82.67 92.57 81.94 86.94 90.02 91.01 83.51 Yes 20.56 17.33 7.43 18.06 13.06 9.98 8.99 16.49 Obesity at 28 No 61.97 63.07 68.82 61.78 64.61 75.18 78.75 67.35 Yes 38.03 36.93 31.18 38.22 35.39 24.82 21.25 32.65 Gender Men 62.56 45.74 40.53 31.68 36.10 38.69 53.95 68.04 Women 37.44 54.26 59.47 68.32 63.90 61.31 46.05 31.96 Pregnant at 28 No 95.33 91.19 81.53 86.78 86.38 88.56 94.41 95.53 Yes 4.67 8.81 18.47 13.22 13.62 11.44 5.59 4.47 The career-family clusters are coded using a the following scheme: CO college education, E (stable) employed, UE unemployed or unstable employment, M married, UC unmarried cohabitation, S single, P = living in the parental home, CH = having (had) a child(ren) Table 4 Family background and career-family sequence cluster membership UE-P UE-UC-CH CO-E-M UE-S-CH UE-M-CH CO-E-UC CO-E-S UE-S Parental education < High school 18.37 21.31 8.39 25.39 19.66 7.79 4.22 8.59 High school 32.57 42.90 23.26 41.75 31.18 29.93 17.03 31.96 Some college 27.21 22.44 24.94 19.76 24.86 23.84 19.62 29.90 4-year col. 17.48 7.95 39.57 9.29 17.70 35.04 55.59 26.12 Missing 4.37 5.40 3.84 3.80 6.60 3.41 3.54 3.44 Parental income Quartile 1 19.46 26.42 7.91 33.12 20.22 11.68 8.86 16.15 Quartile 2 20.16 25.85 16.55 22.64 19.24 18.00 10.35 21.99 Quartile 3 18.57 19.03 24.70 12.57 21.63 23.84 18.80 19.93 Quartile 4 16.48 8.52 30.22 6.02 14.89 25.79 38.42 22.34 Missing 25.32 20.17 20.62 25.65 24.02 20.68 23.57 19.59 Race White 43.79 36.93 74.82 25.52 57.3 73.72 68.80 57.39 Black 29.89 32.10 8.15 56.81 13.76 10.71 20.16 22.68 Hispanic 25.32 29.55 15.83 16.88 28.23 14.84 9.95 18.21 Other 0.99 1.42 1.20 0.79 0.70 0.73 1.09 1.72 Family structure Both parents 56.21 38.35 66.43 28.53 54.35 59.37 66.21 45.70 1 bio 1 step 9.33 17.05 12.71 13.74 13.62 14.84 9.13 12.71 Single parent 30.09 36.93 17.75 48.43 26.83 24.33 21.80 35.05 Other 4.37 7.67 3.12 9.29 5.20 1.46 2.86 6.53 The career-family clusters are coded using a the following scheme: CO college education, E (stable) employed, UE unemployed or unstable employment, M married, UC unmarried cohabitation, S single, P = living in the parental home, CH = having (had) a child(ren) Mooyaart et al. Logistic regression results Results of a binary logistic regression model, with obes- ity risk at age 28 as the dependent variable, are pre- sented in Table 5. The analyses in Table 5 include the interaction between gender, and the career-family path- way cluster dummies (results without the interaction are available upon request). Noticeable is the strong effect of obesity at age 17. Respondents who were obese at age 17 are more than 16 times more likely to be obese at age 28 compared with those who were not obese at age 17. Two significant family background effects are observed. First, young adults who have one or more university ed- ucated parents have a lower risk of being obese at age 28 compared to those whose parents do not have more than a high school education. Second, blacks have an in- creased probability of being obese at age 28 compared with whites. There are no significant effects for parental income and family structure. From Table 5 we learn that there are significant differ- ences between some career-family clusters, and that these differences are gendered. Because of the inter- action with gender, the coefficients under “career-family clusters” represent the effects for men. The reference Mooyaart et al. BMC Public Health (2019) 19:1511 Page 9 of 12 Fig. 2 Predicted probability of obesity for each career-family cluster, split by gender, for those who are not obese at age 17 Fig. 2 Predicted probability of obesity for each career-family cluster, split by gender, for those who are not obese at age 17 category for the career family clusters variable in Table 5 is UE-P, i.e. those with unstable employment while still living in the parental home. All coefficients in Table 5 therefore indicate the relative difference with respect to those in the UE-P cluster. Since not all relative differ- ences can be shown in the table, we ran the same ana- lysis with different reference categories in order to reveal all significant differences between each pair of career- family clusters (results available upon request). There is a clear positive effect for the CO-E-M cluster, i.e. those who went to college and are stably employed and mar- ried with no children, showing a higher risk of obesity at age 28 for this cluster compared with men in the UE-P, UE-UC-CH, UE-S-CH, CO-E-UC, and CO-E-S clusters. Men who are married and have children, i.e. Logistic regression results those in the UE-M-CH cluster, have a significantly higher risk of obesity compared with the UE-S-CH and CO-E-S clus- ters. All other differences between clusters for men are not statistically significant. those in the CO-E-UC cluster have a lower obesity risk at age 28 compared to those in the UE-P cluster. In order to facilitate the interpretation of results, in Fig. 2 we show the predicted probability of obesity at age 28 for those who were not obese at age 17, for each of the career-family clusters, split by gender.4 We report the predicted probability of obesity for respondents who were not obese at age 17, because we want to focus on which of the different career-family clusters are related to becoming, rather than to staying, obese. Figure 2 shows substantial gender variation within some of the clusters. Men who are in the CO-E-M cluster have the highest probability of becoming obese (30%). Among men, those following a UE-M-CH type of sequence have a 26% probability. The lowest probability, around 18%, is for men in the UE-S-CH, CO-E-S, and CO-E-UC clus- ters. Men in other clusters have around a 20% probabil- ity of becoming obese. For women, the ordering of career-family clusters in terms of highest to lowest probability of becoming obese is very different from that of men. Women in the UE-P cluster have a 28% probability of becoming obese and thereby have the highest risk among women. Next, the UE-UC-CH cluster has a 26% probability. At the lower end in terms of obesity risk are women in the CO-E-M cluster (19% probability), but the lowest obesity risk of all is found for women in the CO-E-S cluster (13% prob- ability). Women in the other career-family clusters have around a 23–24% probability of becoming obese. The interaction terms show how the cluster effects of women differ from those of men. The negative and sig- nificant effects for CO-E-M and UE-M-CH are similar in size to the positive main effect (for men), meaning that for women, being in these clusters is not related to a higher probability of obesity at age 28 with respect to the reference category (UE-P). The interaction with the CO-E-S cluster also shows a negative effect. However, because the effect for men was already negative, this in- dicates that for women there is a strong negative effect of being in the CO-E-S cluster. Logistic regression results Thus, particularly women who attend college, have stable employment afterwards and remain single have a lower risk of be- coming obese. In fact, women in this cluster have a lower risk of becoming obese than all other groups of women. The only other statistically significant difference between career-family clusters among women is that Discussion In line with previous studies, we found that obesity in adolescence is strongly related to obesity in adulthood [22, 34, 48]. While generally those who attend college and postpone childbearing have lower obesity risk, an important finding of this study is that career-family Mooyaart et al. BMC Public Health (2019) 19:1511 Page 10 of 12 Page 10 of 12 pathways during the transition to adulthood have differ- ent associations with the risks of becoming obese for men and for women. and marriage they exert a positive influence on the diet of their partner, more so than men do on their female partners. Women who typically attend 4-year college education, leave the parental home in their early 20s, but postpone union formation and parenthood, have a much lower risk of becoming obese at age 28 compared to women following other career-family pathways. However, this is not merely because of the postponing effect of education on family formation. Women who postpone family for- mation and forego any postsecondary education, have a significantly higher risk of developing obesity than their peers who follow the same family pathways but do at- tend college. Generally, women who experienced early childbearing had a higher risk of becoming obese, com- pared to those in clusters in which no childbearing took place before age 28. This is in line with the idea that work-family conflict can increase BMI [5, 51]. Yet, somewhat surprisingly, women who stayed in the paren- tal home had the highest risk of developing obesity. It may be that this group of women share particular fea- tures that remain unobserved in our analyses. A poten- tial reason could be that staying in the parental home relatively long compared with their peers increases their level of stress. Women who make off-time delayed tran- sitions report higher levels of stress compared to those going through transition such as leaving the parental home and entering marriage at more normative ages [8]. Stress has been linked to obesity, as it can lead to an un- healthy diet [50]. In addition to the impact of career-family pathways dur- ing the transition to adulthood, we find some family back- ground effects. We find a decreased risk of becoming obese for those with at least one parent with a 4-year col- lege degree or more compared with those whose parents have no more than a high school degree. Discussion This suggests that there is cumulative advantage on the basis of educa- tion, as the advantage of a decreased risk of developing obesity by following a “4-year college” sequence and hav- ing highly educated parents stack up. Furthermore, we find that blacks compared with whites have a higher risk of becoming obese in young adulthood. The reason we do not find other effects of family background could be that these effects are mediated through the career-family se- quences in the transition to adulthood and obesity at adolescence. This study has some limitations. First, BMI was calcu- lated based on self-reported height and weight. There is evidence indicating a small bias in these self-reports be- cause height tends to be over-reported and weight over- estimated by men, while underestimated by women [30]. Second, this study has shown that career-family se- quences in the transition to adulthood are related to the risk of becoming obese, but it has not revealed the exact mechanisms by which these pathways impact the risk of obesity. Future research should therefore examine more specifically the mechanisms, for instance through change in diet and physical activity, by which life-course transi- tions and role combinations and obesity are related. For men, the picture is quite different. Early marriage seems to be the defining characteristic of increased obes- ity risk. Surprisingly, men who marry early but do not have a child appear to have the highest risk of develop- ing obesity. A possible explanation for the increase in BMI after marriage is that those who are still in the ‘marriage market’ may be more keen to maintain a healthy body weight in order to attract a potential mar- riage partner [7, 54]. However, one would expect than expect that those marrying early and having one or more children would also have the same obesity risk, or per- haps higher given the higher family burden, but this is not corroborated by our analyses. Furthermore, results show that those who marry and have children early most often do not enter college, whereas men who marry early but do not have children (yet) often do attend college. Thus, it appears that college education does not buffer the risk of becoming obese among men that marry early. However, one would expect this equally strong for both genders which appears not to be the case. Discussion Perhaps, women who are married with children improve their diet compared with women who are married without children. Lake et al. [24] found that women often change their diet after childbirth and that within cohabitation Conclusion This study has shown that different career-family path- ways are related to different risks for developing obesity during young adulthood. Furthermore, results also show that there is a clear gender component in this relation- ship. For women, a combination of college education and the postponement of family formation clearly buffer elevated obesity risks. Women who have a family to take care of, next to having a job in their early 20s, have a higher risk of developing obesity. Helping women deal with work-family conflict, through better family-oriented work policies may therefore lower their risk of becoming obese. However, women who stay relatively long in the parental home and do not attend postsecondary educa- tion have the highest risk of becoming obese, which may be related to the stress of having the feeling of “lagging behind” in terms of the transition to adulthood with re- spect to their peers. Still, more research is needed to understand why this group in particular have a higher risk of becoming obese. For men, attending college Mooyaart et al. BMC Public Health (2019) 19:1511 Page 11 of 12 Page 11 of 12 Received: 19 April 2019 Accepted: 16 October 2019 Abbreviations AIC Ak ik I f 8. Bell S, Lee C. Transitions in emerging adulthood and stress among young Australian women. Int J Behav Med. 2008. https://doi.org/10.1080/ 10705500802365482. 8. Bell S, Lee C. Transitions in emerging adulthood and stress among young Australian women. Int J Behav Med. 2008. https://doi.org/10.1080/ 10705500802365482. AIC: Akaike Information Criteria; BMI: Body Mass Index; NLSY97: National Longitudinal Survey of Youth 1997 9. Billari FC. Life course analysis: two (complementary) cultures? Some reflections with examples from the analysis of the transition to adulthood. Adv Life Course Res. 2005. https://doi.org/10.1016/S1040-2608(05)10010-0. 9. Billari FC. Life course analysis: two (complementary) cultures? Some reflections with examples from the analysis of the transition to adulthood. Adv Life Course Res. 2005. https://doi.org/10.1016/S1040-2608(05)10010-0. Funding Th 12. Clarke P, O’Malley PM, Johnston LD, Schulenberg JE. Social disparities in BMI trajectories across adulthood by gender, race/ ethnicity and lifetime socio- economic position: 1986-2004. Int J Epidemiol. 2009;38(2):499–509. https:// doi.org/10.1093/ije/dyn214. This research has received monetary support of a European Research Grant. The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007–2013) / ERC Grant Agreement n. 324178 (Project: Contexts of Opportunity. PI: Aart C. Liefbroer). The funding body (ERC) had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. 12. Clarke P, O’Malley PM, Johnston LD, Schulenberg JE. Social disparities in BMI trajectories across adulthood by gender, race/ ethnicity and lifetime socio- economic position: 1986-2004. Int J Epidemiol. 2009;38(2):499–509. https:// doi.org/10.1093/ije/dyn214. 13. Cole TJ, Lobstein T. Extended international (IOTF) body mass index cut-offs for thinness, overweight and obesity. Pediatric Obesity. 2012. https://doi. org/10.1111/j.2047-6310.2012.00064.x. 13. Cole TJ, Lobstein T. Extended international (IOTF) body mass index cut-offs for thinness, overweight and obesity. Pediatric Obesity. 2012. https://doi. org/10.1111/j.2047-6310.2012.00064.x. 14. Dannefer D. Cumulative advantage/disadvantage and the life course: cross-fertilizing age and social science theory. J Gerontol B Psychol Sci Soc Sci. 2003;58(6):S327–37. https://doi.org/10.1093/geronb/58.6.S327. Received: 19 April 2019 Accepted: 16 October 2019 education lowers the risk to become obese, but not when college education is combined with early marriage. Our results provide two general policy implications in battling obesity in young adulthood. First, not only sin- gle factors or events in the transition to adulthood mat- ter. It is fundamental to consider the obesity-related risks of combinations of events and states over the life- course which are related to becoming obese in young adulthood, also through a gendered lens. Second, policy makers should be aware that helping young adults change their life-course pathways can be beneficial in re- ducing obesity in young adulthood, with for instance helping individuals who struggle in the transition to adulthood to leave the parental home, get a full-time job or attend education. This life-course perspective may not only be helpful in informing policy on how to reduce obesity, but can also be useful in reducing other health risks over the life-course. References bb 1. Abbott A, Tsay A. Sequence analysis and optimal matching methods in sociology: review and Prospect. Sociol Methods Res. 2000;29(1):3–33. https://doi.org/10.1177/0049124100029001001. 2. Aisenbrey S, Fasang A. The interplay of work and family trajectories over the life course: Germany and the United States in comparison. Am J Sociol. 2017;122(5):1448–84. https://doi.org/10.1086/691128. 2. Aisenbrey S, Fasang A. The interplay of work and family trajectories over the life course: Germany and the United States in comparison. Am J Sociol. 2017;122(5):1448–84. https://doi.org/10.1086/691128. p g 3. Aisenbrey S, Fasang AF. New life for old ideas: The ‘second wave’ of sequence analysis bringing the ‘course’ Back into the life course. Sociol Methods Res. 2010;38(3):420–62. https://doi.org/10.1177/0049124109357532. 4. Akaike H. Likelihood of a model and information criteria. J Econ. 1981;16(1): 3–14. https://doi.org/10.1016/0304-4076(81)90071-3. p g 3. Aisenbrey S, Fasang AF. New life for old ideas: The ‘second wave’ of sequence analysis bringing the ‘course’ Back into the life course. Sociol Methods Res. 2010;38(3):420–62. https://doi.org/10.1177/0049124109357532. 4. Akaike H. Likelihood of a model and information criteria. J Econ. 1981;16(1): 3–14. https://doi.org/10.1016/0304-4076(81)90071-3. 5. Allen TD, Armstrong J. Further examination of the link between work-family conflict and physical health: The role of health-related behaviors. Am Behav Sci. 2006. https://doi.org/10.1177/0002764206286386. 6. Amato PR, Landale NS, Havasevich-Brooks TC, Booth A, Eggebeen DJ, Schoen R, McHale SM. Precursors of young Women’s family formation pathways. J Marriage Fam. 2008;70(5):1271–86. https://doi.org/10.1111/j.1741-3737.2008.00565.x. 7. Averett SL, Sikora A, Argys LM. For better or worse: relationship status and body mass index. Econ Hum Biol. 2008;6(3):330–49. https://doi.org/10.1016/j. ehb.2008.07.003. 7. Averett SL, Sikora A, Argys LM. For better or worse: relationship status and body mass index. Econ Hum Biol. 2008;6(3):330–49. https://doi.org/10.1016/j. ehb.2008.07.003. Consent for publication Not applicable. 19. Furstenberg FF. On a new schedule : adulthood and transitions to family change. Futur Child. 2010;20(1):67–87. https://doi.org/10.1353/foc.0.0038. 19. Furstenberg FF. On a new schedule : adulthood and transitions to family change. Futur Child. 2010;20(1):67–87. https://doi.org/10.1353/foc.0.0038. Competing interests The authors declare that they have no competing interests. 20. Gauthier J-A, Widmer ED, Bucher P, Notredame C. Multichannel sequence analysis applied to social science data. Sociol Methodol. 2010;40(1):1–38. https://doi.org/10.1111/j.1467-9531.2010.01227.x. Authors’ contributions Thi i i k f This manuscript is work from JM’s PhD dissertation. AL and FB are his supervisors. JM restructured the data to prepare for the analysis. The paper was written by JM and co-written by AL and FB. JM designed and ran the analyses and AL and FB co-designed the analyses and provided corrections for the analyses. All authors read and approved the manuscript. 10. Brown WJ, Trost SG. Life transitions and changing physical activity patterns in young women. American Journal of Preventive Medicine. 2003;25(2):140– 3. 10. Brown WJ, Trost SG. Life transitions and changing physical activity patterns in young women. American Journal of Preventive Medicine. 2003;25(2):140– 3. 11. Carmichael F, Ercolani MG. Unpaid caregiving and paid work over life-courses: different pathways, diverging outcomes. Soc Sci Med. 2016;156:1–11. https:// doi.org/10.1016/j.socscimed.2016.03.020. 11. Carmichael F, Ercolani MG. Unpaid caregiving and paid work over life-courses: different pathways, diverging outcomes. Soc Sci Med. 2016;156:1–11. https:// doi.org/10.1016/j.socscimed.2016.03.020. Ethics approval and consent to participate Ethics approval and consent to participate Not applicable as this research uses data from the NLSY97, an anonymized dataset. For more information go to https://www.nlsinfo.org/content/ cohorts/nlsy97. 17. Elder G. The life course as developmental. Child Dev. 1998. https://doi.org/ 10.1111/j.1467-8624.1998.tb06128. 17. Elder G. The life course as developmental. Child Dev. 1998. https://doi.org/ 10.1111/j.1467-8624.1998.tb06128. 18. Elstgeest LEM, Mishra GD, Dobson AJ. Transitions in Living Arrangements Are Associated with Changes in Dietary Patterns in Young Women. The Journal of Nutrition. 2012;142(8):1561–7. 18. Elstgeest LEM, Mishra GD, Dobson AJ. Transitions in Living Arrangements Are Associated with Changes in Dietary Patterns in Young Women. The Journal of Nutrition. 2012;142(8):1561–7. Availability of data and materials This research uses open access data. For more information go to https:// www.nlsinfo.org/content/cohorts/nlsy97 This research uses open access data. For more information go to https:// www.nlsinfo.org/content/cohorts/nlsy97 This research uses open access data. For more information go to https:// www.nlsinfo.org/content/cohorts/nlsy97 15. Dupre ME. Educational differences in health risks and illness over the life course: a test of cumulative disadvantage theory. Soc Sci Res. 2008;37(4): 1253–66. https://doi.org/10.1016/j.ssresearch.2008.05.007. g y The data can be downloaded from the following repository af h l f l The data can be downloaded from the following repository after making an account: https://www.nlsinfo.org/investigator/pages/login.jsp The data can be downloaded from the following repository after making an account: https://www.nlsinfo.org/investigator/pages/login.jsp 16. Elder GH. Time, human agency, and social change: perspectives on the life course. Soc Psychol Q. 1994;57(1):4–15. https://doi.org/10.2307/2786971. 16. Elder GH. Time, human agency, and social change: perspectives on the life course. Soc Psychol Q. 1994;57(1):4–15. https://doi.org/10.2307/2786971. Competing interests Competing interests The authors declare that they have no competing interests. Author details 1 Early childhood poverty, cumulative risk exposure, and body mass index trajectories through young adulthood. 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STUDIES IN COLLOIDAL ARSENIC TRISULPHIDE. PART II. AGEING OF THE SOL
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[Jour. ludiau Cbe111. Soc., Vol. 33, No. 1, 1956] [Jour. ludiau Cbe111. Soc., Vol. 33, No. 1, 1956] BY A. K. M; 'I'RIVKDI AND M. J. PATANI BY A. K. M; 'I'RIVKDI AND M. J. PATANI S•1nHght as well as ox,,·geli is necessary for the ageing of arsenic trisulphide sol. When arsenie trisulphide sol is allmnd to age, only arsenious acid and sulphuric acid are formed. The charge on the micelles decreases on ageing. Sulphuric acid, e•·en in tbe- mo~t faYo1uable circumstances, cau atc01111t for oulr a part of tl:te hydrogen sulphide, which is formed on hydrolysis of arsenic trisulpbide. Arsenic trisulphide sots are fairly stable when kept in the dark. 'I'hf' antllors have found a ·sample· of concentrat~d arsenic trisulphide sol, kept in the dark for 4 years, not to suffer any apparent change, exc-epting deposition of a few particles in the bottom. The sol, kept in SiiLnligh~ on the other hand undergoes a qukk change and ultimately coag.ulates. The effect of sunlight c.1onot be ascribed to the var:ation in temperature, as the sol can be boiled without a11~apparent significant change. The ageing of the sol in presence of light' has been studied by a number of workers {Young and Pingree,/. Ph:ys. Chem., r9 13, 17, 657; Freundlich and Nathansohn, Ko!loid l., 1921, 28, 258; Domanski, ibid., Igzs, 35, 98; Murphy and Mathews, J. A111-er. Clletn. Soc., 1923, 4!B, 16; Joshi, Barve and Desai, Curr. Sti., 1934, 3, 105}. The destabilising action of light anl the increa'Se in conductivity on ng~ing were ascribed to the formation of colloidai sulphur, stabiHsed by pnlytbionk acid. ,~{haria (K.o!Loid-ZI&ur, 1948, 10, 159), on thl! other hand, reports the absence of polythiouic acid (hence of colloidal sulphur also) and attributes the destabilisiog action of light and the increase in acidity on ageing to the formation of sulphuric acid. In the present investigatiQn, the absence of polythionic acid and the formation of sulphuric acid on ageing arsenic trisnlphide sol ha,re been confirmed by the study of the ageing of the sol, kept in the dark. lind also by keeping it in sunlight for different intervals o! tilne and in presence and absence of oxygen. A. K. M. TRIVEDI AND M. J. PATANI A. K. M. TRIVEDI AND M. J. PATANI Agei11g on Dilt1tio11 In this series of experiments, tl1e sJl was kept (i) in darkness, (ii) in sl1nlight for 70 hours and (iii) in sunlight for. 95 hours. The sols in three different dilutio11s w'ere placed in Pyrex test tubes and tightly c rked. As before, tbe concentrations of AsaOs and H,so~ were also determiued (Table ITI). TAnu: Ill Sol E. Expt. S~leoutent. Arsenious acid (t{I.e./litrel. eulphuric ac:id,lw.e./litre). I. II. lii. 11. III. IIJO.O~. ~0 5? 3·! 8.I " 5') 0 21 45 65 '·7 7·6 16.!';• 3 25-0 12 40 42 J,l 7·4 1<'-4 12-5 7 22 61 o.6 s-8 15·9"' 1-ln the dark for 40 days. H-ID sunlight for ;o hollis. III-In san light for 95 boars. "At the time of the aDalysis, the suls were ou the n·rge uf roagula.tion. Ageing in Sunlight on passing Hydrogen and Oxyge11 EXP£R1MEN'l'AI, Ageiug of the sol was followed by analysing ~olloid, arseuious acid and sulplnnic add contents of the sol as well as by determining its conductivity under different conditions. - '.fabl(£ I r.!cords the analysis of the different sols studied .. _ TABr.B I Sol No. Cot:o:d content. .1\ rseuious acid (m.e./litr~). Sulphur1c add (m.e./litrel. C• 130·,5 g./litre 40 J.I D fJ0·5 40 3·2 E uB.o 40 J.I - F 73·4 27 r.o 14 Ageing in Sunlight In tllese experiments about 300 ~ntl. of tlie sol was kept in a soo ml. conical flask and tigntly ~or ked. The sols were, exposed to direct sunlight for about 4 hours a day and for the remaining time they were exposed to day light. The sols werE!ket11; for a total o£ about Ioo hours in sunlight. The a1eing of the sol was followed by measuring at intervals the conductivity as well as the concentrations of As20a !llld H~so.. Two sets of experiments were performed (Table II). 'rime. chr. a 30 3 65 4 ss TABI,E l[ Sol C. Sp. co:1dy. A•11l3 cone.· 1I1S04 cone. (KXJo3). (m.l'.flitl'e). (m.e./litrc). 2-3 40 l·l 40 57 8.0 5·6 79 13-? 6.8 JOO f7-5 Rol )). 1'imc. Stl- candy. ,\~~oj eonc. H:P01 c:onc. (K x IO ). • ru.e./litre). (DJ.e./Htre). o h.r. 2.4 40 3-2 2.) 1-6 6t 9-I 70 4-Q 76 U-7 JO., 6.7 162 17-6 Agei11g on Dilt1tio11 Ageing in Sunlight on passing Hydrogen and Oxyge11 According to Krestins.kaya and Yakovleva (Kolioid Z., 1933, 68, x&7), oxygen i:l necessary for the ageing of the sol. This was tested as follows.. Oxyg~n from oxygen · cylinder or hydrogen from Kipp's, purified through KMnO~ solution, ,was bubbled through the sbl. kept iu a wash-bottle for five bburs in sunlight. No attempt was maile to M\$sure the. rate of paSGing the gases. 'I'he contents of arsenious and sulpl:mric acids are shown in Table IV. . Bl'UDIES lN COJ"'L01DAL ARSENIC TRI!:ItJLl?liiDl!l 15 RKpt. 2 3 1 Duration of passing thegns . .5 hrs 5 .5 .'i TABLE IV Sol F. Ox•'gea. _......., ___ _._ _ _....., ;\R,Of H~O, (in.e.Ilitre). im.e./JHre:. 127 17.2 119 14·9 121 IS-4 IZ7 ·~·7 D I 5 c us 5 I 0 ~ Hydrogel!.·. -------- As203 (ln.e./litre). 34 33 33 35 1.1 J.r l.I I.~ It ~ppears fr.:m Table JII -that when the liol is kept io darkness, practically no ag·ehtg takes place even 011 dilution, and wh~:n it is kept in sunlight, ageing is promoted to an extent depending directly upon the c~urotion of t xposure to sunlight. When it is diluled to different ,;,xtents, tl:e formation of both arsenious as well sulphuric acid is promoted ; the rate of forn~atiOJl of sulp\nuic acis also appears to be much greater than that of <1.rse11ious acid. 'Ch!ls, wlll·n the sot was diluted e1ght times and kept in sunli~.ht for seventy !JOurs, the formation of arsenious acid increased three times, while the formation of sulphuric acid increased nearly eight times. Hence, one may cotJclude that suulig·ht promo:e~ ageing, particularly the formation of ~nlphuric acid. Dilq,tion accelerates the effect of ageing. The cffCl't o( oxygen and hydrogen can be seen from the data in Table IV. In the absence of oxygen l-'ractically no ageiflg takes place. Oxygen promotes ageing. Here again sulphur;c acid is formed to a .much greater extent than arsenio.1s acid. "fABI.E v (/) Sol c. (ii} Sol D. Ti111e. ~pec:ific conductam e nr Time. Specific conJuctance of F.xpt. Sol · H,so~ :'\Ihe!lu. Sol. H2so, 1\licellf.s ([{ >< ro3). (K'x Io3). [ (K-1\'1 X ro3]. (I'> x •c'J. (K'x 1331. [ {r.'"-Ji:"'J >< 1c3]. 0 hr. 2.J I.J J.O o hr. . 2 4. I-4 r.o 2 30 4·"' 3·~ o.S 2<) 4·1j .;.6 t.o J 6s s.~> 4·9 0.7 ;o 4 9 4·5 "·4 4 Bs 6.11 6.4 04 JCO 6.;- 6.5 <'.:! Ageing in Sunlight on passing Hydrogen and Oxyge11 Table V shows the d1ta on the specific conductivities of the sol, of sulphuric acid and of that due to the micelh:!t"" (vide Part I, this issue, p. gl. The conductivity of arsenious acid is very low (Zowldzki, Be,· .• 1903, 86, 1429) nnd hence, it is not .taken into account. lt is opvious t.bat in both the sets of experiments, althc.ugh the specific COll- ducti\lity of the sol increases, .that due to the micelles d~creases. This shows that the reSidual conductivity is due ro the mict:lles only. The decre.ase of charge.ob ageing i~ a normal feature .-f tl1e c::.olloidal system (see for example, •Joshi, Barv!! ~nd De!ai, loc. cit,.). The impor!auce of the data lies pa1·ticularly in the fact that the sol on ie A. lL M. ,.TRIVl~D! AND M. J. PATANI a.gei1zg forms sulplun·ic acid" and arsenious acid and no otheT electrolyte. For, it any other electrolyte were formed, the residt1al couductivhy, reported in 'the last column, could not have decreased as it actually has done. The significance of this observat_ion fs with· regard to tbe calculations of tb:e equivalent conductivity due to the micelles (vide Part I, loc. dt.). a.gei1zg forms sulplun·ic acid" and arsenious acid and no otheT electrolyte. For, it any other electrolyte were formed, the residt1al couductivhy, reported in 'the last column, could not have decreased as it actually has done. The significance of this observat_ion fs with· regard to tbe calculations of tb:e equivalent conductivity due to the micelles (vide Part I, loc. dt.). ) We may now consider the relation betw~n the arsenious acid (a) and the sulphuric acid (b) formed on ageing under different conditions. TABI.R VI No. Rate a/b. Rema~ks. I 28 In p·esl!n<"e of h~·drogen 2 JI and Hunlight for five hours 3 29 (1'ablor lV). 4 ~9 -~ 13 (j 12 In rlark for ·1" day" 7 ·. Jl ['fable HI iii]. M p 9 7 Tn presen<'P. of o:orygen nn•l 10 8 •snnliF!ht for fh·e hours JI 8 (Table I-v). 12 7 lJ 13 14 7 In •mnl;ght Co~ diff,rent IS 6 perind11 (Table TI). 16 6 17 14 18 7 Do 19 7 .20 6 :n 7 Tn sunlight for 70 hours; 22 li sol$ dilut<."d to different 113 !i extents [1'able III (Ill]. Z4 'I 25 4 !16 4 In sanliJ:ht for 9.5 houri! 'n1 PUTNEST.OF ('ai!MHiTI<\01 I,.D, ARTS C'OL~JtGI! & M.G. Scn:KCF. JNS'I'I'nTTJl, !o M~II(J)AJII\n·9· Ageing in Sunlight on passing Hydrogen and Oxyge11 ; 27 4 sols dilated to rlifferent eKten ts ['!'able III (til) ] . TABI.R VI Tn sunlight for 70 hours; sol$ dilut<."d to different extents [1'able III (Ill]. In sanliJ:ht for 9.5 houri! ; sols dilated to rlifferent eKten ts ['!'able III (til) ] . Three moles of sulphuric acid per mole oE arst:nious aGid 11hould be formed, provided that all the hydrogen sulphide formed on hyl1rolysis of the !!ol is t:onverted into sulphu1ic acid (vide Part I, loc. ciU. The data in T<~ble VI show that e\'en iu the mo~t favourable circumstaQce~, formation of sulphuric acid can accouut kr only a part of hydrogen sulphide. The fate of the n•maining hydrogen sulphide aw;Jits further exredmental data, sin-;:e either free sulphur or colloidal sulphur, stabilised by p.olythioni: acid, DJlpears to be ab~ent. The ~uthors' thEmks arc d ~1c to the Ahm~clabarl Educa'l:i~n Soci~ty for a gcner•~ns grant townrqs cht>micals and app:n·atu~.
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Deep Functional Profiling Facilitates the Evaluation of the Antibacterial Potential of the Antibiotic Amicoumacin
Antibiotics
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Article Deep Functional Profiling Facilitates the Evaluation of the Antibacterial Potential of the Antibiotic Amicoumacin Stanislav S. Terekhov 1,2,* , Anton S. Nazarov 1,2 , Yuliana A. Mokrushina 1,2 , Margarita N. Baranova 1,2, Nadezhda A. Potapova 3, Maja V. Malakhova 4, Elena N. Ilina 4, Ivan V. Smirnov 1,2 and Alexander G. Gabibov 1,2,5,* Stanislav S. Terekhov 1,2,* , Anton S. Nazarov 1,2 , Yuliana A. Mokrushina 1,2 , Margarita N. Baranova 1,2, Nadezhda A. Potapova 3, Maja V. Malakhova 4, Elena N. Ilina 4, Ivan V. Smirnov 1,2 and Alexander G. Gabibov 1,2,5,* 1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow 117997, Russia; nazarov.ngu@gmail.com (A.S.N.); yuliana256@mail.ru (Y.A.M.); Baranova@ibch.ru (M.N.B.); smirnov@ibch.ru (I.V.S.) 1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow 117997, Russia; nazarov.ngu@gmail.com (A.S.N.); yuliana256@mail.ru (Y.A.M.); 2 Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia 3 Institute for Information Transmission Problems (Kharkevich Institute) of the Russian Academy of Sciences Moscow 127051, Russia; nadezhdalpotapova@gmail.com p p g 4 Federal Research and Clinical Centre of Physical-Chemical Medicine of Federal Medical Biologi Moscow 119435, Russia; maja_m@mail.ru (M.V.M.); ilinaen@gmail.com (E.N.I.) p p g Federal Research and Clinical Centre of Physical-Chemical Medicine of Federal Medical Biological Agency M 119435 R i j @ il (M VM ) ili @ il (E N I ) j ( ) g ( ) Department of Life Sciences, Higher School of Economics, Moscow 101000, Russia * Correspondence: sterekhoff@gmail.com (S.S.T.); gabibov@ibch.ru or gabibov@gmail.com (A.G.G.)   Academic Editor: Jorge H. Leitão Academic Editor: Jorge H. Leitão Academic Editor: Jorge H. Leitão Received: 12 March 2020; Accepted: 30 March 2020; Published: 2 April 2020   Received: 12 March 2020; Accepted: 30 March 2020; Published: 2 April 2020 Received: 12 March 2020; Accepted: 30 March 2020; Published: 2 April 2020 Abstract: The global spread of antibiotic resistance is forcing the scientific community to find new molecular strategies to counteract it. Deep functional profiling of microbiomes provides an alternative source for the discovery of novel antibiotic producers and probiotics. Recently, we implemented this ultrahigh-throughput screening approach for the isolation of Bacillus pumilus strains efficiently producing the ribosome-targeting antibiotic amicoumacin A (Ami). Proteomics and metabolomics revealed essential insight into the activation of Ami biosynthesis. Here, we applied omics to boost Ami biosynthesis, providing the optimized cultivation conditions for high-scale production of Ami. Article Deep Functional Profiling Facilitates the Evaluation of the Antibacterial Potential of the Antibiotic Amicoumacin Ami displayed a pronounced activity against Lactobacillales and Staphylococcaceae, including methicillin-resistant Staphylococcus aureus (MRSA) strains, which was determined using both classical and massive single-cell microfluidic assays. However, the practical application of Ami is limited by its high cytotoxicity and particularly low stability. The former is associated with its self-lactonization, serving as an improvised intermediate state of Ami hydrolysis. This intramolecular reaction decreases Ami half-life at physiological conditions to less than 2 h, which is unprecedented for a terminal amide. While we speculate that the instability of Ami is essential for Bacillus ecology, we believe that its stable analogs represent attractive lead compounds both for antibiotic discovery and for anticancer drug development. Keywords: deep functional profiling; ultrahigh-throughput screening; amicoumacin; antibiotic activity spectrum; amide stability toward hydrolysis; single-cell; multi-omics antibiotics antibiotics antibiotics www.mdpi.com/journal/antibiotics antibiotics antibiotics 1. Introduction While classical platforms for antibiotic discovery have been successfully applied, providing the majority of antibiotics on the market [3], a new paradigm of studying microbiome function at the single-cell level is required [4]. Recently, we proposed a concept of deep functional microbiome profiling based on single-cell cultivation of microorganisms encapsulated in isolated compartments of a double water-in-oil-in-water emulsion, combined with subsequent probing of their functionality [5]. The origin of Ami biosynthesis has been of interest for a long time [14]. However, Ami biosynthetic gene cluster was firstly identified in Bacillus subtilis not so long ago [18]. Recently, we described a related Ami cluster in B. pumilus, clarifying the mechanisms mediating Ami biosynthesis in Bacilli [5]. A multi-omics approach applied to discover the regulation of Ami production revealed an increased level of the putative AmiP transporter, activating the peptidase AmiB [18–20], and a reversed balance between the kinase AmiN and the phosphatase AmiO upon activation of Ami biosynthesis [5]. Finally, we showed that AmiN (EC 2.7.1.230) and AmiO (EC 3.1.3.107) are responsible for self-resistance and corresponding Ami activation. p g In this paper, we focused on the optimization of B. pumilus cultivation conditions for improved high-scale production of Ami based on the proteomics and metabolomics data obtained previously [5]. A combination of increased aeration, high-carbohydrate medium, and calcium carbonate microparticles mimicking the thin-layer cultivation previously described was efficiently applied to activate Ami biosynthesis and boost its production. The optimized cultivation conditions were implemented for high-scale preparation of Ami, simplifying the downstream purification based on reversed-phase solid-phase extraction (SPE) and HPLC. The purified Ami was used for the evaluation of its antibacterial potential using standard cultivation assays and AR-based deep functional profiling of microbiomes. Ami activity spectrum was studied previously [7,10,13,15], indicating potent activity of Ami against H. pylori, Vibrio, and S. aureus strains, including MRSA. We expand this data illustrating that Ami is active against numerous Gram-positive bacteria, including the overwhelming majority of pathogenic Enterococcus and Staphylococcus strains. However, we suggest that the application of Ami as an antibiotic or anticancer agent is problematic, since it has low stability in aqueous solutions at physiological pH. We observed the sequential transformation of Ami into AmiC and AmiB, which are inactive metabolites of Ami (lactone and carboxylic acid, respectively). The outstanding instability of the terminal amide of Ami originates from its intramolecular self-lactonization. 1. Introduction The global spread of antibiotic resistance (AR) is one of the most urgent problems faced by humanity, particularly relevant to the so-called ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). Globally, antibiotic-resistant infections cause 31.5 million sepsis cases resulting in 5.3 million deaths annually [1]. Hence, the World Health Assembly endorsed a global action plan to tackle AR [2]. www.mdpi.com/journal/antibiotics Antibiotics 2020, 9, 157; doi:10.3390/antibiotics9040157 2 of 12 Antibiotics 2020, 9, 157 The efficient counteraction of the AR problem demands the creation of new molecular tools with antibacterial activity. While classical platforms for antibiotic discovery have been successfully applied, providing the majority of antibiotics on the market [3], a new paradigm of studying microbiome function at the single-cell level is required [4]. Recently, we proposed a concept of deep functional microbiome profiling based on single-cell cultivation of microorganisms encapsulated in isolated compartments of a double water-in-oil-in-water emulsion, combined with subsequent probing of their functionality [5]. Previously, ultrahigh-throughput profiling of bacterial communities for anti-Staphylococcus activity enabled isolating particular clones with the highest level of antibiotic production [6]. The application of this technology to the screening of the exotic microbiome of the East Siberian brown bear (Ursus arctos collaris) resulted in the isolation of Bacillus pumilus strains efficiently producing the antibiotic amicoumacin A (Ami) [5]. While Ami was discovered in the 1980s [7–9], its mechanism of action and molecular target remained ambiguous for a long time [10] and were precisely determined just recently [11]. Ami efficiently targets both pro- and eukaryotic ribosomes [11,12], resulting in similar minimum inhibitory concentrations (MICs) and IC50 values for Ami-sensitive bacterial strains and human cell lines, respectively [12]. This cytotoxicity restricts its application as an antibiotic on the one hand and provides a perspective agent targeting mammalian translation on the other. However, Ami was reported as an effective anti-inflammatory and antiulcer agent [7], which was subsequently associated with its anti-Helicobacter pylori activity [13]. Numerous Bacillus strains produce Ami [14] and are applied as probiotics to enhance gastrointestinal health [13] or to prevent Vibrio infections in aquaculture [15,16]. Whereas the immunostimulatory effects of Bacillus resulting in an increase in IgA level were reported [17], the antibiotic activity of Ami is a more straightforward and documented reason for the efficacy of Ami-producing Bacillus probiotics [15,16]. The efficient counteraction of the AR problem demands the creation of new molecular tools with antibacterial activity. 2.1. High-Scale Ami Production and Purification 2.1. High-Scale Ami Production and Purification The deep functional microbiome profiling method reported recently [5] consists of encapsulating single microorganisms of exotic microbiomes in isolated biocompatible microcompartments, estimating their biological or biochemical activity with fluorescent probes, and selecting subpopulations of interest based on a combination of fluorescent reporters (Figure 1). This technology is based on the application of droplet microfluidics allowing the generation of double water-in-oil-in-water (W/OW) emulsion droplets with precisely controlled size, in particularly mild emulsification conditions. Droplets serve as microbioreactors in which cultivation, co-cultivation, biocatalytic assays, or vital staining can be carried out. Specific combinations of fluorescent reporters indicate the functionality of the encapsulated species. The combinations of fluorescent reporters trigger FACS-assisted selection of the populations of interest. The selected populations are analyzed by classical cultivation-based assays including downstream activity profiling, proteomics, and metabolomics or examined by whole-genome sequencing and bioinformatics to trace unculturable or slow-growing species. The deep functional microbiome profiling method reported recently [5] consists of encapsulating single microorganisms of exotic microbiomes in isolated biocompatible microcompartments, estimating their biological or biochemical activity with fluorescent probes, and selecting subpopulations of interest based on a combination of fluorescent reporters (Figure 1). This technology is based on the application of droplet microfluidics allowing the generation of double water-in-oil-in-water (W/OW) emulsion droplets with precisely controlled size, in particularly mild emulsification conditions. Droplets serve as microbioreactors in which cultivation, co-cultivation, biocatalytic assays, or vital staining can be carried out. Specific combinations of fluorescent reporters indicate the functionality of the encapsulated species. The combinations of fluorescent reporters trigger FACS-assisted selection of the populations of interest. The selected populations are analyzed by classical cultivation-based assays including downstream activity profiling, proteomics, and metabolomics or examined by whole-genome sequencing and bioinformatics to trace unculturable or slow-growing species Figure 1. Pipeline of deep functional microbiome profiling. An exotic microbiota undergoes single- cell encapsulation in droplets of a biocompatible microfluidic double emulsion with the respective fluorogenic reporters. A fluorescence-based assay enables probing of the biological or biochemical activity of the encapsulated microorganisms. FACS-based screening classifies the microbiome based on the phenotype assayed. The selected subpopulations are analyzed subsequently by a combination of omics technologies for a detailed characterization of their phenotypes. NGS: Next Generation Sequencing. Figure 1. Pipeline of deep functional microbiome profiling. An exotic microbiota undergoes single-cell encapsulation in droplets of a biocompatible microfluidic double emulsion with the respective fluorogenic reporters. 2.1. High-Scale Ami Production and Purification 2.1. High-Scale Ami Production and Purification A fluorescence-based assay enables probing of the biological or biochemical activity of the encapsulated microorganisms. FACS-based screening classifies the microbiome based on the phenotype assayed. The selected subpopulations are analyzed subsequently by a combination of omics technologies for a detailed characterization of their phenotypes. NGS: Next Generation Sequencing. Fi 1 Pi li f d f ti l i bi fili A ti i bi t d i l Figure 1. Pipeline of deep functional microbiome profiling. An exotic microbiota undergoes Figure 1. Pipeline of deep functional microbiome profiling. An exotic microbiota undergoes single- cell encapsulation in droplets of a biocompatible microfluidic double emulsion with the respective fluorogenic reporters. A fluorescence-based assay enables probing of the biological or biochemical activity of the encapsulated microorganisms. FACS-based screening classifies the microbiome based on the phenotype assayed. The selected subpopulations are analyzed subsequently by a combination of omics technologies for a detailed characterization of their phenotypes. NGS: Next Generation Sequencing. Figure 1. Pipeline of deep functional microbiome profiling. An exotic microbiota undergoes single-cell encapsulation in droplets of a biocompatible microfluidic double emulsion with the respective fluorogenic reporters. A fluorescence-based assay enables probing of the biological or biochemical activity of the encapsulated microorganisms. FACS-based screening classifies the microbiome based on the phenotype assayed. The selected subpopulations are analyzed subsequently by a combination of omics technologies for a detailed characterization of their phenotypes. NGS: Next Generation Sequencing. Figure 1. Pipeline of deep functional microbiome profiling. An exotic microbiota undergoes single- cell encapsulation in droplets of a biocompatible microfluidic double emulsion with the respective fluorogenic reporters. A fluorescence-based assay enables probing of the biological or biochemical activity of the encapsulated microorganisms. FACS-based screening classifies the microbiome based on the phenotype assayed. The selected subpopulations are analyzed subsequently by a combination of omics technologies for a detailed characterization of their phenotypes. NGS: Next Generation Sequencing. Figure 1. Pipeline of deep functional microbiome profiling. An exotic microbiota undergoes single-cell encapsulation in droplets of a biocompatible microfluidic double emulsion with the respective fluorogenic reporters. A fluorescence-based assay enables probing of the biological or biochemical activity of the encapsulated microorganisms. FACS-based screening classifies the microbiome based on the phenotype assayed. The selected subpopulations are analyzed subsequently by a combination of omics technologies for a detailed characterization of their phenotypes. NGS: Next Generation Sequencing. The described functional profiling platform was applied for the isolation of Ami-producing B. 1. Introduction This process is exceptionally efficient in the case of Ami, decreasing its half-life to less than 2 h at physiological conditions. We speculate that this self-lactonization is so productive because of the formation of the energetically favorable five-membered transition state. Moreover, the proximate protonated amine group enhances this effect. Hence, we consider that self-lactonization serves as an improvised intermediate state, substantially 3 of 12 f 12 Antibiotics 2020, 9, 157 decreasing the energetic barrier of amide hydrolysis. While the exact mechanism of this reaction should be detailed in the future, a balance between antibiotic potency and stability seems to be necessary for Ami-producing Bacillus. However, it represents a great obstacle for the practical application of Ami as an antibiotic and anticancer drug. barrier of amide hydrolysis. While the exact mechanism of this reaction should be detailed in the future, a balance between antibiotic potency and stability seems to be necessary for Ami-producing Bacillus. However, it represents a great obstacle for the practical application of Ami as an antibiotic and anticancer drug. 2.1. High-Scale Ami Production and Purification 2.1. High-Scale Ami Production and Purification Hence, we reasoned that stimulation of cell–cell contacts is essential for high-scale Ami production, and this results in differences in Ami production during cultivation in liquid and semi-liquid growth medium. We stimulated cell–cell interactions and adhesion by the addition of calcium carbonate microparticles. The resulting SYC medium allowed a more than eightfold increase in Ami production in comparison with 2YT medium (Figure 2D). The concentration of Ami in 2YT medium could be increased by cultivation in shaking flasks in a decreased volume. However, this will not be convenient for Ami high-scale production. Taking this into account, we suggested boosting the activation of Ami biosynthesis by high carbohydrate concentrations, in turn mediating the activation of IMP synthesis and facilitating the formation of peptidoglycan [21]. Previously, we associated flagellum organization in B. pumilus with biofilm formation. Hence, we reasoned that stimulation of cell–cell contacts is essential for high-scale Ami production, and this results in differences in Ami production during cultivation in liquid and semi-liquid growth medium. We stimulated cell–cell interactions and adhesion by the addition of calcium carbonate microparticles. The resulting SYC medium allowed a more than eightfold increase in Ami production in comparison with 2YT medium (Figure 2D). The concentration of Ami in 2YT medium could be increased by cultivation in shaking flasks in a decreased volume. However, this will not be convenient for Ami high-scale production. Figure 2. The selected Bacillus pumilus efficiently produce Ami. Cultivation of Ami-producing B. pumilus on agar causes the appearance of substantial zones of clearance. The clearance zones were observed with the agar overlay assay using a reporter Staphylococcus aureus strain producing GFP in visible light (A) and by fluorescence analysis of GFP (B). The colonies around B. pumilus (center) are the representative strains picked from the oral microbiome of brown bear, randomly. The data illustrate the representative view of three independent repeats. (C) Cultivation of B. pumilus in limited (grey) or high (aquamarine) aeration conditions. (D) Cultivation of B. pumilus at high aeration in SYC medium (red) and 2YT medium in the same volume (yellow) or in a twice reduced volume (orange). Ami concentration (dots) was estimated by an antibacterial activity assay of culture medium in triplicate. Data represent mean ± SD. Figure 2. The selected Bacillus pumilus efficiently produce Ami. Cultivation of Ami-producing B. pumilus on agar causes the appearance of substantial zones of clearance. 2.1. High-Scale Ami Production and Purification 2.1. High-Scale Ami Production and Purification pumilus strains, and we exploited it to select the most efficient Ami producers from the same source of the microbiota. We isolated more than 50 Ami-producing B. pumilus clones totally, which were evaluated for Ami production. About 28% of them had a similar level of antagonistic activity, and we associated them with the same strain of origin. The isolated B. pumilus strains produced remarkable (>40 mm) zones of clearance that were observed using a reporter S. aureus (MRSA) strain (Figure 2A, 2B). However, cultivation in a nutritionally rich 2YT medium with limited aeration resulted in mediocre Ami production (Figure 2C), while increased aeration facilitated Ami production by more than sixfold (Figure 2C). Hence, we concluded that the cultivation conditions are particularly important for efficient Ami production. Previous proteomics observations [5] indicated that the activation of inosine monophosphate (IMP) biosynthesis, peptidoglycan synthesis, as well as flagellum organization correlate with cultivation in the thin layer associated with Ami production. Taking this into account, we suggested boosting the activation of Ami biosynthesis by high The described functional profiling platform was applied for the isolation of Ami-producing B. pumilus strains, and we exploited it to select the most efficient Ami producers from the same source of the microbiota. We isolated more than 50 Ami-producing B. pumilus clones totally, which were evaluated for Ami production. About 28% of them had a similar level of antagonistic activity, and we associated them with the same strain of origin. The isolated B. pumilus strains produced remarkable (>40 mm) zones of clearance that were observed using a reporter S. aureus (MRSA) strain (Figure 2A, 2B). However, cultivation in a nutritionally rich 2YT medium with limited aeration resulted in mediocre Ami production (Figure 2C), while increased aeration facilitated Ami production by more than sixfold (Figure 2C). Hence, we concluded that the cultivation conditions are particularly important for efficient Ami production. Previous proteomics observations [5] indicated that the activation of inosine monophosphate (IMP) biosynthesis, peptidoglycan synthesis, as well as flagellum organization correlate with cultivation in the thin layer associated with Ami production. Taking this into account, we suggested boosting the activation of Ami biosynthesis by high carbohydrate concentrations, in 4 of 12 4 of 12 Antibiotics 2020, 9, 157 Antibiotics 2020, 9, x FOR turn mediating the activation of IMP synthesis and facilitating the formation of peptidoglycan [21]. Previously, we associated flagellum organization in B. pumilus with biofilm formation. 2.1. High-Scale Ami Production and Purification 2.1. High-Scale Ami Production and Purification The clearance zones were observed with the agar overlay assay using a reporter Staphylococcus aureus strain producing GFP in visible light (A) and by fluorescence analysis of GFP (B). The colonies around B. pumilus (center) are the representative strains picked from the oral microbiome of brown bear, randomly. The data illustrate the representative view of three independent repeats. (C) Cultivation of B. pumilus in limited (grey) or high (aquamarine) aeration conditions. (D) Cultivation of B. pumilus at high aeration in SYC medium (red) and 2YT medium in the same volume (yellow) or in a twice reduced volume (orange). Ami concentration (dots) was estimated by an antibacterial activity assay of culture medium in triplicate. Data represent mean ± SD. Figure 2. The selected Bacillus pumilus efficiently produce Ami. Cultivation of Ami-producing B. pumilus on agar causes the appearance of substantial zones of clearance. The clearance zones were observed with the agar overlay assay using a reporter Staphylococcus aureus strain producing GFP in visible light (A) and by fluorescence analysis of GFP (B). The colonies around B. pumilus (center) are the representative strains picked from the oral microbiome of brown bear, randomly. The data illustrate the representative view of three independent repeats. (C) Cultivation of B. pumilus in limited (grey) or high (aquamarine) aeration conditions. (D) Cultivation of B. pumilus at high aeration in SYC medium (red) and 2YT medium in the same volume (yellow) or in a twice reduced volume (orange). Ami concentration (dots) was estimated by an antibacterial activity assay of culture medium in triplicate. Data represent mean ± SD. Figure 2. The selected Bacillus pumilus efficiently produce Ami. Cultivation of Ami-producing B. pumilus on agar causes the appearance of substantial zones of clearance. The clearance zones were observed with the agar overlay assay using a reporter Staphylococcus aureus strain producing GFP in visible light (A) and by fluorescence analysis of GFP (B). The colonies around B. pumilus (center) are the representative strains picked from the oral microbiome of brown bear, randomly. The data illustrate the representative view of three independent repeats. (C) Cultivation of B. pumilus in limited (grey) or high (aquamarine) aeration conditions. (D) Cultivation of B. pumilus at high aeration in SYC medium (red) and 2YT medium in the same volume (yellow) or in a twice reduced volume (orange). Ami concentration (dots) was estimated by an antibacterial activity assay of culture medium in triplicate. 2.1. High-Scale Ami Production and Purification 2.1. High-Scale Ami Production and Purification Data represent mean ± SD. The subsequent purification was simplified by solid-phase extraction (SPE) with a polystyrene- based resin (Figure 3A) followed by subsequent polishing on a C18 HPLC column (Figure 3B–D). The subsequent purification was simplified by solid-phase extraction (SPE) with a polystyrene-based resin (Figure 3A) followed by subsequent polishing on a C18 HPLC column (Figure 3B–D). 5 of 12 f 12 Antibiotics 2020, 9, 157 Antibiotics 2020, 9, x FOR PEER REVIEW 5 of Figure 3. Optimized procedure of Ami purification. (A) Solid-phase extraction (SPE) with the polystyrene-based resin LPS 500 (Technosorbent, Russia). The portion of Ami eluted by the corresponding gradient step is indicated. (B,C) Sequential purification of Ami-containing fraction using a C18 RP-HPLC column. (D) Chromatogram of the purified Ami sample. Ami yield was estimated by an antibacterial activity assay in triplicate. Data represent mean ± SD. SPE resulted in a highly Ami-enriched fraction eluted with 35% acetonitrile (ACN), which w Figure 3. Optimized procedure of Ami purification. (A) Solid-phase extraction (SPE) with the polystyrene-based resin LPS 500 (Technosorbent, Russia). The portion of Ami eluted by the corresponding gradient step is indicated. (B,C) Sequential purification of Ami-containing fraction using a C18 RP-HPLC column. (D) Chromatogram of the purified Ami sample. Ami yield was estimated by an antibacterial activity assay in triplicate. Data represent mean ± SD. SPE resulted in a highly Ami-enriched fraction eluted with 35% acetonitrile (ACN), which w fficiently purified by C18 RP HPLC resulting in pure Ami (Figure 3D) These steps are scalable an Figure 3. Optimized procedure of Ami purification. (A) Solid-phase extraction (SPE) with the polystyrene-based resin LPS 500 (Technosorbent, Russia). The portion of Ami eluted by the corresponding gradient step is indicated. (B,C) Sequential purification of Ami-containing fraction using a C18 RP-HPLC column. (D) Chromatogram of the purified Ami sample. Ami yield was estimated by an antibacterial activity assay in triplicate. Data represent mean ± SD. Figure 3. Optimized procedure of Ami purification. (A) Solid-phase extraction (SPE) with the polystyrene-based resin LPS 500 (Technosorbent, Russia). The portion of Ami eluted by the corresponding gradient step is indicated. (B,C) Sequential purification of Ami-containing fraction using a C18 RP-HPLC column. (D) Chromatogram of the purified Ami sample. Ami yield was estimated by an antibacterial activity assay in triplicate. Data represent mean ± SD. Figure 3. Optimized procedure of Ami purification. (A) Solid-phase extraction (SPE) with the polystyrene-based resin LPS 500 (Technosorbent, Russia). 2.1. High-Scale Ami Production and Purification 2.1. High-Scale Ami Production and Purification The portion of Ami eluted by the corresponding gradient step is indicated. (B,C) Sequential purification of Ami-containing fraction using a C18 RP-HPLC column. (D) Chromatogram of the purified Ami sample. Ami yield was estimated by an antibacterial activity assay in triplicate. Data represent mean ± SD. Figure 3. Optimized procedure of Ami purification. (A) Solid-phase extraction (SPE) with the polystyrene-based resin LPS 500 (Technosorbent, Russia). The portion of Ami eluted by the corresponding gradient step is indicated. (B,C) Sequential purification of Ami-containing fraction using a C18 RP-HPLC column. (D) Chromatogram of the purified Ami sample. Ami yield was estimated by an antibacterial activity assay in triplicate. Data represent mean ± SD. SPE resulted in a highly Ami-enriched fraction eluted with 35% acetonitrile (ACN), which was efficiently purified by C18 RP-HPLC, resulting in pure Ami (Figure 3D). These steps are scalable and could be adopted for extensive bioproduction. SPE resulted in a highly Ami-enriched fraction eluted with 35% acetonitrile (ACN), which was efficiently purified by C18 RP-HPLC, resulting in pure Ami (Figure 3D). These steps are scalable and could be adopted for extensive bioproduction. SPE resulted in a highly Ami-enriched fraction eluted with 35% acetonitrile (ACN), which was efficiently purified by C18 RP-HPLC, resulting in pure Ami (Figure 3D). These steps are scalable and could be adopted for extensive bioproduction. SPE resulted in a highly Ami-enriched fraction eluted with 35% acetonitrile (ACN), which was efficiently purified by C18 RP-HPLC, resulting in pure Ami (Figure 3D). These steps are scalable and could be adopted for extensive bioproduction. 2.2. The Activity Spectrum of Ami 2.2. The Activity Spectrum of Ami Sensitive, intermediate, and resistant bacteria are indicated according to the color bar and marked as S, I, and R, respectively. The phylogenetic tree was reconstructed based on 16S rRNA. Bacteria analyzed solely using a bioinformatics-based deep functional profiling approach and not confirmed by standard cultivation assays are colored in light Figure 4. Comprehensive overview of Ami activity spectrum. The phylogeny of bacteria is presented with the respective mean minimum inhibitory concentrations (MICs). The color and area of circle markers indicate the average MIC value. Sensitive, intermediate, and resistant bacteria are indicated according to the color bar and marked as S, I, and R, respectively. The phylogenetic tree was reconstructed based on 16S rRNA. Bacteria analyzed solely using a bioinformatics-based deep functional profiling approach and not confirmed by standard cultivation assays are colored in light gray. gray. Generally, Ami is active against Gram-positive non-spore-forming bacteria, including Lactobacillales and Staphylococcaceae. Ami is inactive toward Bacillus that we associate with homologs of AmiN kinase reported previously. Ami displayed antibacterial activity against some laboratory strains of Escherichia coli [11]. However, it showed only mediocre activity against E. coli wild strains. Moreover, Ami was ineffective against an especially valuable Gram-negative cohort of ESKAPE pathogens (K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter species), while it was highly active against the overwhelming majority of Enterococcus and Staphylococcus strains (referred to as “E” and “S” ESKAPE pathogens, respectively). These bacteria represent the most clinically relevant fraction of Gram-positive pathogens, particularly associated with AR. Distinct Bacteroides, including Bacteroides dorei and Bacteroides vulgatus, were also sensitive to Ami (Figure 4). However, the majority of strains representing mutualistic gastrointestinal microbiota components were resistant to Ami, Generally, Ami is active against Gram-positive non-spore-forming bacteria, including Lactobacillales and Staphylococcaceae. Ami is inactive toward Bacillus that we associate with homologs of AmiN kinase reported previously. Ami displayed antibacterial activity against some laboratory strains of Escherichia coli [11]. However, it showed only mediocre activity against E. coli wild strains. Moreover, Ami was ineffective against an especially valuable Gram-negative cohort of ESKAPE pathogens (K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter species), while it was highly active against the overwhelming majority of Enterococcus and Staphylococcus strains (referred to as “E” and “S” ESKAPE pathogens, respectively). These bacteria represent the most clinically relevant fraction of Gram-positive pathogens, particularly associated with AR. Distinct Bacteroides, including Bacteroides dorei and Bacteroides vulgatus, were also sensitive to Ami (Figure 4). 2.2. The Activity Spectrum of Ami 2.2. The Activity Spectrum of Ami However, the majority of strains representing mutualistic gastrointestinal microbiota components were resistant to Ami, indicating Ami limited potential against this critical microbiota component. 2.2. The Activity Spectrum of Ami 2.2. The Activity Spectrum of Ami The purified Ami was used to characterize its activity spectrum using standard cultivation assays and deep functional profiling of microbiomes for Ami resistance. Deep functional profiling of microbiomes for Ami resistance is based on the cultivation of single bacteria from a particular microbiome in droplets with various concentrations of Ami, followed by their viability staining, FACS-based selection, metagenomic sequencing, and quantitative estimation of live bacteria via bioinformatics [5]. Here, we used a broad panel of clinical isolates to provide a more comprehensive description of the Ami activity spectrum (Figure 4). The purified Ami was used to characterize its activity spectrum using standard cultivation assays and deep functional profiling of microbiomes for Ami resistance. Deep functional profiling of microbiomes for Ami resistance is based on the cultivation of single bacteria from a particular microbiome in droplets with various concentrations of Ami, followed by their viability staining, FACS-based selection, metagenomic sequencing, and quantitative estimation of live bacteria via bioinformatics [5]. Here, we used a broad panel of clinical isolates to provide a more comprehensive description of the Ami activity spectrum (Figure 4). 6 of 12 Antibiotics 2020, 9, 157 ntibiotics 2020, 9, x FOR PEER REVIEW 6 o Figure 4. Comprehensive overview of Ami activity spectrum. The phylogeny of bacteria is presented with the respective mean minimum inhibitory concentrations (MICs). The color and area of circle markers indicate the average MIC value. Sensitive, intermediate, and resistant bacteria are indicated according to the color bar and marked as S, I, and R, respectively. The phylogenetic tree was reconstructed based on 16S rRNA. Bacteria analyzed solely using a bioinformatics-based deep functional profiling approach and not confirmed by standard cultivation assays are colored in light gray Figure 4. Comprehensive overview of Ami activity spectrum. The phylogeny of bacteria is presented with the respective mean minimum inhibitory concentrations (MICs). The color and area of circle markers indicate the average MIC value. Sensitive, intermediate, and resistant bacteria are indicated according to the color bar and marked as S, I, and R, respectively. The phylogenetic tree was reconstructed based on 16S rRNA. Bacteria analyzed solely using a bioinformatics-based deep functional profiling approach and not confirmed by standard cultivation assays are colored in light gray. Figure 4. Comprehensive overview of Ami activity spectrum. The phylogeny of bacteria is presented with the respective mean minimum inhibitory concentrations (MICs). The color and area of circle markers indicate the average MIC value. indicating Ami limited potential 2.3. Exceptional Instability of Ami (A) General scheme, describing the sequential transformation of biologically active Ami (AmiA) into inactive amicoumacins AmiC and AmiB, respectively. (B) pH dependence of Ami decomposition. The residual portion of Ami over time is presented. Color indicates pH. (C) Representative time course of the conversion of AmiA into AmiC and AmiB, taking place at pH 7.5. The corresponding percentages of AmiA, AmiC, and AmiB in the reaction mixtures are presented. Amicoumacins were analyzed by HPLC (dots). The data were approximated by a two-step sequential reaction model (lines). The concentrations of amicoumacins were analyzed in triplicate. Data represent mean ± SD. (D) pH dependence of the rate-limiting step of Ami decomposition. The kinetic constants of the self-conversion of AmiA into AmiC are presented for pH 5–9. Data represent mean ± SD Figure 5. Ami is a particularly unstable amide, decomposing via intramolecular lactonization in physiological conditions. (A) General scheme, describing the sequential transformation of biologically active Ami (AmiA) into inactive amicoumacins AmiC and AmiB, respectively. (B) pH dependence of Ami decomposition. The residual portion of Ami over time is presented. Color indicates pH. (C) Representative time course of the conversion of AmiA into AmiC and AmiB, taking place at pH 7.5. The corresponding percentages of AmiA, AmiC, and AmiB in the reaction mixtures are presented. Amicoumacins were analyzed by HPLC (dots). The data were approximated by a two-step sequential reaction model (lines). The concentrations of amicoumacins were analyzed in triplicate. Data represent mean ± SD. (D) pH dependence of the rate-limiting step of Ami decomposition. The kinetic constants of the self-conversion of AmiA into AmiC are presented for pH 5–9. Data represent mean ± SD. Ami has a terminal amide bond that is exceptionally unstable, and we decided to study this feature in detail. We observed that Ami decomposition is pH-dependent, accelerating rapidly from low acidic to neutral pH and reaching a maximum at pH 7–9 (Figure 5B, 5D). The analysis of Ami decomposition kinetics revealed that Ami spontaneously converts into lactone AmiC, which subsequently hydrolyzes to AmiB (Figure 5C). Our data indicate that even if direct conversion of Ami into AmiB was observed, it should be more than an order of magnitude slower than the transition of Ami into AmiC. Hence, the rate-limiting step of Ami hydrolysis is the first reaction, resulting in AmiC formation by intramolecular lactonization. indicating Ami limited potential 2.3. Exceptional Instability of Ami 2.3. Exceptional Instability of Ami It was previously reported [9,20], and we also observed [5], that Ami (AmiA) is not stable in aqueous solutions and transforms into biologically inactive products, i.e., amicoumacin B (AmiB) and It was previously reported [9,20], and we also observed [5], that Ami (AmiA) is not stable in aqueous solutions and transforms into biologically inactive products, i.e., amicoumacin B (AmiB) and amicoumacin C (AmiC) (Figure 5A). 7 of 12 7 of 12 Antibiotics 2020, 9, 157 Figure 5. Ami is a particularly unstable amide, decomposing via intramolecular lactonization in physiological conditions. (A) General scheme, describing the sequential transformation of biologically active Ami (AmiA) into inactive amicoumacins AmiC and AmiB, respectively. (B) pH dependence of Ami decomposition. The residual portion of Ami over time is presented. Color indicates pH. (C) Representative time course of the conversion of AmiA into AmiC and AmiB, taking place at pH 7.5. The corresponding percentages of AmiA, AmiC, and AmiB in the reaction mixtures are presented. Amicoumacins were analyzed by HPLC (dots). The data were approximated by a two-step sequential reaction model (lines). The concentrations of amicoumacins were analyzed in triplicate. Data represent mean ± SD. (D) pH dependence of the rate-limiting step of Ami decomposition. The kinetic constants of the self-conversion of AmiA into AmiC are presented for pH 5–9. Data represent mean ± SD. Figure 5. Ami is a particularly unstable amide, decomposing via intramolecular lactonization in physiological conditions. (A) General scheme, describing the sequential transformation of biologically active Ami (AmiA) into inactive amicoumacins AmiC and AmiB, respectively. (B) pH dependence of Ami decomposition. The residual portion of Ami over time is presented. Color indicates pH. (C) Representative time course of the conversion of AmiA into AmiC and AmiB, taking place at pH 7.5. The corresponding percentages of AmiA, AmiC, and AmiB in the reaction mixtures are presented. Amicoumacins were analyzed by HPLC (dots). The data were approximated by a two-step sequential reaction model (lines). The concentrations of amicoumacins were analyzed in triplicate. Data represent mean ± SD. (D) pH dependence of the rate-limiting step of Ami decomposition. The kinetic constants of the self-conversion of AmiA into AmiC are presented for pH 5–9. Data represent mean ± SD. Figure 5. Ami is a particularly unstable amide, decomposing via intramolecular lactonization in physiological conditions. indicating Ami limited potential 2.3. Exceptional Instability of Ami We suggest that Ami structure determines Ami instability by the formation of the energetically favored five-membered ring transition state, accelerating its self- inactivation by lactonization (Figure 5A). Hence, AmiC lactone acts as an improvised intermediate state, substantially decreasing the energetic barrier of amide hydrolysis. We suppose this self- lactonization step provides the main contribution to Ami instability, decreasing its half-life from years, as reported for other terminal amides [22], to less than 2 h at pH 8 and 37 °C. The observed pH dependence indicates the potential impact of the Ami amine group on self-lactonization. This amine Ami has a terminal amide bond that is exceptionally unstable, and we decided to study this feature in detail. We observed that Ami decomposition is pH-dependent, accelerating rapidly from low acidic to neutral pH and reaching a maximum at pH 7–9 (Figure 5B,D). The analysis of Ami decomposition kinetics revealed that Ami spontaneously converts into lactone AmiC, which subsequently hydrolyzes to AmiB (Figure 5C). Our data indicate that even if direct conversion of Ami into AmiB was observed, it should be more than an order of magnitude slower than the transition of Ami into AmiC. Hence, the rate-limiting step of Ami hydrolysis is the first reaction, resulting in AmiC formation by intramolecular lactonization. We suggest that Ami structure determines Ami instability by the formation of the energetically favored five-membered ring transition state, accelerating its self-inactivation by lactonization (Figure 5A). Hence, AmiC lactone acts as an improvised intermediate state, substantially decreasing the energetic barrier of amide hydrolysis. We suppose this self-lactonization step provides the main contribution to Ami instability, decreasing its half-life from years, as reported for other terminal amides [22], to less than 2 h at pH 8 and 37 ◦C. The observed pH dependence indicates the potential impact of the Ami amine group on self-lactonization. This 8 of 12 Antibiotics 2020, 9, 157 amine group has an estimated pKa of ~7.2. We suggest its protonation is critical for the self-conversion of Ami into AmiC, accelerating when pH increases from 5 to 7 and reaching a maximum at pH 7–9. We speculate that the effect of the protonated amine is mediated by intramolecular stabilization of the –OHδ- nucleophile. Alternatively, the protonated amine could serve as an intramolecular proton donor for the ammonia leaving group. 3. Discussion Since its discovery at the beginning of the 1980s [7–9], Ami raised only limited practical interest, predominantly in academia. Recently, the ribosome-targeting mechanism of Ami was elucidated both for pro- [11] and for eukaryotes [12]. Furthermore, the molecular mechanisms of Ami activation/attenuation [20] and self-resistance [5] were identified. Hence, we suggest that these recent advances provide new applications of Ami in the fields of synthetic biology, biotechnology, and biomedicine. The high-level production of Ami is of interest in these cases, and our results indicate how it could be achieved by the application of state-of-the-art microbiological technologies. Deep functional profiling facilitates the isolation of potent Ami producers, while proteomics and metabolomics provide an essential basis for boosting Ami production. Ami is active against both bacteria and mammalian cells, and this is the fundamental problem limiting its broad application. Taking into account the antibiotic potential of Ami, we classify it as a potent agent against Gram-positive pathogenic strains of Enterococcus and Staphylococcus, including MRSA. Ami was also reported as an efficient anti-H. pylori antibiotic, providing attractive gastroprotective action [13]. However, the major obstacle to the practical application of Ami is its low stability at physiological conditions. This is also relevant to H. pylori, which neutralizes the acidic conditions of the stomach via urease activity. Ami has a half-life of ~2 h in neutral and slightly alkaline conditions. That is more than 104 times lower than the respective values obtained for terminally amidated peptides [23]. Hence, the rate of Ami hydrolysis at physiological conditions is similar to that of spontaneous amide hydrolysis at 170 ◦C [24] and close to the hydrolysis rate achieved by artificial enzymes, i.e., proteolytic antibodies [23,25]. We suggest that the low stability of the terminal amide bond in Ami is an exclusive feature originating from its unique structure. The decomposition of Ami is a two-step process, including its self-lactonization, followed by lactone hydrolysis. The self-lactonization of Ami is the rate-limiting step that makes the main contribution to the acceleration of Ami hydrolysis. This is achieved by the formation of the lactone AmiC, simulating the transition state and reducing the energetic barrier of amide hydrolysis. We consider that the potential of Ami has been underestimated for a long time because of the decreased stability of Ami in biological fluids. While the instability of Ami is a stumbling block for its clinical application, we suggest this feature is not accidental. indicating Ami limited potential 2.3. Exceptional Instability of Ami However, the detailed molecular mechanism of this particular Ami instability should be thoroughly investigated in the future and supported by quantum mechanics simulations and calculation of energetic profiles of Ami self-lactonization. 3. Discussion We speculate that Ami serves as an efficient biocontrol agent providing the benefit for Bacillus over its competitors in the wild. The instability of Ami is advantageous in this case, as it enables a precisely time-resolved control over its inhibitory activity. The total synthesis of natural products of the amicoumacin family was reported recently [26,27]. Hence, total organic synthesis could provide a vital source of new Ami analogs tailored for antimicrobial or anticancer drug discovery, reviving the interest in this antibiotic. 4.2. Deep Functional Profiling The selection of bacteria displaying anti-S. aureus activity was described in detail previously [6]. Briefly, target S. aureus cells producing a GFP reporter were vitally stained with sulfo-Cyanine5 NHS (Lumiprobe, Russia), washed, filtered using 20 µm solvent filters (A-313, IDEX, USA), and co-encapsulated with a microbiota suspension in droplets of microfluidic double emulsion (MDE), using 20 µm microfluidic chips produced via soft lithography. Microbiota samples were unfrozen directly before encapsulation, resuspended in BHI broth (BD, USA), and filtered through 40 µm cell strainers (Greiner Bio-One, USA). After overnight incubation at 35 ◦C, Calcein Violet AM (Thermo Fisher Scientific, Waltham, MA, USA) was added to the droplet emulsion to the final concentration of 10 µM. Subsequently, the droplets with simultaneous sCy5high, GFPlow, and Calcein Violethigh fluorescence were sorted using a FACSAria III cell sorter (BD, USA). Bacterial colonies were regenerated after plating on BHI–agar (BD, USA) and tested for anti-S. aureus activity using the agar overlay assay. Bacterial clones of B. pumilus demonstrating inhibition of S. aureus growth were identified by mass spectrometry. The procedure of deep profiling of Ami activity was described in detail previously [5]. Briefly, microbiota samples were co-encapsulated with Ami inside the MDE droplets, followed by cultivation, Calcein Violet AM staining, and FACSAria III sorting. The collected droplets were frozen in liquid nitrogen, freeze-dried, and analyzed by whole-genome sequencing and bioinformatics, resulting in the quantification of bacteria in the samples. The abundance of bacteria in the samples was used for the estimation of MIC, as it was described previously [5]. 4.3. NGS Sequencing and Bioinformatics The selected MDE droplets were freeze-dried, and total DNA was isolated using the QIAamp DNA Investigator Kit (Qiagen, USA). Whole-genome amplification was performed using the REPLI-g Single Cell Kit (Qiagen, USA). Fragment libraries were prepared using the NEBNext® DNA Library Prep Reagent Set for Illumina and the NEBNext® Multiplex Oligos for Illumina® (96 Index Primers) (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions. The sequencing of libraries was performed using the genetic analyzer HiSeq2500, the HiSeq PE Cluster Kit v4 cBot and the HiSeq SBS Kit v4 (250 cycles) (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions. Genome assemblies were performed using SPAdes 3.9.0 [28]. Bacteria abundance in metagenome samples was assessed with Metaphlan2 [29]. The bacterial phylogeny was reconstructed based on 16S rRNA. We used Muscle [30] for sequence alignment. Quality control of alignment was done in UGENE [31]. RaxML was used for phylogenetic tree reconstruction [32]. FigTree was used for phylogenetic tree visualization. 4.1. Microbiota Collection and Storage All human donors provided written informed consent and were examined before collecting the fecal sample. The examination included: general blood test, biochemical blood test, ELISA blood screening for Lamblias, Toksokara, Opisthorchis, Ascaris, and Trichinella, hepatitis B and C test, HIV test, syphilis test, urinalysis, and stool screening for Clostridium difficile, Campylobacter, Salmonella, 9 of 12 Antibiotics 2020, 9, 157 enteroinvasive E. coli (EIEC), Shigella, Rotavirus, Norovirus, Adenovirus, Cryptosporidium, Cyclospora, Giardia, and Isospora. The results were negative for the pathogens tested. Stool samples were suspended in sterile medium for microbiota cryopreservation, composed of Brain Heart Infusion (BHI) medium (BD, USA), 20% glycerol, and 30% heat-inactivated fetal bovine serum (FBS) (Gibco, USA). The oral microbiota of East Siberian brown bear (Ursus arctos collaris) was collected previously [5] and stored in liquid nitrogen. 4.4. Identification of Bacteria using Mass Spectrometry Bacterial cells were spotted on a sample spot of a MALDI target plate (MSP 96 target, ground steel; Bruker Daltonics, Billerica, MA, USA) and were overlaid with HCCA matrix solution (saturated solution of α-4-cyano-hydroxycinnamic acid; Bruker Daltonics, Billerica, MA, USA) in 50% acetonitrile (Sigma-Aldrich, St. Louis, MO, USA) and 2.5% trifluoroacetic acid solution (Sigma-Aldrich, St. Louis, MO, USA). Mass spectra profiles were acquired using a Microflex spectrometer (Bruker Daltonics, Billerica, MA, USA). The molecular ions were measured automatically in linear positive ion mode with the instrument parameters optimized for a range of 2,000–20,000 m/z. The software packages flexControl 3.0 (Bruker Daltonics, Billerica, MA, USA) and flexAnalysis 3.0 (Bruker Daltonics, Billerica, MA, USA) 10 of 12 10 of 12 Antibiotics 2020, 9, 157 were used for mass spectra recording and processing. Spectra identification and analysis were carried out using the MALDI Biotyper 3.0 (Bruker Daltonics, Billerica, MA, USA). The identification was performed by comparing the obtained spectra with those in the MALDI Biotyper 3.0 library (version 3.2.1.1). 4.5. Antimicrobial Activity A bacterial collection of clinical isolates was kindly provided by Lytech Co. Ltd. The MICs for the bacteria were determined by serial two-fold dilution (0.1–256 µg/mL) in Mueller–Hinton Broth (BD, USA) or Anaerobe Basal Broth (Oxoid, USA) in anaerobic conditions for Bacteroides. MIC was defined as the lowest concentration of amicoumacins that prevented the growth of the test organism in a 96-well plate after 10 h of cultivation. Prolonged cultivation is undesirable due to spontaneous Ami self-hydrolysis reaction and leads to overestimated MICs. The bacterial growth time course was monitored at 800 nm, using a Varioskan Flash multimode reader (Thermo Fisher Scientific, Waltham, MA, USA). 4.6. Ami Production and Purification Ami producing B. pumilus was cultivated in 2YT medium (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl) or SYC medium containing 40 g/L sucrose, 5 g/L yeast extract, 4 g/L CaCO3, 1.5 g/L K2HPO4, 2 g/L glucose, 2 g/L NaCl, 1.5 g/L MgSO4, 2 g/L (NH4)2SO4, 0.01 g/L FeSO4, 0.01 g/L MnCl2, at 28 ◦C. B. pumilus was inoculated from an overnight culture (using 1:100 dilution) and cultivated using 750 mL flasks in 100 mL with 250 rpm shaking. The cells were centrifuged at 10,000× g for 10 min, and the supernatant was filtered using a Millistak + HC Pod Depth Filter (Millipore, Billerica, MA, USA). Ami purification included three steps of chromatography. In the first step, the supernatant was purified by SPE with LPS-500 sorbent (Technosorbent, Russia) on column XK 26 (GE Healthcare Life Sciences, Pittsburgh, PA, USA), using buffer A (10 mM NH4OAc pH 5.0, 5% ACN), buffer B (10 mM NH4OAc pH 5.0, 80% ACN), flow rate 6 mL/min, step gradient 0–10 min (0% B), 10–20 min (20% B), 20–36 min (40% B), and 36–45 min (100% B). The fractions containing Ami (40% B) were freeze-dried, dissolved in DMSO, and fractionated twice on an RP-HPLC Zorbax ODS 62 × 250 mm (Agilent, Santa Clara, CA, USA) column using buffer A and B, flow rate 5 mL/min, gradient 0–10 min (0% B), 10–24 min (0%–70% B), 24–25 min (70%–100% B). Finally, Ami was purified on a Symmetry C18 5 µm 4.6 × 150 mm (Waters) RP-HPLC column, using buffer A and B, flow rate 1 mL/min, gradient 0–5 min (0% B), 5–20 min (0%–100% B). Amicoumacin and its derivatives were monitored by absorbance at 315 nm. The concentration of Ami was measured using εMeOH 315nm = 4380 M−1cm−1. Author Contributions: S.S.T., I.V.S., E.N.I., and A.G.G. designed the research; S.S.T., A.S.N., M.V.M., and I.V.S. performed the research; A.S.N., Y.A.M., M.N.B., and N.A.P. contributed new analytic tools; S.S.T., A.S.N., I.V.S., N.A.P., M.V.M., E.N.I., and A.G.G. analyzed the data; S.S.T., I.V.S., and A.G.G. wrote the paper. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Grant 19-34-70021 (for SST) and 18-29-08054 (for IVS) from the Russian Foundation for Basic Research, Grant 19-14-00331 from the Russian Science Foundation. Conflicts of Interest: The authors declare no conflict of interest. References 1. Fleischmann, C.; Scherag, A.; Adhikari, N.K.J.; Hartog, C.S.; Tsaganos, T.; Schlattmann, P.; Angus, D.C.; Reinhart, K. Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Current Estimates and Limitations. Am. J. Respir. Crit. Care Med. 2016, 193, 259–272. [CrossRef] [PubMed] 2. 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Agricul. Biolog. Chem. 1982, 46, 2659–2665. [CrossRef] 10. Lama, A.; Pané-Farré, J.; Chon, T.; Wiersma, A.M.; Sit, C.S.; Vederas, J.C.; Hecker, M.; Nakano, M.M. Response f M thi illi R i t t St h l t A i i A PL S ONE 2012 7 34037 [C R f] 0. Lama, A.; Pané-Farré, J.; Chon, T.; Wiersma, A.M.; Sit, C.S.; Vederas, J.C.; Hecker, M.; Nakano, M.M. Respo of Methicillin-Resistant Staphylococcus aureus to Amicoumacin A. PLoS ONE 2012, 7, e34037. 4.7. Ami Hydrolysis Ami hydrolysis was performed at 37 ◦C in 20 mM buffers: NaOAc pH 5.0, NaOAc pH 6.0, Na-phosphate pH 7.0, Na-phosphate pH 7.5, Na-phosphate pH 8.0, Bis-Tris-Propane pH 9.0. The reaction mix was analyzed by RP-HPLC using the Symmetry C18 (Waters, USA) column (1 mL/min, linear gradient 5%–53% ACN with 20 mM NH4OAc pH 6.0, in 10 min). Amicoumacin and its derivatives were monitored by absorbance, assuming εMeOH 315nm = 4380 M−1 cm−1. The retention times were as follows: AmiB –8.47 ± 0.03 min, Ami (AmiA)–9.00 ± 0.03 min, AmiC–9.90 ± 0.03 min. Author Contributions: S.S.T., I.V.S., E.N.I., and A.G.G. designed the research; S.S.T., A.S.N., M.V.M., and I.V.S. performed the research; A.S.N., Y.A.M., M.N.B., and N.A.P. contributed new analytic tools; S.S.T., A.S.N., I.V.S., N.A.P., M.V.M., E.N.I., and A.G.G. analyzed the data; S.S.T., I.V.S., and A.G.G. wrote the paper. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Grant 19-34-70021 (for SST) and 18-29-08054 (for IVS) from the Russian Foundation for Basic Research, Grant 19-14-00331 from the Russian Science Foundation. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 11 of 12 11 of 12 Antibiotics 2020, 9, 157 References [CrossR 11. Polikanov, Y.S.; Osterman, I.A.; Szal, T.; Tashlitsky, V.N.; Serebryakova, M.V.; Kusochek, P.; Bulkley, D.; Malanicheva, I.A.; Efimenko, T.; Efremenkova, O.V.; et al. Amicoumacin a inhibits translation by stabilizing mRNA interaction with the ribosome. Mol. Cell 2014, 56, 531–540. [CrossRef] 12. Prokhorova, I.V.; Akulich, K.A.; Makeeva, D.S.; Osterman, I.A.; Skvortsov, D.A.; Sergiev, P.V.; Dontsova, O.A.; Yusupova, G.; Yusupov, M.M.; Dmitriev, S.E. Amicoumacin A induces cancer cell death by targeting the eukaryotic ribosome. Sci. Rep. 2016, 6, 27720. [CrossRef] [PubMed] 13. Pinchuk, I.V.; Bressollier, P.; Verneuil, B.; Fenet, B.; Sorokulova, I.B.; Mégraud, F.; Urdaci, M.C. In vitro anti-Helicobacter pylori activity of the probiotic strain Bacillus subtilis 3 is due to secretion of antibiotics. Antimicrob. Agents Chemother. 2001, 45, 3156–3161. [CrossRef] 14. Pinchuk, I.V.; Bressollier, P.; Sorokulova, I.B.; Verneuil, B.; Urdaci, M.C. Amicoumacin antibiotic production and genetic diversity of Bacillus subtilis strains isolated from different habitats. Res. Microbiol. 2002, 153, 269–276. [CrossRef] 15. Gao, X.-Y.; Liu, Y.; Miao, L.-L.; Li, E.-W.; Hou, T.-T.; Liu, Z.-P. Mechanism of anti-Vibrio activity of marine probiotic strain Bacillus pumilus H2, and characterization of the active substance. AMB Express 2017, 7, 23. [CrossRef] [PubMed] 16. Zidour, M.; Chevalier, M.; Belguesmia, Y.; Cudennec, B.; Grard, T.; Drider, D.; Souissi, S.; Flahaut, C. Isolation and Characterization of Bacteria Colonizing Acartia tonsa Copepod Eggs and Displaying Antagonist Effects against Vibrio anguillarum, Vibrio alginolyticus and Other Pathogenic Strains. Front. Microbiol. 2017, 8, 1919. [CrossRef] [PubMed] 17. Lefevre, M.; Racedo, S.M.; Ripert, G.; Housez, B.; Cazaubiel, M.; Maudet, C.; Jüsten, P.; Marteau, P.; Urdaci, M.C. Probiotic strain Bacillus subtilis CU1 stimulates immune system of elderly during common infectious disease period: A randomized, double-blind placebo-controlled study. Immun. Ageing 2015, 12, 24. [CrossRef] 18. Li, Y.; Li, Z.; Yamanaka, K.; Xu, Y.; Zhang, W.; Vlamakis, H.; Kolter, R.; Moore, B.S.; Qian, P.-Y. Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis. Sci. Rep. 2015, 5, 9383. [CrossRef] 19. Reimer, D.; Pos, K.M.; Thines, M.; Grün, P.; Bode, H.B. A natural prodrug activation mechanism in nonribosomal peptide synthesis. Nat. Chem. Biol. 2011, 7, 888–890. [CrossRef] 12 of 12 Antibiotics 2020, 9, 157 20. Park, H.B.; Perez, C.E.; Perry, E.K.; Crawford, J.M. Activating and Attenuating the Amicoumacin Antibiotics. Molecules 2016, 21, 824. [CrossRef] 21. Niu, H.; Wang, J.; Zhuang, W.; Liu, D.; Chen, Y.; Zhu, C.; Ying, H. Comparative transcriptomic and proteomic analysis of Arthrobacter sp. References CGMCC 3584 responding to dissolved oxygen for cAMP production. Sci. Rep. 2018, 8, 1246. [CrossRef] 22. Mahesh, S.; Tang, K.-C.; Raj, M. Amide Bond Activation of Biological Molecules. Molecules 2018, 23, 2615. [CrossRef] [PubMed] 23. Gao, C.; Lavey, B.J.; Lo, C.-H.L.; Datta, A.; Wentworth, P.; Janda, K.D. Direct Selection for Catalysis from Combinatorial Antibody Libraries Using a Boronic Acid Probe: Primary Amide Bond Hydrolysis. J. Am. Che. Soc. 1998, 120, 2211–2217. [CrossRef] 24. Robinson, B.A.; Tester, J.W. Kinetics of alkaline hydrolysis of organic esters and amides in neutrally-buffered solution. Int. J. Chem. Kinet. 1990, 22, 431–448. [CrossRef] 25. Martin, M.T.; Angeles, T.S.; Sugasawara, R.; Aman, N.I.; Napper, A.D.; Darsley, M.J.; Sanchez, R.I.; Booth, P.; Titmas, R.C. Antibody-Catalyzed Hydrolysis of an Unsubstituted Amide. J. Am. Chem. Soc. 1994, 116, 6508–6512. [CrossRef] 26. Suzuki, T.; Nagasawa, T.; Enomoto, M.; Kuwahara, S. Stereoselective total synthesis of amicoumacin C. Tetrahedron 2015, 71, 1992–1997. [CrossRef] 27. Tsukaguchi, S.; Enomoto, M.; Towada, R.; Ogura, Y.; Kuwahara, S. Unified Total Synthesis of Hetiamacins A–D. Eur. J. Org. Chem. 2019, 2019, 6110–6116. [CrossRef] 28. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.A.; Dvorkin, M.; Kulikov, A.S.; Lesin, V.M.; Nikolenko, S.I.; Pham, S.; Prjibelski, A.D.; et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012, 19, 455–477. [CrossRef] 29. Truong, D.T.; Franzosa, E.A.; Tickle, T.L.; Scholz, M.; Weingart, G.; Pasolli, E.; Tett, A.; Huttenhower, C.; Segata, N. MetaPhlAn2 for enhanced metagenomic taxonomic profiling. Nat. Methods 2015, 12, 902–903. [CrossRef] 30. Madeira, F.; Park, Y.M.; Lee, J.; Buso, N.; Gur, T.; Madhusoodanan, N.; Basutkar, P.; Tivey, A.R.N.; Potter, S.C.; Finn, R.D.; et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic. Acids Res. 2019, 47, W636–W641. [CrossRef] 31. Okonechnikov, K.; Golosova, O.; Fursov, M.; Ugene Team. Unipro UGENE: A unified bioinformatics toolkit. Bioinformatics 2012, 28, 1166–1167. [CrossRef] 32. Stamatakis, A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014, 30, 1312–1313. [CrossRef] [PubMed] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Self-regulation and learning: evidence from meta-analysis and from classrooms
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Running head: Self regulation and learning Running head: Self regulation and learning Running head: Self regulation and learning Word count: 4,616 (excluding the abstract, reference list, tables and figures) Word count: 4,616 (excluding the abstract, reference list, tables and figures) *Requests for reprints should be addressed to Professor Steve Higgins, School of Education, Leazes Road, Durham, DH1 1TA, UK (e-mail: s.e.higgins@durham.ac.uk). 1Durham University 1Durham University Steve Higgins 1Durham University Steve Higgins Aims. This paper has two main aims. The first is to make a case for the relative benefits of metacognitive and self-regulatory approaches for improving learning. This paper has two main aims. The first is to make a case for the relative benefits of metacognitive and self-regulatory approaches for improving learning. Comparative data from over 50 meta-analyses of interventions in schools indicates such approaches are more beneficial than other interventions. The second aim is to present data about different kinds of metacognitive thinking in classrooms which was elicited with cartoon templates. Acknowledgements This paper was based on a keynote presentation prepared for the “Self-Regulation and Dialogue in Primary Classrooms” Psychological Aspects of Education Current Trends Conference organised by the British Journal of Educational Psychology and held in Cambridge, June 2nd- 3rd, 2011. The analysis and findings from the review of meta- analyses was funded by the Sutton Trust, the classroom research was undertaken as part of the ‘Learning to Learn in Schools’ project funded through the Campaign for Learning, an independent charity working for a society where learning is at the heart of social inclusion. Thanks also go to Kate Wall and Emma Mercier for help with the development of the paper and to Richard Remedios for the analysis of the data from the pupil views templates. 1 Background. Research indicates that supporting self-regulation and metacognition in learners improves their attainment and wider learning capabilities. However, using this knowledge effectively is challenging. Results. There was an increase in all kinds of thinking recorded on the templates with younger children (4 -11 year olds), but contrary to expectation more complex kinds of thinking were identified less frequently with the 11-15 year olds. Sample and methods. Data about thinking in classrooms is drawn from a sample of 355 pupils, aged 4- 15, in 12 schools who were involved in a project which promoted meta-cognition and self-regulation. The completed templates were coded for different kinds of thinking, including metacognitive knowledge and metacognitive skillfulness. A two way ANOVA (gender and age) was conducted to examine the development of thinking across age groups (4-7 year olds; 7-11 year olds; 11-15 year olds). 2 Conclusion. There is strong evidence from meta-analysis that metacognition and self- regulation are key dimensions in supporting learning, but there are also indications from classrooms that such approaches are not routinely embedded in schools in a way which takes advantage of learners’ developing capabilities. 3 3 3 Introduction There is increasing evidence that supporting meta-cognition and the self- regulation of school-age learners is effective in improving their attainment and their wider learning capabilities. The aims of this paper are to strengthen the case for the development and use of such approaches by identifying the relative benefits of metacognitive and self-regulatory approaches, using data from a number of meta-analyses. Not only are meta-cognitive and self-regulatory approaches beneficial, but they are also amongst the most effective of the interventions in education which have been researched with experimental designs. Section 1: the importance of self-regulation and metacognition for learning Section 1: the importance of self-regulation and metacognition for learning Drawing on constructivist theories of learning, the idea of self-regulation is that students should take active responsibility for aspects of their own learning (Zimmerman, 2001). Although there is a wide range of theoretical perspectives about self-regulation (Zeidner, Boekaerts & Pintrich, 2000), definitions tend to focus on a combination of metacognitive and motivational strategies (Zimmerman, 1986) and a more pro-active approach by learners (Winne, 2011). Boekaerts (1999) identifies three aspects of self-regulated learning in terms of cognition, metacognition, and motivation/affect. Cognition relates to the range of cognitive strategies that learners use, with metacognitive strategies used to manage, control and regulate this cognition, such as through planning, 4 monitoring and evaluating specific cognitive strategies and the motivational and volitional components supporting affective and conative aspects of behaviour such as resilience and perseverance (Boekaerts & Corno 2005). As Winne (2005) observes, all learners in academic contexts must to some extent self- regulate. The question is how to ensure that this is optimal, with the challenge for practice being whether teachers can change their classrooms to encourage in self-regulated learning among their students (Zimmerman, 2008). These ideas are also related to contemporary ideas from policy and practice about ‘learning to learn’ (Higgins, Wall et al., 2007) and also reflect a shift in policy, particularly in Europe, where the documents such as the ‘Framework of Life-long Learning’ (EU Council 2002), argues that we need students who can learn in a self-regulated way during school, and even more importantly after schooling and throughout their working life. A number of meta-analytic reviews have consistently indicated the effectiveness of meta-cognitive and self-regulation approaches (Abrami, Bernard et al., 2008; Chiu 1998; Dignath, Buettner & Langfeldt, 2008; Haller, Child & Walberg, 1988; Higgins, Baumfield & Moseley, 2005; Klauer & Phye 2008). There is also growing evidence that not only are they effective, but that they are relatively more promising than other approaches. This idea builds on the work of researchers such as Fraser, Walberg, Welch and Hattie (1987), Sipe and Curlette (1997), Marzano (1998) and Hattie (2008) who have argued that one of the benefits of meta-analysis is that we can draw comparative inferences between meta- 5 analyses across different areas of research. analyses across different areas of research. analyses across different areas of research. Meta-analysis aggregates data across research studies by using a common metric, effect size, to compare studies. As a statistical technique to look at similar studies on a particular topic it is relatively uncontroversial. However it is also tempting to look at results across different kinds of studies with a common population, so to provide more general or more comparative inferences. This approach is, of course, even more vulnerable to the classic “apples and oranges” criticism of meta-analysis which claims you can’t really make a sensible comparison between different kinds of things. However, as Gene Glass (2000, p. 6) said, “Of course it mixes apples and oranges; in the study of fruit nothing else is sensible; comparing apples and oranges is the only endeavor worthy of true scientists; comparing apples to apples is trivial.” A number of studies have attempted to take meta-analysis to this further stage, by synthesising the results from a number of existing meta-analyses and producing what has been called a ‘meta-meta-analysis’ (Kazrin, Durac & Agteros, 1979), a ‘mega-analysis’ (Smith 1982), ‘super-analysis’ (Dillon, 1982) or ‘super- synthesis’ (e.g. Sipe & Curlette, 1997). There are some differences apparent in researchers’ intentions in this further aggregation. Some use each meta-analysis as the unit of analysis in order to say something about the process of conducting a quantitative synthesis and identifying statistical commonalities which may be of importance (e.g. Bloom, Hill, Black & Lipsey, 2008; Ioannidis & Trikalinos, 2007; Lipsey & Wilson, 2001), particularly in terms of the effect of factors such as 6 research design or effect of the choice of different outcome measures. Others, however, attempt to combine different meta-analyses into a single message about a more general topic than each individual meta-analysis can achieve. Even here, there appear to be some differences. Some retain a clear focus, either by using meta-analyses as the source for identifying original studies (e.g. Marzano, 1998) in effect producing something that might best be considered as a larger meta-analysis rather than a meta-meta-analysis. Others, though, make claims about broad and quite distinct educational areas by directly combining results from different meta-analyses (e.g. Fraser et al. 1987; Sipe & Curlette, 1997). 7 The most ambitious of these ‘super-syntheses’ to date is a collation of more than 800 meta-analyses (Hattie, 2008) which produces some interesting conclusions. analyses across different areas of research. First of all, it identifies that most things in education ‘work’, as the average effect size is about 0.4. Hattie then uses this to provide a benchmark for what works above this ‘hinge’ point as particularly beneficial approaches. There are, of course, some reservations about this ‘hinge’ as small effects may be valuable if they are either cheap or easy to obtain, to tackle an otherwise intractable problem. Similarly large effect sizes may be less important if they are unrealistic and if they cannot be replicated easily in classrooms by teachers. Further reservations about combining effect sizes of different kinds suggest that intervention effects should be distinguished from maturational differences or correlational effects sizes. The distributions in these studies may be of different kinds, so that unlike comparing fruit, it is more like comparing an apple with a chair (Higgins & Simpson, 2011): the effect size of the difference between the 7 performance of girls and boys (0.12) is of a different kind from the impact of approaches to individualise instruction (0.2). With a narrower focus on approaches to improving learning, however, approaches like reciprocal teaching (0.74), feedback (0.72) and meta-cognitive strategies (0.67) are all identified as particularly valuable in Hattie’s (2008) analysis. performance of girls and boys (0.12) is of a different kind from the impact of approaches to individualise instruction (0.2). With a narrower focus on approaches to improving learning, however, approaches like reciprocal teaching (0.74), feedback (0.72) and meta-cognitive strategies (0.67) are all identified as particularly valuable in Hattie’s (2008) analysis. A report (Higgins, Kokotsaki & Coe, 2011a) for the Sutton Trust, a UK charity which focuses on underachievement in education, has developed this approach in order to identify ‘best bets’ for schools based on the evidence from meta- analysis. The policy context for this was the allocation of the Pupil Premium in England for each of their disadvantaged pupils allocated as part of their funding. These pupils were identified as those who were eligible to receive free school meals or who had previously received free school meals in the past six years. The inclusion criteria for the review aimed to produce a set of meta-analyses sufficiently similar for comparison to enable the cost-effectiveness to be estimated across the different interventions and approaches. These criteria included similar research designs and broadly similar intervention fields (i.e. analyses across different areas of research. interventions in schools with similar outcome measures: Hill, Bloom, Black & Lipsey, 2007) with the population clearly specified, as effect sizes are likely to differ for various sub-populations and to reduce with age (Bloom et al., 2008). The intervention approaches selected were those identified by the government, such as one-to-one tutoring or reducing class sizes or the adoption of school uniforms. Initially the review focused on the cost-benefit of these suggestions, and further areas were added in response to teachers’ ideas about how they The intervention approaches selected were those identified by the government, such as one-to-one tutoring or reducing class sizes or the adoption of school uniforms. Initially the review focused on the cost-benefit of these suggestions, and further areas were added in response to teachers’ ideas about how they 8 would be likely to spend the Pupil Premium, such as the appointment of additional teaching assistants, and additional categories were drawn from research summaries of effective approaches (e.g. Hattie, 2008; Sipe & Curlette, 1997). The analysis therefore tried to identify which approaches were more likely to be beneficial than others and looked at over 50 meta-analyses as a source of comparative data. Our conclusions were, similar to Hattie’s (2008), that the most successful interventions focus on the process of teaching and learning and the quality of teaching and learning interactions either involving the teacher (particularly when providing feedback) or supporting the learner to monitor themselves (metacognition and self-regulation) or each other (peer tutoring and peer-assisted learning). All of these also involve the learner in working harder at learning, often requiring increased cognitive effort. By contrast structural or organizational approaches (such as forms of grouping or individualized instruction) tended to have smaller effects (see Figure 1). For each area an indicative effect size was selected from the meta-analyses or other studies available. For full details of the studies, methodology and distribution of effects see Higgins, Kokotsaki and Coe (2011b). Figure 1: About here There are, of course, some limitations and caveats to this approach. Effect size is a standardized metric, usually the difference between two groups divided by the pooled standard of these groups. As such it is vulnerable to a number of issues 9 related to the methods of calculation and in particular the standard deviation chosen. The basic concept is a powerful one as it focuses on improvement, relative to the distribution or spread of scores. However, the underlying comparability is crucial to the meaningfulness of the result. So, if you are looking at similar populations of school pupils with similar underlying circumstances then such comparisons may be valid. Comparing approaches in other contexts such as approaches which work with young children compared with which those which work with older pupils may not be reasonable as the distribution of scores changes with age (Bloom et al. 2008). Studies of interventions with younger children tend to have higher effect sizes as the standard deviations tend to be smaller. Another issue with meta-analysis is that it looks at averages. All of the approaches have a range of effects which are combined into a pooled average. Some examples of the technique or approaches will have been extremely successful, others less so. What meta-analyses tells you is how effective an approach is on average, indicating which interventions are more or less likely to be productive in other settings and contexts. However there are no guarantees that the findings will transfer to a new context. There is also a problem in terms of the changing nature of the context, such as with information and communications technology (ICT). Technology has advanced rapidly over the last 30 years so the findings of the effects of technology supported learning from the 1980s may well not apply in 2013. Also 10 although ICT is easily identifiable as an approach, the type of technology and they way that it is used may make a difference. Averaging technology effects over time may therefore be misleading. Overall the approach has a number of strengths, particularly as it is the only way to compare effects across areas of research using quantitative data. Considerable caution is needed in interpreting the differences without understanding the limitations of the technique. Figure 1: About here Whilst some tentativeness is clearly needed in drawing conclusions, metacognitive and self-regulatory approaches are at the high end of the distribution of effects suggesting that their use is likely to be productive for improving learning in schools. Considerable caution is needed in interpreting the differences without Another strength of meta-analysis is that it can identify features associated with greater or smaller improvement by looking at factors (or ‘moderator variables’) associated with the pattern of effects across a comparable set of studies, so the final part of this section looks at what the messages are from meta-analysis of intervention research in education about self-regulation and metacognition. 11 Dignath et al. (2008) summarise the most effective characteristics of interventions and suggest (p. 121) that programmes should be based on social- cognitive theories, should focus on strategy training: particularly elaboration and problem solving strategies at the cognitive level, planning strategies at the metacognitive level, and planning and feedback strategies at the motivational level, with a focus on providing knowledge to learners about use of strategies and about their benefits. Group work is challenging and primary school pupils in 11 particular will need support to develop their skills in this area. Haller et al.’s (1988) early work on reading comprehension indicated that teaching self- questioning strategies are particularly important. Chiu’s (1998) analysis of reading interventions suggests that meta-cognitive approaches are more effective with low attainers, work better with slightly older students (9-11 year olds) in small groups (rather than individual or class teaching) and that less intensive programmes are more effective than more intensive. Two of these findings are counter-intuitive. Usually what works well for low attaining students works as well or even better for higher attaining pupils and usually more intensive programmes of short duration are more effective than longer ones with less intensity (Hattie, 2008). Perhaps what works for low attainers is that meta-cognitive approaches make aspects of learning more explicit, while high performing learners can work out strategies for themselves. Perhaps time is also needed for all young learners to transfer or apply newly acquired skills for them to have a lasting effect. Findings from Abrami et al. (2008) and Higgins et al. (2005) indicate that direct teaching of thinking is more effective with a meta-cognitive component than a purely cognitive approach. Figure 1: About here A mixed approach is beneficial which allows for direct teaching combined with an infused approach (Effect size (ES) = 0.94 compared with 0.38 for general thinking skills and 0.54 for infusion: Abrami et al., 2008, p. 1118). Again the evidence seems to indicate the effectiveness of explicit teaching of strategies with an emphasis on conscious application and use. 12 Section 2: Meta-cognition and self-regulation in schools: older pupils can but don’t Section 2: Meta-cognition and self-regulation in schools: older pupils can but don’t but don’t This section of the paper turns to look at some evidence about what kinds of thinking learners in school typically tend to show in class when given the opportunity to think about their learning. Some aspects of self-regulation can be thought of as part “doing school”, learning to behave in accordance with school expectations and norms, such as lining up, conforming to routines and routines. For young children this may be a more implicit introduction to the culture of formal schooling than for older learners where it tends to be more explicit (Campbell & Ramey, 1995). There is also evidence that maturation is more important than direct experience of school (Skibbe, Connor, Morrison & Jewkes 2011) though approaches which focus on teaching self-regulation explicitly show potential for boosting young children’s skills in this area (Bodrova & Leong, 2005; Winne, 2011). As part of the Learning to Learn in Schools (Phase 4) project, teachers administered pupil views templates (Wall & Higgins, 2006; Wall, Higgins & Packard, 2007) to pupils in their classes (see Figure 2 for an example of a completed template). These have been shown to elicit children and young people’s thinking about their learning (Wall & Higgins, 2006). In total, templates from 355 pupils from the first year of this project were analysed. The completed templates were from a total of 12 schools comprising both primary and secondary age pupils from a variety of geographical and socio-economic regions 13 across England (see Wall, Hall et al., 2009 for further details). The age range of pupils who completed templates was from 4 to 15 years old. The teachers administered the templates as part of their professional enquiries into learning (Baumfield, Hall, Higgins & Wall 2009), and they received support in their use through the professional development and research support strand of the research project (Wall et al. 2009). Figure 2: About here The written content of each template was transcribed and imported into NVivo8 for analysis using a deductive coding procedure (described below). A code was applied based on the sense and meaning of a pupil’s response with a judgment made by the researchers as to the intended meaning, and a category code applied accordingly. A category could therefore be applied to a single word, to a sentence fragment, a full sentence or a paragraph. Results are presented in terms of total words coded as the most sensitive output of NVivo (both proportionally and in relation to the research aims). 14 In the first stage of analysis, documents were coded according to the following variables: school, length of school’s involvement in the project, gender, age and which year of Phase 4 the templates were collected in. We were restricted to gender and age as variables about the pupils in terms of the permissions for use of the data across all of the schools involved. Ideally further areas such as current level of attainment or socio-economic status would have been interesting to explore. The text units were also tagged at this stage with whether they were 14 written in the speech bubble or thought bubble. In the second stage of the analysis the statements were categorised using Moseley and colleagues model of thinking (Moseley, Elliot, Gregson & Higgins, 2005; Moseley, Baumfield et al. 2005: see Figure 3). Figure 3: About here This model of thinking was chosen as it is based on an inclusive synthesis of 42 taxonomies and frameworks of thinking (Moseley, Baumfield et al. 2005), and has been used to create categories with a high reliability across different coders (Wall, Higgins et al, 2012). The statements were categorised as to whether they were predominantly evidence of cognitive skills (information gathering, building understanding, or productive thinking); and/or whether they were evidence of metacognitive thought (strategic and reflective thinking in Moseley, Baumfield et al.’s 2005 model). The following definitions based on this analysis were used. Information gathering is characterised by recall of ideas and processes and recognition or basic comprehension of information they have been told or have read. Building understanding requires some organisation of ideas and recollections, some idea of relationships or connections, with some development of meaning about implications and/or patterns which could be applied or interpreted. Productive thinking comments tended to show more complex thinking such as reasoning, problem solving and some movement of understanding beyond the concrete and towards the abstract. Ideas that were more clearly generalisable or creative were placed also in this category. Strategic 15 and reflective thinking comments represented an awareness of the process of learning, including a reflective or strategic element to the statement or explicit thinking about learning (metacognitive awareness of learning). The statements which were labeled as strategic and reflective, and therefore indicative of metacognition, were then further analysed for evidence of metacognitive knowledge and metacognitive skillfulness (Veenman, Kok & Blöte, 2005). These categories were characterised in the following ways. Metacognitive knowledge comments showed an understanding that the learner could think about learning, and could talk about some of the processes which supported their own learning (declarative knowledge). Metacognitive skillfulness comments involved the procedural application and translation of thinking and learning skills across different contexts or for different purposes (for definitions and further clarification see also Veenman & Spaans (2005), p 160). This coding system was checked for inter-rater reliability with an agreement of 82%. Exemplification of the coding can be seen in Table 1 where examples of each coding category are given. All the examples were taken from the same school where teachers were focusing their professional enquiry on how Circle Time (a classroom strategy to support children’s reflections on their learning) could support children in talking about their learning experiences. Table 1: About here Table 1: About here Statistical analysis was conducted using a fully between-subjects 3 (age) x 2 (gender) two-way factorial ANOVA. Sample sizes are shown in Table 2. Figure 3: About here These templates come from a class including Year 1 and 2 pupils (age 5, 6 and 7 years old). It should be noted that the categories used were not necessarily mutually exclusive and a single text unit could be classified as fitting under more than one 16 heading so percentages in the following graphs do not necessarily total to 100%. Table 1: About here Table 2: About here The purpose of the analysis was to find out whether there would be differences in five dependent variables based on rater’s scores of pupils’ ability to use different cognitive skills in their descriptions of their learning. These skills were Information gathering (IG), Building understanding (BU), Productive thinking (PT), Meta- cognitive knowledge (MK) and Meta-cognitive skillfulness (MS). These five dependent variables were mapped against two factors, age (three levels: age 4-7 years, age 7-11 years and age 11-15 years (these age bands correspond with the ‘Key Stages’ of schooling in England) and against gender (male and female). The hypothesis was that more complex, productive and meta-cognitive thinking would be more evident in older learners (Skibbe et al., 2011, p 47) and that young children would show limited meta-cognitive knowledge and skillfulness (Veenman, Kok & Blöte 2005, p 197). Veenman and Spaans (2005, p 162) argue that metacognitive awareness may start at the age of 4–6 years as an inclination that something is wrong and that metacognitive knowledge grows gradually thereafter, but the suggest development of metacognitive skills does not usually appear until the age of 11–12 years. By contrast, Whitebread and colleagues 17 argue that meta-cognitive knowledge and meta-cognitive regulation are observable in 3-5 year old children (Whitebread et al., 2008) and Wall (2012) describes both meta-cognitive knowledge and skillfulness reported by 4-5 children. The existing literature does not provide any clear expectations regarding different developmental trajectories of meta-cognition by gender. In terms of cognitive development, gender differences are typically small, argue that meta-cognitive knowledge and meta-cognitive regulation are observable in 3-5 year old children (Whitebread et al., 2008) and Wall (2012) describes both meta-cognitive knowledge and skillfulness reported by 4-5 children. The existing literature does not provide any clear expectations regarding different developmental trajectories of meta-cognition by gender. In terms of cognitive development, gender differences are typically small, accounting for only 1-3% of the variance in performance (Ardila, Rosselli, Matute & Inozemtseva, 2011), though there is also some evidence that girls tend to be better at self-regulation and self-discipline (Duckworth & Seligman, 2006; von Suchodoletza, Gestsdottir et al., 2012). Due to the lack of clarity in the research literature, we therefore also undertook an exploratory analysis by gender. Table 2: About here accounting for only 1-3% of the variance in performance (Ardila, Rosselli, Matute & Inozemtseva, 2011), though there is also some evidence that girls tend to be better at self-regulation and self-discipline (Duckworth & Seligman, 2006; von Suchodoletza, Gestsdottir et al., 2012). Due to the lack of clarity in the research literature, we therefore also undertook an exploratory analysis by gender. Summary of findings Before presenting the specific findings, Table 3 summarises the results of the analysis across the five dependent variables. Table 3: about here This reveals that there were very few main effects for gender but there were consistent main effects for age. Only one significant interaction effect was observed (Building Understanding), though the interaction effects for Productive Thinking and Metacognitive knowledge were only marginally non-significant. The next section examines the findings for the individual measures in more detail. 18 18 For the category of Information Gathering, the two-way ANOVA revealed a main effect for age but no effects for gender, nor was there an interaction effect (see Table 3). The patterns of means are shown in Fig 4. For the category of Information Gathering, the two-way ANOVA revealed a main effect for age but no effects for gender, nor was there an interaction effect (see Table 3). The patterns of means are shown in Fig 4. Figure 4: about here This shows that contrary to expectation, 7-11 year old pupils used the strategy of information gathering more compared with 4-7 year olds and the 11-15 year olds more than both younger groups. Simple main effects analyses revealed that these differences were significant (see Table 3). For the measure of Building Understanding, the findings were more complicated. Similar to Information Gathering, the two-way ANOVA revealed a main effect but here there was also an effect for gender and, importantly, there was also an interaction effect. Figure 6: About here Figure 6 shows a similar pattern observed for Building Understanding, whereby 7-11 year old pupils described using this kind of thinking significantly more compared with both other age groups. Also, although the interaction effect was not statistically significant (p = .06), simple main effects analysis revealed that the means for 4-7 year old boys and 11-15 year old boys were significantly different from both 7-11 year old boys at KS2 and girls. This difference should be interpreted with caution, however, as the interaction effect was not statistically significant. No other conditions were significantly different from each other. For the measure of Metacognitive Knowledge, the findings were again similar to Building Understanding, but this time main effects were observed for both age and gender. The interaction effect was marginally non-significant (p = .07). The patterns of means are shown in Figure 7. Figure 5: About here Figure 5 show that in line with expectations, 7-11 year old pupils used the cognitive thinking skills involved in Building Understanding more compared with 4-7 year olds (see Table 3). Contrary to expectations the oldest pupils (11-13 years) described using Building Understanding less. Simple main effects analysis of the interaction (Ho, 2006, p 64-71) indicated that the mean for 7-11 year old girls was significantly different to all the other means. No other conditions were significantly different from each other. 19 For the measure of Productive Thinking, the findings were similar to Building Understanding, with the two-way ANOVA revealing a main effect for age. There were no effects for gender nor was there an interaction effect. The patterns of means are shown in Fig 6. Figure 6: About here 20 This reveals the now familiar pattern observed for Building Understanding 20 This reveals the now familiar pattern observed for Building Understanding 20 whereby 7-11 year old pupils described these kinds of thinking significantly more compared with both other age groups. This was true for both boys and girls. Because the interaction effect was marginally non-significant (p = .06), a simple effects analysis was carried out. This analysis revealed that the mean rating for 7-11 year old girls was significantly different from all other means. In addition, the mean rating for 4-7 year old boys was significantly different from 7-11 year olds girls. No other conditions were significantly different from each other. whereby 7-11 year old pupils described these kinds of thinking significantly more compared with both other age groups. This was true for both boys and girls. whereby 7-11 year old pupils described these kinds of thinking significantly more compared with both other age groups. This was true for both boys and girls. Because the interaction effect was marginally non-significant (p = .06), a simple effects analysis was carried out. This analysis revealed that the mean rating for 7-11 year old girls was significantly different from all other means. In addition, the mean rating for 4-7 year old boys was significantly different from 7-11 year olds girls. No other conditions were significantly different from each other. For the measure of Metacognitive Skillfulness, the findings were similar to Productive Thinking, namely a main effect for age but no effects for gender, nor an interaction effect. The patterns of means are shown in Figure 8. Figure 8: About here This shows the by now relatively consistent pattern for more complex kinds of thinking where skill usage is significantly more prevalent for 7-11 year olds compared with both younger and older children. The results are something of a puzzle. The expected pattern of reported thinking increasing with age only occurred for the least complex kind of thinking, Information Gathering. For Building Understanding and Productive Thinking the 11-15 year olds described these kinds of cognitive skills less frequently than the 7-11 year olds. Even more puzzling is that this pattern was repeated for both Metacognitive Knowledge and Metacognitive Skillfulness. The schools were relatively comparable in terms of attainment and were all seeking actively to 21 support pupils’ learning through the Learning to Learn project. The teachers were all similarly engaged in undertaking enquiries into their professional practice. It could be that the opportunity sample of schools was not sufficiently could be that the opportunity sample of schools was not sufficiently representative to produce an accurate picture or that some of the teachers of the 7-11 year olds have been more successful in developing learning to learn and metacognitive talk. We know that the kinds of thinking that pupils express is related to their expectations about what they should do in school: a feature of “doing school” (Pope, 2003). Our tentative interpretation at this stage is that, at least for these schools, the older students did not expect to have to demonstrate more complex thinking in the task. It may be that the template format was less successful at eliciting different kinds of thinking from older learners because of the cartoon style of presentation and so provides a less valid or less reliable measure. We know that there are particular challenges of this kind in the assessment of metacognition (Veenman, Van Hout-Wolters & Afflerbach, 2006). Or it may be that the demands in lessons at secondary level are not challenging the students to engage in more complex thinking and that they therefore do not routinely expect to demonstrate it. This, in turn, may relate to the nature of the curriculum and its assessment with a focus on information gathering and building understanding, rather than more complex aspects of thinking. Figure 8: About here Whatever the explanation, it is certainly the case that the primary schools involved were more successful at providing a context where meta-cognition and self-regulation were more readily or more fluently expressed by their pupils. 22 Taken with the evidence from the first part of this paper, this suggests that they 22 are being more successful at supporting some of the more effective approaches to improving learning through metacognition and self-regulation. By contrast, secondary learners may not be being challenged in a way in which they are thinking so productively about their learning, even though they are at an age where they are capable of doing this. 23 23 References Abrami, P.C., Bernard, R.M., Borokhovski, E., Wade, A., Surkes, M.A., Tamim, R. & Zhang, D. (2008). Instructional Interventions Affecting Critical Thinking Skills and Dispositions: A Stage 1 Meta-Analysis. 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DOI: 10.3102/0002831207312909 32 32 Figure 1: Approaches and effect sizes -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8Effective feedbackMeta-cognition and self-regulationPeer tutoring/ peer-assisted…Early interventionOne-to-one tutoringHomeworkICTAssessment for learningParental involvementSummer schoolsReducing class sizesSports participationAfter school programmesLearning stylesIndividualised instructionArts participationTeaching assistantsAbility groupingSchool uniforms Effect size Approach Approaches and effect size estimates 33 33 Figure 2: An example of a completed Pupil Views Template Figure 2: An example of a completed Pupil Views Template 34 34 Figure 3: Moseley et al.’s model of thinking 35 35 Table 1: Coding categories Table 1: Coding categories Table 1: Coding categories Information gathering In Circle Time we share our thoughts and smiles Building understanding I like Circle Time because you tell other children about you Productive thinking I didn’t feel nervous because I got to know the other children and new friends. Strategic & reflective thinking Metacognitive knowledge Circle Time is a bit scary because sometimes you have to speak in front of everyone. Metacognitive skilfulness If people are stuck on a work (sic), asking the person or a friend to help you. 36 36 Table 2: Sample sizes broken down by Age and Gender Age 4-7 years 7-11 years 11-15 years Totals Male 80 38 49 167 Female 87 39 62 188 Totals 167 77 111 355 37 37 Table 3: Summary of main effects and interactions for the five dependent variables Dependent variable Main Effect Gender Main Effect Age Gender x Age interaction F p η2 F p η2 F p η2 Information Gathering 1.96 (1, 349) .88 59.58 (2, 349) <.001 .25 46.85 (1, 349) .58 Building understanding 19.22 (1, 350) <.001 .05 23.66 (2, 350) <.001 .12 5.19 (2, 350) <.05 .03 Productive thinking 3.72 (1, 350) .06 81.94 (2, 350) <.001 .07 2.64 (2, 350) .06 Meta-cognitive knowledge 29.84 (1, 349) <.001 .08 20.58 (2, 350) <.001 .11 2.76 (2, 350) .07 Metacognitive skilfulness 0.70 (1, 349) .40 7.31 (2, 349) <.001 .04 1.42 (2, 350) .24 F= F value, p= p value, η2 = partial eta squared effect size Table 3: Summary of main effects and interactions for the five dependent variables 38 38 Figure 4: Means for Information Gathering by Age and Gender 39 39 Figure 5: Means for Building Understanding by Age and Gender 40 Figure 6: Means for Productive Thinking by Age and Gender 41 Figure 7: Means for Metacognitive Knowledge by Age and Gender 42 42 Figure 8: Means for Metacognitive Skilfulness by Age and Gender Figure 8: Means for Metacognitive Skilfulness by Age and Gender 43 43
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Fundamentalabbildung und Potentialbestimmung gegebener Riemannscher Fl�chen
Mathematische Zeitschrift
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Fundamentalabbildung und Potentialbestimmung gegebener Riemannscher Fl~chen. Von Paul Koebe in Jena. Es sei F ein endlich-vielbliittriger, endlich-vielfaeh zus&mmenhiingender, analytisch begrenzter Bereich mit endlich vielen Windungspunkten im Innern. Wiihrend die alten Methoden die Abbildungsaufgabe als Anwendung potentialtheoretiseher ~) Entwicldungen behandeln, gest&tten die neuen, rein ~nktionentheoretischen Methoden der konformen Abbildung eine Umkehrung der Reihenfolge der Problemstellungen vorzunehmenl~). 1. Riinderzuordnung. Wir k6nnen die Fundamentalabbildung der Fliiche F rein funktionentheoretiseh ausfiihren, d. i. die eineindeutige konforme Abbildung der zu F geh6renden einfaeh zusammenhiingenden relativ unverzweigten Uberage~rungsfl~iche F ~ auf die Fl~iehe eines ~-Einheitskreises. Denken wit uns diese Abbildung zuniiehst nur fiir die inneren Punkte des Bereiehs erkliirt, so kann nunmehr durch eine besondere Untersuchung dargetan werden, daft die Abbildungsfunktion ~ (z) auoh liings der Randlinien regul~, in den E~kpunkten zwischen den einzelnen regul~ analytisehen Begrenzungsteilen jedenfalls stetig ist. Dies ergibt sich nnter Einfiihrung einer bemerkenswerten Modifikation gegeniiber meinen friiheren Entwicklungen zur Frage der Riinderzuordnung~) bei anslytisch oder allgemeiner stetig begrenzten Bereiehen folgendermaflen. 1) R i e m a n n - H i l b e r t : Dirichletsohes Prinzip; S c h w a r z und C. N e u m a n n : Alternierendes Veffahren; P o i n c a r 6 : Methode de balayage. 11). Vgl. eine Bemerkung am Schlusse meines Artikels, ,Uber eine neue Methode der konformen Abbildung und Uniformisierung ~, GStt. Naohr. 1912, S. 848. 2) S. meinen Artikel, ,R~inderzuordnung bei konformer Abbildung", GStt. Ns~hr. 1918, S. 286--288 und besonders ,Abhandlungen zur Theorie der konformen Abbildung. I", dritter Teil, Journ f. M~th. 14~ (1915), S. 205---219.~ P. Koebe. Fundamentalabbildung u. Potentialbestimmung Riemsnnscher Fl~chen. 249 Die Fl~ehe F kann als Teil einer Fliiche F' aufgefaflt werden, die aus F dutch iiuflere Anfiigung zweifach zusammenhiingender schmaler Fl~henstreifen entsteht~ Die Funktion t ' (z), die die Fundamentalabbildung der Fl~iche F' leisteL liefert gleichzeitig eine v~llig regul~re Ubertragung der Fliiche F ~| auf einen schlichten einfach zusammenh~ingenden Bereich ~. Die Untersuchung der R~uderzuordnung fiir die Fundamentalabbildung der Fliiche F i s t dadurch auf die Untersuchung der Riinderzuordnung fiir die Abbildung des Bereiches ~ auf das Inhere des ~-Einheitskreises zuriickgefiihrt, d.h. auf die Untersuchung der Abbi!dungsfunktion ~(~'). Sei ~ ein analytisches Begrenzungsstiick des Bereichs ~. Die Funktion ~(~') mul~ sich dann in jedem Begrenzungspunkte t0 von 2 zun/iehst stetig verhalten. !Denn andernfalls wiirde man innerhalb ~5 in beliebiger N~ihe der Ste]le ~0 Linienstiicke beliebig kleiner L~inge angeben k6nnen, denen in der t-Ebene Linien entsprechen wiirden, die sich gleichmiil~ig der Peripherie des Einheitskreises ann~ihern und dabei kmplitudenschwankungen h~itten, die fiir alle diese Linien oberhalb einer yon Null verschiedenen Schranke y bleiben. Dies bedingt, daft es ein endliches Peripheriestiick des t-Einheitskreises gibt, l~ings dessen ganzer Ausdehnung solche Linien in beliebiger N~ihe verlaufen wiirden. Bedenkt man nun, daft die Funktion ~'(t) innerhalb des ~-Einheitskreises beschriinkt ist, daft ferner die Funkfionswerte auf den genannten Linien sich schliel31ich auf den Wert to reduzieren, so folgt, daft die Funktion t ' (t) iiberhaupt eine Konstante wiire~a). Da dies jedoch nicht der Fall ist, ergibt sich somit die Stetigkei~t der Funktion t ( ~ ' ) und folglich auch der Funktion ~(z) liings jedes analytischen Begrenzungsteiles, otienbar aueh in den Eeken, die yon analytischen Begrenzungsteilen gebildet werden, ferner iiberhaupt auch fiir Begrenzungsteile vom Charakter Jordanseher Kurvenstiieke. Wir wollen die angedeutete Verallgemeinerung auf Jordansche Begrenzungsstiicke hier nieht welter veffolgen, verweisen diesbeziiglieh vielmehr auf unsere oben zitierte Abhandlung. Wir setzen jetzt die Betrachtung fiir ein analytisches B%oTenzungsstiick in folgender Weise fort. Die Funktion t(~'), yon der wir bereits wissen, daft sie liings eines analytischen Begrenzungsstiiekes ,~tetig ist, kann niemals liings eines solchen Teils oder eines Stiickes davon konstant sein. Denn dies wiirde wiederum zur Folge haben, daft die Funktion t (t') sich iiberhaupt auf eine Konstante reduziert. Somit wird ein analytisehes Begrenzungsstiick ~ des Bereiches und damit aueh ein analytisches Begrenzungsstiick l des Bereiches F unter Aufrechterhaltung der Anordnung der Punkte stetig eineindeutig auf ein entsprechendes Stiick der Peripherie des ~-Einheitskreises iibertragen. ~a) Siehe meine genannte Abhandlung in Journ. f. Math. 14[5, S. 213. 250 P. Koebe. Um nun weiter die Regutaritdt der Abbildungsfunktion ~(z)l~ngs l naehzuweisen, gehen wir yon dem gewonnenen Ergebnis der Stetigkeit dieser Abbildung aus. Wit maehen eine reguliire Hilfsabbi]dung eines 1 einbettenden Fliichenteiles, bei der l selbst in ein Stiiek BC der Aehse des Reellen iibergeht. Es sei z'(z) die Abbildungdunktion; es sei ferner A ein Punkt innerhalb des Inter*calls BC. Dann beschreiben wir um A als Mittelpunkt einen kleinen Kreis, der eine ganz in dem gefundenen Fl~henstreifon enthaltene Kreisfliiche K einsclfliel~t. Die obere Halbkreisfl~iche K ' ersohei'nt jetzt auf ein zweieckf6rmiges Teilgebiet ~o der ~-Einheitskreisfl~iche abgebildet, dessen Begrenzung aus zwei analytisehen Linienstiicken besteht, einem innerhalb des ~-Einheitskreises verlaufenden und einem Stiick fl~, des ~-Einheitskreises selbst. Durch lineare Transformation verwandeln wit das Gebiet ~ in ein anderes 90', indem wir dem Intervall f17 ein Intervalt fl'~,P der Achse des Reellen entsprechen lassen. Spiegeln wit ~ an der Achse des ReeUen, so entsteht ein grSl~erer symmetrischer Bereich (~'-~-~v'). Es ist dann eine Abbildungsbeziehung zwischen ~der Kreisfl~iche K und dem Gebiete ( ~ ' ~ ~,r) hergestellt, bei der dem reellen Intervalt B C das reeUe Intervall fit 7' stetig entspricht. Nun kann man abet eine solche Abbildung zwischen K und ( ~ ' ~ ~") auch direkt dadurch gewinnen, da~ man die Fundamentalabbildung des Bereiches ( ~ r ~ ~,,) auf die F l ~ h e K vornimmt, wobei auch der Symmetrie geniigt werden kann. Diese Abbildung ist innerhalb des reellen Intervalls jedenfalls regular. Sie mul~ mit der vorbetrachteten Abbildung iibereinstimmend werden, wenn man abgesehen von der bereits richtig beibehaltenen Zuordnung der Intervallendpunkte auch noch von einem inneren P u n ~ e des Intervalls den zugeordneten Punkt in derselben Weise bestimmt wie bei jener Abbildung. Man hat dann n~imlieh zwei Abbildungen der F l ~ h e ~' auf die Halbkreisfl~iehe K ~, bei der drei Randpunktpaare in gleicher Weise zugeordnet sind. 2. Gew~hnliehe Randwertaufgabe. Es sei nunmehr die gew~hnliehe Randwe~taufgabe fiir den Bereich F zu 15sere Wir denken uns dazu die Fl~iche F zweckm~i~ig l~ngs q ~ 2p Querschnitten yon Rand zu Rand zu einer einfach zusammenh~ngenden Fl~che -~o aufgesehnitten. Die Fl~che F (~) entsteht durch sukzessive relationenfreie Aneinanderheltung unend!ich vieier Exemplare F o. Dem Grundexemplar F o entspricht in der $-Ebene ein Bereich q5o mit 2p Querschnittseiten, die paarweise dutch hyperbolisehe lineare Substitutionen einander zugeordnet sind. Diese Substitutionen erzeugen die Fundamentalgruppe U. Bei Ausiibung dieser :Fundamentalgruppe auf ~o ergeben sieh unendlich viele Bilder yon ~o, deren auf der Peripherie des $-Einheitskreises lie- Fundamentalabbildung und Potentialbestimmung Riemannscher Fl~chen. 251 gende Randteile diesen Kreis bis aui unendlieh viele diskret liegende Punkte ausfiillen. Diese Grenzpunkte lassen sich in endlich viele Inter. valle yon beliebig kleiner Gesamtliinge einsehliel~en, well die Summe der L~ngen der Bilder aller Querschnittseiten yon ~o wegen der eigentlichen Diskontinuifiit tier Gruppe /" auf der Peripherie des Einheitslcreises konvergiertS). Uberpflanzt man jetzt die gegebenen Randwerte des Bereiches F auf die Peripherie des ~-Einheitskreises, wo sie in unendlieh h~iufiger WiederhoIung gem~ifl I" erscheinen, sa l~ann man nunmehr ohne weiteres das Poissonsehe Integral flit diese Randwerte ansetzen und erh~ilt eine Potentialfunktion innerhalb des ~-Einheitskreises, die mit Riicksicht auf die Geltung des Unit~tssatzes flit die Randwertauigabe (trotz der unendlich vielen Grenzpunkte wegen der Beschr~inktheit der vorgegebenen Randwerte und wegen der erw~hnten EinschlieBungsmSgliehkeit der Grenzpunkte) gegeniiber den Substitutionen der Gruppe 1" unge~ndert bleibt, daher in der Ubertragung auf F eindeuti~ wird, wie verlangt. 3. Einfithrung yon polaren, logarithmischen, ArcustangensUnstetigkeiten, yon inneren und Rand-PeriodizitiitsmoduIn. Schreibt man aufler den Randwerten auf z~ auch noch inhere polare oder logarithmische Unstetigkeiten vor, so kann diese Aufgabe nach dem soeben gewonnenen Ergebnis auf den Fall reduziert werden, dab die Randwerte identisch null sind. Nach dieser Redul~ion lassen sieh dann die betreffenden Potentiale sofort dutch unendliche Reihen darstellen, l~an sucht im Bereiche ~o die entspreehenden Unstetigkeitsstellen auf und schreibt dort die der Ubertragung gem~i~ bestimmten Unstetigkeiten vor. Es gibt dann eine elementare Potentialfunktion, die diese Unstetigkeiten und nur diese im Innern des ~-Einheitskreises bzw. der im folgenden start der Kreisfli~che setzbaren zugrunde gelegten oberen $-Halbebene hat. Diese Potentialfunk~ion unterwifft man s~mtlichen durch die Gruppe U vorgeschriebenen Verpflanzungen und summiert die so entstehenden unendlieh vielen Potentialen. Das Resultat ist ein automorphes Potential~ das, auf F iiberpflanzt, die verlangte Potentialfunk'tion liefert4). Gibt man die Randwerte null und im Innern yon F zwei Arcustangens-Unstetigkei~en mit entgegengesetzt gleichen Periodizit~itsmoduln vor, wobei man sieh zweckm~l~ig die Fl~iehe F bzw. F o li~ngs einer die YSnstetigkeitspunkte verbindenden Linie aufgeschnitten denkt, urn die 15otentialfunktion eindeutig zu ~machen, so fiihrt das gleiche Verfahren zum a) Siehe hierzu ,Abhandlungen zur Theorie der konformen Abbildung. IV", w 6, Aeta math. 4 | (1918), S. 326--328. 4) Siehe hierzu I. c. Abh. IV der genannten Serie, w167 4, 6, 7. 252 P. Koebe. Ziele. ])as Elementarpotential in der ~-Ebene hat die geometrische Bedeutung der Summe der scheinbaren GrSl~e des BildPunktepaares iener Unstetigkeitsstellen in ~o plus der scheinbaren GrSl3e seines Spiegeipaares in bezug auf die Achse des Reellen, gesehen yore Punkte ~ aus. Das gesuchte Potential wird gefunden als Summe der scheinbaren GrSBen aller mit den beiclen genannten iiquivalenten Punktpaare, ebenfalls yore Punkte aus gesehen. Um die 2 p - ~ q - 1 auf F existierenden iiberaU endlichen Pote~tiale erster Art zu gewinnen, hat m a n einerseits das Potential zu bestimmen, das l~ngs einer Randlinie den konstariten Weft 1, l~ings der iibrigen Rand]inien den konstanten Wert 0 bat. Diese Aufgabe subsumiert sich unter die erste oben besprochene Randwertaufgabe, ist aber direkt durch Reihenblldung liisbar. E~ ergiht sich eine Darstellung als Summe der scheinbaren Gr61~en yon unendlich vielen Fixpunktepaaren (s. 1. c. w4). Zweitens mul~ man ein Potential konstruieren, das tiings aller Randlinien den konstanten Wert null annimmt, liings eines vorgegehenen nicht zerfiillenden inneren Riickkehrschnittes jedoch einen konstanten Sprung aufweist. Diese Funktion kann man aus Funktionen mit je zwei Arcustangens-Unstetigkeiten gewinnen, indem man l~ngs des Riiekkehrschnittes eine endliche Anzahl yon Unstetigkeitspuukten einfiihrt und nunmehr je zwei in der geschlossenen Reihe aufeinander folgende clieser Punkte als Paar flit zwei entgegengesetzte Arcustangens-Unstetigkeiten benutzt uncl die erhaltenen Potentiale addiert, wobei d~e Unstetigkeiten zum Wegfall und der gewiinschte Wertesprung l~ings des geschlossenen Riickkehrschnittes zum Vorschein kommt. Sind aueh Periodizitiitsmoduln l~ngs der Randlinien vorgeschrieben, so kann man, um keine Randwerte zu bevorzugen, die Randbedingung ,,normale Ableitung gleich null '''~) zweckmi~Sig heranziehen. Sind nun keine inneren Arcustangens-Unstetigkeiten vorgeSchriehen, so kann man jetzt die Randperiodizitiitsmoduln zum Versehwinden brlngen, indem man die zu den oben konstruierten Potentialen erster Art mit konstanten Randwerten konjugierten Potentiale, die dutch die betreffenden unendlichen Reihen mitgeliefert werden, herstellt und eine geeignete Kombination derselben yon der zu bestimmenden Funktion in Abzug bringt. Sind jedoch aueh Arcustangens-Unstetigkeiten in endlicher Zahl im Innern gegeben~ so kann im besonderen die Summe der dazugehiirenden Periodizitiitsmoduln nulI sein. Dann ist auch die Summe der Randperiodizitiitsmoduln gleieh null und man kann alle Rand[periodizitiitsmoduln zum Wegfall bringen, wenn man erstens eine lineare Kombination oben genannter ~) vgl. ~8 1. c. Fundamentalabbildung und Potentialbestimmung Riemannseher Fliiehen. 253 konjugierter Potentiale yon der zu bestimmenden Funktion abzieht und welter eine lineare Kombination von Potentialen mit je zwei ArcustangensUnstetigkeiten. Ist aber die Summe der zu den Areustangens-Unstetigkeiten gehSrenden Periodizit~itsmoduln nicht gleich null, "so wird man durch das Abzugsveffahren auf die Bestimmung eines Potentiales gefiihrt, das nur eine Areustangens-Unstetigkeit im Innern und einen gleich groflen Randperiodizit~itsmodul besitzt. Eine solche Funktion mit der normalen Ableitung Null am Rande l~iflt sieh dann aber als Summe yon seheinbaren Gr61~en in der ~-Ebene aufbauen. Die Stammgr61~e ist hierbei die scheinbare Gr61~e desjenigen Punktepaares, das yore korrespondierenden Punkte der Unstetigkeitsstelle und seinem Spiegelpunktbilde in .bezug auf die Aehse des Reellen der ~-Ebene gebildet wird. 4. Bildung einer zu F geh~irenden reellen algebraisehen Kurve. Die vorstehenden Entwicklungen geben auch die Grundlage ab, um dem Bereiche F eine reelle algebraische Kurve (x, y) in demselben Sinne zuzuordnen, wie dies S c h o t t k y in seiner Dissertation (Journ. f. Math. 83) iiir endlieh-vielfach zusammenh~ngende schlichte Bereiehe g~tan hat. Die GrSl~en x und y werden als zwei solche Funktionen der Variablen z bestimmt, die in $' abgesehen yon endlich vielen Polen eindeutig und regulgr :sind, ferner am Rande nur reelle Werte annehmen, ferner die Eigenschaft ihaben, dal3 alle iibrigen Funktionen w (z) mit denselben genannten Eigen:sehaften sich rational durch x und y ausdriicken lassen. Um die Funktionen x und y zu bilden, geniigt es offenbar, Potentiale' mit nur polaren Unstetigkeiten heranzuziehen, aus denen sieh durch lineare Kombination sofort solche Potentiale ergeben, deren konjugierte Potentiale sicher eindeutig sind. Man kann zwecks Bildung solcher Funktionen aueh mit Poincar6sehen O-Quotienten operieren. Hat man etwa auf solche Weise eine Kurve (x, y) gewonnen und damit den zur Fl/iche ~F gehSrenden xeellen algebraischen FunktionenkSrper, so kann man natiirlich auch von hier aus die Bildung der Potentiale bzw. deren Differentiale nach bekannten Methoden vornehmen. 5. Behandlung geschlossener Riemann.~eher Fl~ichen. Es sei F eine geschlossen~ R i e m a n n s c h e Fl~ehe. Hier bieten sich der'Behandlung zwei Wege. Entweder man konstruiert zun~ichst zu F geh6rige algebraische Funk~i0nen und damit eine zugehSrige algebraische Kurve (x, y), indem man d i e Fl~iehe F einer Fundamentalabbildung (zweckrh~13ig mit Relativverzweigung) 6) unterwirft und d~nn Poinear~'~) Siehe ,Abhandlungen zur Theorie der konformen Abbildung. 1I", w 8, Acta math. 40 (1916), S: 287--290. 254 P. Koebe. Fundamentalabbildung u. Potentialbestimmung Riemannscher FIKchenr sche O-Quotienten in Ansatz bringt, darnach die gewiinsehten Potentiale nach bekannten Methoden herstellt; oder abet, man verwandelt die Fliiche F zuniichst in eine berandete Fl~iche F ' dutch Entfernung etwa einer kleinen Kreisseheibe k~ Mit den zu F ' gehSrenden Potentialen kann man dann unschwer durch Grenziiber.gang, indem man k sich auf einen Punkt reduzieren li~l]t, zu den .Potentlalen der Fl~iche F gelangen, wie dies yon mir in w4 der Abh. III der wiederholt genannten Serie in einem bestimmten Falle dargelegt ist~). Es maeht dabei niehts aus, aueh mehrere 0ffnungen gleichzeitig einztdiiltren. Von diesen ]etzten Bemerkungen kann man zweckmiit~ig Gebraueh maehen, indem man aus der Riemannschen Fi~iehe F durch alle Bliitter hindureh Kreisseheiben ausgestanzt denkt, so, daft dabei s~mtliche Windungsptmkte zum Fortfall kommen. Die zur so entstandenen oi~enen Fliiehe F gehSrige Fundamentalveriinderliche ohne relative Verzweigung ist dann identiseh mit der Fundamentalver~inderliehen des schliehten mehrfaeh zusammenh~ingenden Bereiches, iiber dem die Fl~iehe F ausgebreitet isiS). Sehliel]lieh sei noch bemerkt, dal3 jede gesehlossene R i e m a n n s c h e Fl~iche F durch elementare eineindeutige Abbildung in eine andere gesehlossene Riemannsche Fliiche mit lauter reellen Windungspunkten verwandelt werden kann. Ist n~imlich etwa a-q-bi ein nichtreeller Windungspunkt, so wird die Flhehe F dutch die Transformation (z -- a) ~ = z r auf eine andere Fl~che abgebildet, deren Anzahl niehtreeller Windungspunkte um mindestens eine Einheit verringert worden ist. Dutch wiederholte Anwendung solcher Transformationen kommt man also schliel~lich zu einer Fl~iche mit nut reellen Windungspunkten. Die Gesamtzahl dieser Windungspunkte hat sieh dabei im allgemeinen vergrSt3ertg). 7) Journ. f. Math. 147 (1917), S. 77--83. 8) Diese Fundamentalveriinderlichen werden in A'bh. II der genannten Serie mittels eines Sehmiegungsveffahrens bestimmt. 9) Vgl. auch Poincar~ in Acta math. 4 ('1884), S. 246--250. Eingegangen am 10. September 192t.)
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Editorial: Sustainability assessment of chemicals in consumer products
Frontiers in sustainability
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TYPE Editorial PUBLISHED 20 February 2024 DOI 10.3389/frsus.2024.1376817 TYPE Editorial PUBLISHED 20 February 2024 DOI 10.3389/frsus.2024.1376817 TYPE Editorial PUBLISHED 20 February 2024 DOI 10.3389/frsus.2024.1376817 frontiersin.org OPEN ACCESS O CC SS EDITED AND REVIEWED BY Shabbir H. Gheewala, King Mongkut’s University of Technology Thonburi, Thailand *CORRESPONDENCE Li Li lili@unr.edu RECEIVED 26 January 2024 ACCEPTED 05 February 2024 PUBLISHED 20 February 2024 CITATION Li L, Fantke P and Huang L (2024) Editorial: Sustainability assessment of chemicals in consumer products. Front. Sustain. 5:1376817. doi: 10.3389/frsus.2024.1376817 COPYRIGHT © 2024 Li, Fantke and Huang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice No use, distribution or reproduction is permitted which does not comply with these terms. EDITED AND REVIEWED BY Shabbir H. Gheewala, King Mongkut’s University of Technology Thonburi, Thailand *CORRESPONDENCE Li Li lili@unr.edu RECEIVED 26 January 2024 ACCEPTED 05 February 2024 PUBLISHED 20 February 2024 CITATION Li L, Fantke P and Huang L (2024) Editorial: Sustainability assessment of chemicals in consumer products. Front. Sustain. 5:1376817. doi: 10.3389/frsus.2024.1376817 COPYRIGHT © 2024 Li, Fantke and Huang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practic No use, distribution or reproduction is permitted which does not comply with these terms. EDITED AND REVIEWED BY Shabbir H. Gheewala, King Mongkut’s University of Technology Thonburi, Thailand *CORRESPONDENCE Li Li lili@unr.edu RECEIVED 26 January 2024 ACCEPTED 05 February 2024 PUBLISHED 20 February 2024 CITATION Li L, Fantke P and Huang L (2024) Editorial: Sustainability assessment of chemicals in consumer products. Front. Sustain. 5:1376817. doi: 10.3389/frsus.2024.1376817 EDITED AND REVIEWED BY Shabbir H. Gheewala, King Mongkut’s University of Technology Thonburi, Thailand Li Li1*, Peter Fantke2 and Lei Huang3 1School of Public Health, University of Nevada, Reno, NV, United States, 2Quantitative Sustainability Assessment, Department of Environmental and Resource Engineering, Technical University of Denmark, Kongens Lyngby, Denmark, 3California Department of Toxic Substances Control, Sacramento, CA, United States KEYWORDS consumer products, chemicals, sustainability, life cycle, product use, supply chain, recycling COPYRIGHT © 2024 Li, Fantke and Huang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision. Focusing on textile industry as well, Schumacher and Forster examine the transition from a linear to a circular economy model within the U.S. textile industry, highlighting both challenges and opportunities. They emphasize the critical need for collaboration, system harmonization, and the exchange of data and information. In addition, they propose a series of necessary actions, including the development of standards, advancements in labeling, design modifications, the introduction of alternative business models for brands and retailers, the expansion of end markets for recyclers, community engagement and educational initiatives, research and development, and the influence of policy and regulation. This analysis and the suggested strategies offer a unique perspective in the field, particularly with respect to the U.S. context. The handling editor SG declared a past collaboration with the author PF. Funding Sharkey and Coggins discuss the impact of chemical additives in textiles on recycling processes. They identify chemical additives that render textiles environmentally hazardous and hence non- recyclable. The authors found that presence of these chemical additives, as well as the lack of effective recycling systems, contribute to the increase in waste textiles. These challenges underline the need for transitioning to a circular economic model, focusing on sustainable waste management practices. The authors offer a perspective on textile waste management, particularly in relation to the European Union’s waste policies and practices, combined with reducing consumption. The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Author contributions LL: Writing – original draft. PF: Writing – review & editing. LH: Writing – review & editing. Editorial on the Research Topic Their approach enhances decision-making in chemical selection, ensuring that alternative chemicals are effective while posing minimal health and environmental risks. The integration of CEA into AA is particularly notable for its potential to significantly improve the safety and sustainability of chemical alternatives. also highlight state-of-the-art aspects in CEA and AA, such as transparency, uncertainty, chemical mixtures, and sensitive receptors. Their approach enhances decision-making in chemical selection, ensuring that alternative chemicals are effective while posing minimal health and environmental risks. The integration of CEA into AA is particularly notable for its potential to significantly improve the safety and sustainability of chemical alternatives. With these contributions, this Research Topic not only presents relevant research but also sets the stage for future inquiries and solutions in more sustainable chemicals management. It serves as a crucial resource for researchers, policymakers, and industry professionals seeking to understand and address the multidimensional challenges associated with the sustainability of chemicals in consumer products. The insights and methodologies developed in these contributions have the potential to guide more sustainable practices across the consumer products industry, paving the way for a future where chemical use is aligned with environmental and health considerations. Overcash et al. assess the natural resource energy use and related carbon footprint of producing L-tryptophan, a chemical manufactured for a wide range of medicinal and dietary applications. The work highlights significant energy consumption and carbon emissions associated with its production. As a consequence, the authors emphasize the urgent need for more sustainable and energy-efficient production procedures. Notably, the authors presented the first detailed life cycle inventory for L- tryptophan, exemplifying the important role of detailed supply chain data in promoting more sustainable manufacturing of chemicals, using carbon footprint as illustrative impact category. Editorial on the Research Topic Editorial on the Research Topic Sustainability assessment of chemicals in consumer products Chemicals are the building blocks of today’s consumer products, serving varied functional performance roles in materials, products and technologies. Recent data reveal that ∼350,000 chemicals and chemical mixtures have been registered globally for production and use (Wang et al., 2020). Common examples, such as plasticizers, flame retardants, and preservatives, are prevalent in various consumer products. Exposure to several chemicals has been associated with detrimental effects on human health and on ecological integrity. Consumer products can be a significant source of human exposure to their chemicals constituents due to close contact during use; they can further harm ecosystems and wildlife when they enter natural environments. Overall, chemicals in consumer products can be released from multiple life cycle stages, enter multiple environmental compartments, and expose humans and ecological receptors through multiple pathways and routes (Fantke et al., 2021; Li et al., 2021). Such a “multidimensionality” of the issue of chemicals in consumer products necessitates a thorough understanding and effective management in the context of environmental sustainability. Research on safer and more sustainable chemical usage in consumer products is critical for improving manufacturing methods and enhancing the sustainability of consumer products throughout their life cycle. This Research Topic, titled “Sustainability assessment of chemicals in consumer products,” seeks to examine the multidimensionality of chemicals in consumer products, focusing on their exposures and effects on human health and the environment. This Research Topic compiles diverse studies covering various stages of the consumer product life cycle (e.g., manufacturing and supply chain, use, and disposal and recycling), and highlights the need for more sustainable chemicals that pose less harm to human health and the environment throughout the product life cycle. Meng and Zhou present an overview of the role of comparative exposure assessment (CEA) in alternatives assessment (AA) and discuss possibilities for integration of CEA into the overall AA workflow. They emphasize the importance of CEA as a crucial step in AA for selecting safer chemical alternatives in consumer products. This paper advocates for a holistic approach, connecting CEA with other AA components, such as the assessments of chemical hazards, life cycle impacts, and economic burdens. They Frontiers in Sustainability 01 frontiersin.org Li et al. Li et al. 10.3389/frsus.2024.1376817 also highlight state-of-the-art aspects in CEA and AA, such as transparency, uncertainty, chemical mixtures, and sensitive receptors. chemical emissions, fate, exposure, and risk. Environ. Health Perspect. 129:127006. doi: 10.1289/EHP9372 chemical emissions, fate, exposure, and risk. Environ. Health Perspect. 129:127006. doi: 10.1289/EHP9372 Wang, Z., Walker, G. W., Muir, D. C. G., and Nagatani-Yoshida, K. (2020). Toward a global understanding of chemical pollution: a first comprehensive analysis of national and regional chemical inventories. Environ. Sci. Technol. 54, 2575–2584. doi: 10.1021/acs.est.9b06379 chemical emissions, fate, exposure, and risk. Environ. Health Perspect. 129:127006. doi: 10.1289/EHP9372 Wang, Z., Walker, G. W., Muir, D. C. G., and Nagatani-Yoshida, K. (2020). Toward a global understanding of chemical pollution: a first comprehensive analysis of national and regional chemical inventories. Environ. Sci. Technol. 54, 2575–2584. doi: 10.1021/acs.est.9b06379 chemical emissions, fate, exposure, and risk. Environ. Health Perspect. 129:127006. doi: 10.1289/EHP9372 Fantke, P., Chiu, W. A., Aylward, L., Judson, R., Huang, L., Jang, S., et al. (2021). Exposure and toxicity characterization of chemical emissions and chemicals in products: global recommendations and implementation in USEtox. Int. J. Life Cycle Assess. 26, 899–915. doi: 10.1007/s11367-021-01889-y Wang, Z., Walker, G. W., Muir, D. C. G., and Nagatani-Yoshida, K. (2020). Toward a global understanding of chemical pollution: a first comprehensive analysis of national and regional chemical inventories. Environ. Sci. Technol. 54, 2575–2584. doi: 10.1021/acs.est.9b06379 Li, L., Sangion, A., Wania, F., Armitage, J., Toose, L., Hughes, L., et al. (2021). Development and evaluation of a holistic and mechanistic modeling framework for References Fantke, P., Chiu, W. A., Aylward, L., Judson, R., Huang, L., Jang, S., et al. (2021). Exposure and toxicity characterization of chemical emissions and chemicals in products: global recommendations and implementation in USEtox. Int. J. Life Cycle Assess. 26, 899–915. doi: 10.1007/s11367-021-01889-y 02 frontiersin.org Frontiers in Sustainability
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Microstructural evolution and mechanical properties of rheocast Al-7.1WT%Si alloy
Metallurgical & Materials Engineering
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* Corresponding author: Mehdi Raeissia, raeissi@eng.sku.ac.ir Mehdi Raeissia1* and Behzad Niroumandb2 1 Department of Materials Engineering, Faculty of Engineering, Shahrekord University, Shahrekord, 88186-34141,Iran 2 Department of Materials Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran 1 Department of Materials Engineering, Faculty of Engineering, Shahrekord University, Shahrekord, 88186-34141,Iran 2 Department of Materials Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran Received 18.03.2016 Accepted 26.08.2016 Received 18.03.2016 Accepted 26.08.2016 Abstract Effects of Semi-Solid Rheocasting (SSR) process on the structure and mechanical properties of Al-7.1wt%Si alloy were studied. The results showed that the non-dendritic structure could be formed by a short stirring period below liquidus temperature provided that stirring was combined with localized rapid heat extraction from the melt. The effect was examined by calculation of the time needed for detachment of dendrite arms at the initial stages of solidification and was associated with a large particle density in the rheocast slurry. Samples processed in semisolid state had lower porosity, higher tensile strength and higher elongation than that processed in fully-liquid state. The SSR- processed sample had a lower tensile strength but a higher elongation than the sample cooled without localized cooling and stirring. It was suggested that the lower strength of SSR-processed alloy was related to the inability of the α-particles to restrain crack propagation through the brittle eutectic matrix. Keywords: Semisolid rheocasting particle density Dendrite arm detachment Ripening Keywords: Semisolid rheocasting, particle density, Dendrite arm detachment, Ripening model, Tensile strength. Metallurgical and Materials Engineering Association of Metallurgical Engineers of Serbia AMES Metallurgical and Materials Engineering Association of Metallurgical Engineers of Serbia AMES Scientific paper UDC: 669.715'782 MICROSTRUCTURAL EVOLUTION AND MECHANICAL PROPERTIES OF RHEOCAST Al-7.1WT%Si ALLOY Mehdi Raeissia1* and Behzad Niroumandb2 Introduction Researches on rheology of semisolid slurries suggest that the ideal slurry for semisolid metal (SSM) processing is one containing a suitable volume fraction of fine and spheroidal particles uniformly dispersed in a low melting liquid matrix [1]. The processes invented for generating such non-dendritic microstructures can be classified into two routes according to their mechanism of microstructure evolution [2], namely dendrite fragmentation and nucleation mechanisms. In the first type of processes, the liquid metal is cooled to its semisolid temperature to form primary dendrites which are subsequently fragmented, mainly by use of some sort of stirring, into small particles suitable for semi-solid processing. The stirring methods include mechanical stirring, 156 Metall. Mater. Eng. Vol 22 (3) 2016 p. 155-164 electromagnetic stirring, gas bubbling, electric shock agitation, etc [1, 2]. In the second type of processes, formation of dendritic particles at the start of solidification is prevented by controlling the cooling conditions of the liquid. These processes are more time and cost efficient than those based on the dendrite fragmentation mechanism. Several rheocasting processes based on the nucleation mechanism have been developed in recent years, some of which are being commercialized in the casting industry. These include, for example, include the NRC (New RheoCasting) [3], the SEED (Swirled Enthalpy Equilibration Device) [4], Low superheat pouring with a shear field (LSPSF) [5], Gas induced semi-solid (GISS) [6], Cooling slope [7] SSR (Semi- Solid Rheocasting) [8] and RSF (Rapid Slurry Forming) [9] processes. The SSR process is fundamentally different from other rheocasting processes in the view that the molten alloy is converted to semisolid slurry by applying instantaneous rapid cooling and convection for a very short time during the early stages of solidification [8]. In this process, molten alloy is held a few degrees above its liquidus temperature. Solidification starts with the insertion of a rotating cold rod into the melt. The rotating rod is removed after a short interval and the quiescent melt is further cooled to the casting temperature and poured into a die [8]. It has been shown that desirable non-dendritic microstructures can be formed quickly and efficiently just by a short processing of the molten alloy at its liquidus temperature [8, 10-12]. This paper reports parts of the results of an investigation on the effects of SSR process on the structure and mechanical properties of rheocast Al-7.1wt%Si alloy and compares them with those of conventionally cast samples. Introduction Also attempt is made to explain the microstructural changes brought about by the SSR process based on its effect on the density of the primary particles formed during the early stages of solidification. Experimental procedures p p Fig.1(a) shows the rheocasting set-up employed in carrying out the experiments which was comprised of a resistance furnace, a graphite crucible having bottom pouring arrangements and a graphite stirring rod. Casting conditions used in different experiments are presented in Table 1 and explained below. Comparison of SSR-processed and conventionally-solidified samples For the conventionally-solidified sample (No. 1), the alloy was melted and held for 3 minutes at a temperature 10ºC above its measured liquidus temperature and was then allowed to cool down inside the furnace to room temperature The cooing rate of the melt when passing the liquidus temperature (Tl±1°C) and within the entire semisolid zone was about 0.07 and 0.04 °Cs-1, respectively. For the SSR-processed sample (No. 2), the melt was prepared similarly. However, the cold graphite rod rotating at 1000rpm was immersed into the melt at 3ºC superheat removing the superheat almost instantly. The rod was kept in the melt for about 7 seconds cooling the melt to a temperature corresponding to 0.05 fraction solid. The rod was then removed and the alloy was allowed to cool down inside the furnace to room temperature. The cooing rate of the melt when passing the liquidus temperature (Tl±1°C) and within the entire semisolid zone was about 0.13 and 0.04 °Cs-1, respectively. Raeissi et al. - Microstructural Evolution and Mechanical Properties of Rheocast … 157 (a) Fig.1. (a) Schematic of the equipment designed and built for this study and (b) The position of stirring rod in the crucible. (a) (b) (b) (a) Fig.1. (a) Schematic of the equipment designed and built for this study and (b) The position of stirring rod in the crucible. Effects of SSR process on porosity content and mechanical properties of the alloy Effects of SSR process on porosity content and mechanical properties of the alloy ff f p p y p p f y In order to evaluate the effects of SSR process on the porosity content and mechanical properties of the alloy, three samples were produced under different conditions. Sample No. 3 was cast from fully liquid state into a steel die. For sample No. 4, the melt was cooled down in the furnace to 0.3 solid fraction without stirring and cast in the steel die. Table 1. Casting conditions used in different experiments. Characterisation methods Comparison of SSR-processed and conventionally-solidified samples Comparison between the SSR-processed and the conventionally-solidified alloy Sample weight (gr) Cooling rate at Tl±1ºC (°Cs-1) Solidification time (min) Stirring time (sec) Stirring speed (rpm) 1 520 0.07 20 - - 2 520 0.13 20 7 1000 Comparison between various casting processes Sample weight (gr) Cooling rate at Tl±1ºC (°Cs-1) Stirring time (sec) Stirring speed (rpm) Casting condition 3 520 - - - liquid state 4 520 0.07 - - 0.3 solid 5 520 0.13 7 1000 0.3 solid In the third experiment (sample No. 5), the alloy was heated and held in the furnace at approximately 3°C superheat. The graphite rod rotating at 1000rpm was then immersed into the melt. The rod was kept in the melt for about 7 seconds (correspond to 0.05 solid fraction), after which it was removed and the slurry was allowed to cool slowly in the furnace without stirring to 0.3 solid fraction and cast in the steel die. Characterisation methods Table 1. Casting conditions used in different experiments. Comparison between the SSR-processed and the conventionally-solidified alloy Sample weight (gr) Cooling rate at Tl±1ºC (°Cs-1) Solidification time (min) Stirring time (sec) Stirring speed (rpm) 1 520 0.07 20 - - 2 520 0.13 20 7 1000 Comparison between various casting processes Sample weight (gr) Cooling rate at Tl±1ºC (°Cs-1) Stirring time (sec) Stirring speed (rpm) Casting condition 3 520 - - - liquid state 4 520 0.07 - - 0.3 solid 5 520 0.13 7 1000 0.3 solid Table 1. Casting conditions used in different experiments. In the third experiment (sample No. 5), the alloy was heated and held in the furnace at approximately 3°C superheat. The graphite rod rotating at 1000rpm was then immersed into the melt. The rod was kept in the melt for about 7 seconds (correspond to 0.05 solid fraction), after which it was removed and the slurry was allowed to cool slowly in the furnace without stirring to 0.3 solid fraction and cast in the steel die. Characterisation methods 158 Metall. Mater. Eng. Vol 22 (3) 2016 p. 155-164 Microstructural investigations were carried out on polished and etched samples cut from the castings. Comparison of SSR-processed and conventionally-solidified samples Porosity contents of the castings were measured using equation (1): app % Porosity (1- ) 100     (1) app % Porosity (1- ) 100     (1) where the apparent density, ρapp, of each sample was obtained by Archimedes method and the actual density of Al-7.1wt%Si alloy, ρ, was extracted from literature [13, 14]. [ ] Tensile tests were carried out on specimens made according to Fig.2 [15] using a Hounsfield tensile machine (H50KS model) under a strain rate of 0.01 min-1. Fig.2. The tension test specimen according to ASTM E8. Fig.2. The tension test specimen according to ASTM E8. Results and discussion Comparison of SSR-processed and conventionally solidified samples Fig.3(a) shows the microstructure of the conventionally cast alloy with large dendrites and widely spaced secondary arms. The dendrites are surrounded by eutectic constituents which appear darker in the micrograph. Fig.3(b) shows the SSR-processed microstructure consisting of large spheroidal particles surrounded by the eutectic constituents located at particles boundaries. The comparison of these two microstructures indicates the higher density of the nucleated and grown α-particles in the SSR-processed microstructure. In another words, the figures suggest that the formation of non-dendritic structures in rheocast alloys can be the result of a significantly greater particle density than that of the dendritic structure in conventional solidification. In the SSR-processed alloy, immersion of the cold rod induces a region of high local cooling rate in the melt and considerably increases the nucleation intensity of the primary particles on or in the areas adjacent to the rod surface. Rotation of the rod causes a through distribution of the solid particles formed in the melt. Furthermore, the generated melt convection makes the temperature distribution in the melt relatively uniform increasing the possibility of the nuclei survival. These conditions can lead to an increased particles density in the microstructure of the SSR-processed alloy. Study of the mechanism of microstructure formation in the SSR processed alloy requires an understanding of the effects of such region of high local cooling rate on the primary particles density. Therefore an attempt is made to study the effects of such region on the average time for melting off of the dendrite arms and to relate it to the primary particles density. For this purpose, first the heat-transfer coefficient of the rod/melt interface and the average particles size must be estimated. Raeissi et al. - Microstructural Evolution and Mechanical Properties of Rheocast … 159 Fig.3. (a) Conventionally solidified structure and (b) SSR-processed structure of Al-7.1wt%Si alloy (Both samples cooled inside the furnace until the end of the solidification) Fig.3. (a) Conventionally solidified structure and (b) SSR-processed structure of Al-7.1wt%Si alloy (Both samples cooled inside the furnace until the end of the solidification) Ignoring the heat loss from the melt surface and from the crucible walls compared with that through the graphite stirring rod, it can be assumed that the heat flow out of the melt, Qºout, equals the heat flow into the stirring rod, Qºin. Results and discussion The rate of heat flow out of the melt during stirring time of Δt is approximated by equation (2); s out M f H Q t     (2) s out M f H Q t     (2) where M, fs and ΔH are the mass of the melt, the solid fraction formed during stirring and the heat of fusion per unit mass of pure aluminum, respectively. The heat flow into the graphite stirring rod can be approximated by equation (3) [16]; in rod r Q A h ( T )   (3) in rod r Q A h ( T )   (3) where A, hrod and ΔTr are the total rod-melt contact area, the heat-transfer coefficient of the rod/melt interface and the temperature difference between the rod and the melt, respectively. Equating equations (2) and (3) yields: s rod r ( M ) f H h A T ( t )     (4) s rod r ( M ) f H h A T ( t )     (4) The dendrite arm spacing (DAS) of the dendrites which form on or near the rod surface can be estimated using the following approach adapted from Ref. [17]. It is assumed that the heat flow into the cooling rod is interface controlled; the mother dendrites are growing radially on the rod surface and in the opposite direction to the heat flow and the full volumetric heat of fusion of aluminum is removed by the cooling rod for each increment of growth of the dendrite tip. Under these conditions the average dendrites tip velocity, Rs, can be taken as an estimate of the solidification rate and may be expressed as; rod r s h T R H     (5) rod r s h T R H     (5) 160 Metall. Mater. Eng. Vol 22 (3) 2016 p. 155-164 By substituting hrod from equation (4) in equation (5), the final expression for estimating the average dendrites tip velocity is obtained; s s (M) f R A ( t)    (6) s s (M) f R A ( t)    (6) s s (M) f R A ( t)    (6) Substituting fs equal to 0.05 (the solid fraction of sample No. 1/3 DAS 8.73 ( t)   By substituting this value into equation (8), the critical time for arms detachment is approximated to be about 12.5 seconds. These approximations clearly show the significant influence of the insertion of the cold graphite rod into the melt on the number of free moving solid particles during solidification. Furthermore, in the presence of a high local cooling zone, when the dendrites start to grow with an initial tip radius of approximately 1.5µm, stirring accelerates the arm melt off and grain multiplication through the thermal and mechanical influences on the dendrite and carries the detached arms into the bulk of the melt. At the same time, the stirring action of the cooling rod is likely to make the temperature gradient in the melt relatively uniform and eliminates any regions of high local temperature in the bulk of the melt resulting in continuous growth of a large density of the separated particles. As a result the particles spacing becomes very small and the particles will have limited space for growth. On the other hand, in such circumstances, the probability of overlap of the diffusion fields of the adjacent particles increases significantly which leads to the decrease of the concentration gradients in front of the solid-liquid interfaces and removes the driving force for the instability of the growing interfaces. Under these conditions, the particles will have limited but more stable growth and the non-dendritic structure is expected to form in a short period after the start of solidification. Conversely, if the local chilling is not complemented by stirring, solidification of the dendrites would progress with less arm detachment while ripening of the initial dendrite arms proceeds. It is expected that natural convection can carry away only a portion of the detached particles into the bulk of the melt. Even then many of the carried away particles may face regions of high local temperature and re-melt. Thus the relatively small number of the survived particles will have large inter-particle spacing and therefore most of the particles growing away from the cooling rod find enough chances for equiaxed dendritic growth. The final structure would be columnar or equiaxed dendritic. A third scenario can also be envisioned when the density of the separated particles is intermediate between the two aforementioned conditions. This situation may arise when the stirring is not strong enough. Results and discussion 2 after 7 seconds of stirring) and A equal to 48cm2 (using the geometry and the immersion depth of the rod which shown in Fig.1(b)) into equation (6), the average dendrites tip velocity of sample No. 2 is estimated to be slightly greater than 0.3mm/s. Equation (7) shows the relationship between the dendrite tip radius and the dendrites tip velocity [16]; 1/2 l 0 s D r 2 ( ) ( T ) K R     (7) 1/2 l 0 s D r 2 ( ) ( T ) K R     1/2 l 0 s D r 2 ( ) ( T ) K R     (7) (7) where r is the dendrite tip radius, Dl is the solute diffusivity in the melt, Г is Gibbs- Thompson coefficient, ΔT0 is the solidification range for the alloy and K is the solute partition ratio. Fig.4 depicts the relationship for Al-7.1wt%Si using the parametric values extracted from Ref. [13]. Fig.4 shows that at dendrite tip velocity of 0.3 mm/s the corresponding tip radius would be approximately 1.5µm. 0.1 1 10 0.01 0.1 1 10 Dendrite tip velocity ( mm/sec ) Dendrite tip radius ( microns ) Fig.4. Relationship between dendrite tip radius and dendrite tip velocity for Al-7.1wt%Si Fig.4. Relationship between dendrite tip radius and dendrite tip velocity for Al-7.1wt%Si On the other hand, the time required for a dendrite arm to be detached from its mother dendrite by melting off of its root due to ripening, tcrit, can be calculated using equation (8) proposed by Kattamis et al. [18]; 3 0 l crit l H C m (1-K) d t 0.0085 D T     (8) 3 0 l crit l H C m (1-K) d t 0.0085 D T     (8) 161 Raeissi et al. - Microstructural Evolution and Mechanical Properties of Rheocast … where σ is the solid-liquid interfacial energy, d is DAS, C0 is solute content of the alloy and ml is slope of the liquidus line. Assuming the dendrite arm spacing to be about twice the tip radius (3µm for sample No. 2) and that the temperature (T) is just below the liquidus temperature (corresponding to 0.05 solid fraction after 7 seconds of stirring), the critical time for detachment of the arms is worked out to be approximately 0.1 second. Results and discussion For sample No. 1 which was solidified inside the furnace without insertion of the rotating graphite rod, DAS after 7 seconds of solidification is estimated to be 15µm using the empirical ripening equation of the alloy (equation (9)) [11]: (9) 1/3 DAS 8.73 ( t)   1/3 DAS 8.73 ( t)   In this condition, the distances between the primary particles are still large but diffusion fields overlap may take place at later stages of solidification. Therefore primary particles will have more chance and space to start and continue part of their growth with unstable interfaces. They will ripen into rosettes 162 Metall. Mater. Eng. Vol 22 (3) 2016 p. 155-164 and later into spheroids under the effects of stirring and diffusion fields overlap. In such conditions longer stirring times are required for formation of the non-dendritic structure. Thus, it is postulated that the required time for formation of non-dendrite particles after the start of solidification is inversely related to the initial density of the particles formed just below the liquidus temperature. The higher the particles density, the shorter the time needed for the formation of non-dendritic microstructure. Fig.5 depicts the typical microstructures for samples No. 3 to 5 processed at three different conditions. Fig.5. Typical microstructures of the (a) alloy processed in fully-liquid state, (b) alloy processed in semisolid state without stirring and (c) SSR-processed alloy. Fig.5. Typical microstructures of the (a) alloy processed in fully-liquid state, (b) alloy processed in semisolid state without stirring and (c) SSR-processed alloy. For sample No. 3 cast from fully liquid state (Fig.5(a)), the α-phases have dendritic morphology. Sample No. 4 which was cooled to 0.3 solid fraction before casting (Fig.5(b)) contains large primary dendrites with an average DAS of 37µm. The remaining melt solidified in the die (secondary particles) has also dendritic morphology with an average DAS of less than 10µm. In the SSR-processed sample (No. 5), the morphology of the primary particles solidified before casting is non-dendritic while that of the secondary particles is dendritic (Fig.5(c)). Effects of SSR process on porosity content and mechanical properties of the alloy Effects of SSR process on porosity content and mechanical properties of the alloy The porosity content and mechanical properties of samples No. 3 to 5 are shown in Table 2. The comparison between the porosity contents of these samples indicates that the sample cast from the fully liquid state has higher porosity content than those of the samples processed in semisolid state. Considering the fact that in samples No. 4 and 5, which have been cast from the semisolid state, part of the solidification and its associated shrinkage has taken place outside the die, the result seems reasonable. Table 2. Porosity contents and mechanical properties of samples No. 3, 4 and 5. Table 2. Porosity contents and mechanical properties of samples No. 3, 4 and 5. Sample No. Volume Porosity(%) Tensile Strength (MPa) Elongation (%) 3 5.48±0.11 127±7 9.14±0.11 4 4.61±0.07 161±5 9.8±0.15 5 4.36±0.08 150±4 9.97±0.1 Although the casting temperatures were the same for the two semisolid-cast samples, the porosity content of the SSR-processed sample was relatively lower. Viscosity of solidifying melts has been shown to rise gradually with increasing the solid fraction until reaching the dendrite coherency point (DCP) [19] after which it increases abruptly. At DCP a continuous 3D solid skeleton is formed in the casting which makes 163 Raeissi et al. - Microstructural Evolution and Mechanical Properties of Rheocast … the feeding difficult. This marks the transition from liquid and mass feeding to interdendritic feeding during solidification [20]. In semisolid non-dendritic slurries, the DCP is reached at higher solid fractions due to the change in the morphology and the size of the primary particles. Thus it is believed that the lower porosity content of the SSR-processed alloy is due to the more extended and effective operation of liquid and mass feeding mechanisms toward the end of solidification. A comparison between the strengths of samples No. 3 to 5 shows that the sample cast from fully liquid state has the lowest tensile strength. The high porosity of this sample is believed to have reduced its tensile strength. The major constituents present in hypoeutectic A1-Si alloys are the aluminum- rich α- solid solution and the eutectic structure. Semisolid processing of these alloys permits dispersion of ductile particles of proeutectic α-phase in a matrix of brittle eutectic [21]. Such a structure allows them to be considered as brittle matrix ductile dispersoid particulate composites. Effects of SSR process on porosity content and mechanical properties of the alloy For the brittle matrix-ductile particle composites it has been proposed that the role of ductile particles is to restrain the crack propagation through the brittle matrix. The mechanism by which such a restraint is affected is believed to be the compressive stresses which the ligaments of the unbroken particles joining the crack faces exert to restrain the displacement of the crack faces. It has been suggested that for the above mechanism to be operative, particles of cylindrical morphology should be used instead of spheroidal particles. When spheroidal particles are employed, the crack in the matrix simply by-passes the particles and no crack particle interaction results [22]. It appears, therefore, that the lower strength of SSR-processed alloy containing spheroidal particles in comparison with the sample processed in semisolid state without stirring is related to the inability of spheroidal α-particles to restrain crack propagation through the brittle eutectic matrix. The comparison between the elongations of the samples (Table 2) indicates that elongations of the samples are inversely related to the porosity content of the samples. Conclusions 1- Non-dendritic structure can be formed by a short stirring period below liquidus temperature provided that stirring is combined with rapid heat extraction from the melt. This treatment produces significantly larger particle density in the rheocast alloys which result in the formation of non-dendritic structures. 1- Non-dendritic structure can be formed by a short stirring period below liquidus temperature provided that stirring is combined with rapid heat extraction from the melt. This treatment produces significantly larger particle density in the rheocast alloys which result in the formation of non-dendritic structures. 2- The sample cast from fully-liquid state had a higher porosity content and lower tensile strength and elongation in comparison with those processed in semisolid state. The longer operation of liquid and mass feeding mechanisms seems to be a contributor to lower porosity of the SSR-processed sample compared to the sample cast at the same solid fraction without SSR treatment. 2- The sample cast from fully-liquid state had a higher porosity content and lower tensile strength and elongation in comparison with those processed in semisolid state. The longer operation of liquid and mass feeding mechanisms seems to be a contributor to lower porosity of the SSR-processed sample compared to the sample cast at the same solid fraction without SSR treatment. 3- The results indicated that the SSR-processed sample had a lower tensile strength than the sample cast at the same solid fraction without SSR treatment. It was suggested that the lower strength of SSR-processed alloy was related to the inability of the spheroidal α- particles to restrain crack propagation through the brittle eutectic matrix. References [1] M.C. Flemings: Metal. Trans., 1991, 22A, 957-981. References References [1] M.C. Flemings: Metal. Trans., 1991, 22A, 957-981. Metall. Mater. Eng. Vol 22 (3) 2016 p. 155-164 164 [2] Z. Fan: Int. Mater. Rev. 47 (2002) 49-85. [2] Z. Fan: Int. Mater. Rev. 47 (2002) 49-85. [3] H. Kaufmann, P.J. Uggowitzer, Adv. Eng. [3] H. Kaufmann, P.J. Uggowitzer, Adv. Eng. Mater. 3 (2001) 963-967. ann, P.J. Uggowitzer, Adv. Eng. Mater. 3 (2001) 963- [4] S. Nafisi, R. Ghomashchi, Mater. Sci. Eng. 415A (2006) 273-285. [5] H.M. Guo, X.J. Yang, J.X. Wang, Mater. Sci. Forum 628 (2009) 477-482. 5] H.M. Guo, X.J. Yang, J.X. Wang, Mater. Sci. For [6] R. Canyook, J. Wannasin, S. Wisuthmethangkul, M.C. Flemings, Acta Mater. 60 (2012) 3501–3510. ( ) [7] E.C. Legoretta, H.V. Atkinson, H. Jones, J. Mater. Sci. 43 (2008) 5448-5455. [8] R.A. Martinez, A. Karma, M.C. Flemings: Metal. Mater. Trans. 37A (2006) 2807- 2815. [9] M. Payandeh, Anders E.W. Jarfors, M. Wessén, Metal. Mater. Trans. 47A (2016) 1215-1228. ( ) [10] M. Reisi, B. Niroumand: J. Alloys Compd. 475 (2009) 643–647. B. Niroumand: J. Alloys Compd 470 (2009) 413–41 [11] M. Reisi, B. Niroumand: J. Alloys Compd 470 (2009) 413–419. ] M. Reisi, B. Niroumand, , J. Mater. Chem. Phys. 13 [13] Ch. -A. Gandin: Acta Mater. 48 (2000) 2483-2501. [14] G. Heiberg, Ch. -A. Gandin, H. Goerner, L. Arnburg: Metall. Mater. Trans. 35A (2004) 2981-2991. [15] ASTM E8/E8M-08 Standard Test Methods for Tension Testing of Metallic Materials. d [16] R.B. Bird, W.E. Stewart, E.N. Lightfoot: ‘Transport Phenomena’ 2nd edn, Wiley, New York, 2002. [17] W. Kurz, D.J. Fisher: ‘Fundamentals of Solidification’, 4th edn, Trans. Tech. Publication, Switzerland, 1998. [18] T.Z. Kattamis, U.T. Holmberg, M.C. Flemings: J. Inst. Met. 95 (1967) 343-347. [19] D.B. Spencer, R. Mehrabian, M.C. Flemings: Met. Trans. 3 (1972) 1925-1932. [20] A.K. Dahle, D.H. Stjohn: Acta Mater. 47 (1999) 31-41. [21] A. Vogel, R.D. Doherty, B.C. Cantor: Proc. Conf. on ‘Solidification and Casting of Metals’, Metals Society, Sheffield, UK, 1977, p. 518. [22] A.G. Evans, A.H. Heuer, D.L. Porter: Proc. 4th Int. Conf. on Fracture, Waterloo, 1977, University of Waterloo Press, p.529.
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Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg plant diversity
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Corresponding author: Luiza F. A. de Paula (luizafap@gmail.com) Academic editor: Gianniantonio Domina Received: 10 Apr 2020 | Accepted: 07 Jun 2020 | Published: 19 Jun 2020 Citation: de Paula LFA, Azevedo LO, Mauad LP, Cardoso LJT, Braga JMA, Kollmann LJ.C, Fraga CN, Menini Neto L, Labiak PH, Mello-Silva R, Porembski S, Forzza RC (2020) Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg plant diversity. Biodiversity Data Journal 8: e53135. https://doi.org/10.3897/BDJ.8.e53135 Biodiversity Data Journal 8: e53135 doi: 10.3897/BDJ.8.e53135 Data Paper Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg plant diversity Luiza F. A. de Paula , Luísa O. Azevedo , Luana Paula Mauad , Leandro Jorge Telles Cardoso , João Marcelo Alvarenga Braga , Ludovic J.C. Kollmann , Claudio N. Fraga , Luiz Menini Neto , Paulo H. Labiak , Renato Mello-Silva , Stefan Porembski , Rafaela Campostrini Forzza ‡ Universidade Federal de Minas Gerais, Belo Horizonte, Brazil § Jardim Botânico do Rio de Janeiro, Rio de Janeiro, Brazil | Instituto Nacional da Mata Atlântica, Museu de Biologia Prof. Mello Leitão, Santa Teresa, Brazil ¶ Universidade Federal de Juiz de Fora, Juiz de Fora, Brazil # Universidade Federal do Paraná, Curitiba, Brazil ¤ Universidade de São Paulo, São Paulo, Brazil « Universität Rostock, Rostock, Germany Corresponding author: Luiza F. A. de Paula (luizafap@gmail.com) Academic editor: Gianniantonio Domina Received: 10 Apr 2020 | Accepted: 07 Jun 2020 | Published: 19 Jun 2020 Citation: de Paula LFA, Azevedo LO, Mauad LP, Cardoso LJT, Braga JMA, Kollmann LJ.C, Fraga CN, Menini Neto L, Labiak PH, Mello-Silva R, Porembski S, Forzza RC (2020) Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg plant diversity. Biodiversity Data Journal 8: e53135. https://doi.org/10.3897/BDJ.8.e53135 ‡ ‡ § § § | § ¶ # ¤ « § Biodiversity Data Journal 8: e53135 doi: 10.3897/BDJ.8.e53135 Data Paper Biodiversity Data Journal 8: e53135 doi: 10.3897/BDJ.8.e53135 Data Paper © de Paula L et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Keywords Atlantic Forest, granite outcrops, rock outcrops, rupicolous plants Background Isolated monoliths of granitic and/or gneissic rock rising abruptly from the surrounding landscape are known as inselbergs. Dome-shaped inselbergs are common throughout the Atlantic Forest in south-eastern Brazil, a region known as Sugarloaf Land (SLL). This study aimed to create the first checklist of vascular plant species occurring on lowland inselbergs in SLL, with a focus on vegetation islands. We used information from online databases, our own field sampling and data from previously-published studies. We found 548 vascular plant species (505 angiosperms; 43 ferns and lycophytes) belonging to 69 families and 212 genera. Of all identified species, 536 are native and 12 are naturalised. © de Paula L et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. de Paula L et al de Paula L et al de Paula L et al 2 New information We updated the information currently available in Flora do Brasil 2020, as 59% of the angiosperms and 63% of the ferns and lycophytes on our checklist were not previously characterised as occurring on rock outcrops. As a first step towards generating a Virtual Herbarium of lowland inselberg vascular plants, we added barcode vouchers with images available online for 75% of the total number of vascular species. In the official lists of endangered species, 115 angiosperms and five ferns and lycophytes are mentioned. However, the conservation status of many species have not yet been evaluated (77% angiosperms; 88% ferns and lycophytes), thus this list is an important step towards their conservation. The information provided herein is essential for management programmes related to rock outcrops in Brazil as they are facing serious threats to conservation. Introduction Brazil contains the richest seed plant diversity in the world (BFG 2018). Surprisingly, inventories in well-studied areas, such as the Atlantic Forest in south-eastern (SE) Brazil, are still documenting new records and identifying species new to science (Sobral and Stehmann 2009). This is especially true for overlooked habitats within this domain, such as inselbergs (Barthlott and Porembski 2000), isolated monoliths of granitic and/or gneissic rock, where dozens of new species belonging to different families have been identified over the last decade (e.g. Kollmann and Fontana 2010, Leme et al. 2010a, Leme et al. 2010b, Viana and de Paula 2013, Fraga and Guimarães 2014, de Oliveira and Sobrado 2016, Gonçalves and de Paula 2016, Gouvêa et al. 2018, Meyer et al. 2018, Fraga et al. 2019, Morales and Kollmann 2019, Valadares et al. 2019). These ancient rock outcrops are common elements in various landscapes around the world (Hopper et al. 2016) and are especially biodiverse in SE Brazil, the central highlands of Madagascar and in south- western Australia (Porembski 2007). They are characterised by extreme edaphic and microclimatic conditions, leading to their ecological isolation from the surrounding matrix and their island-like characteristics (Porembski et al. 2000). Consequently, their vegetation is distinct from the surrounding area (Parmentier et al. 2005, Porembski 2007). In many parts of Brazil, inselbergs are characteristic elements of the landscape, particularly in the Atlantic Forest domain where they occur in large numbers and at varying sizes, altitudes and degrees of isolation (Safford and Martinelli 2000). The term “Sugarloaf” (pão de açúcar) is associated with lowland, isolated and dome-shaped inselbergs in the rainforest in SE Brazil (Ab’Sáber 1967). As the core area of lowland inselbergs seems to form a particular phytogeographical region, it has been named Sugarloaf Land (de Paula et al. 2016). Literature on the flora and vegetation of inselbergs is sparse and data on their ecological characteristics are mostly descriptive. Despite the lack of detailed knowledge Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... 3 about Brazilian inselbergs, previous regional studies have suggested extraordinarily high floristic richness (Meirelles et al. 1999, Safford 1999, Caiafa and Silva 2005, Ribeiro et al. 2007, Couto et al. 2017) which is further corroborated by the occurrence of high levels of beta diversity amongst inselbergs from SE Brazil (Meirelles et al. 1999, Safford and Martinelli 2000, de Paula et al. 2019b). Introduction It is also common for inselberg plant communities to be endemic to a specific region, often with species isolated to a single outcrop (de Paula et al. 2016, de Paula et al. 2017a, Couto et al. 2017). Additionally, recent phylogeographic studies show that inselberg endemics display strong genetic differentiation amongst individual rock outcrops, indicating low degrees of gene flow (e.g. Palma-Silva et al. 2011, Hmeljevski et al. 2015, Hmeljevski et al. 2017). Although no specific reference to mountains, including rock outcrops, exists in Brazilian legislation on biodiversity (Martinelli 2007), these environments have been highlighted as an important aspect of the Convention on Biological Diversity (2002), in which the Mountain Work Programme (MWP) was proposed to reduce global, regional and local loss of mountain biodiversity (Martinelli 2007). The lack of specific legislation has resulted in significant gaps in knowledge related to the flora of several mountain regions throughout Brazil (Safford and Martinelli 2000, Carmo et al. 2018). This scenario is worrying since inselbergs and rock outcrops, in general, are amongst the most threatened and neglected environments in the world (Porembski et al. 2016). Considering the lack of research on, and rapid destruction of, these unique landscape features and, in order to pay particular attention to the high levels of biodiversity in these ecotonal habitats, this study presents a list of vascular plants occurring on lowland inselbergs in the Atlantic Forest in SE Brazil. We aim to provide comprehensive and updated information regarding taxonomic nomenclature, life forms and conservation status, while also illustrating the unique diversity of Sugarloaf Land. Project description Study area description: Inselbergs are found in large concentrations in eastern Brazil (Vieira et al. 2015). In SE Brazil in particular, two main types of granite outcrops occur: highland and lowland inselbergs (Safford and Martinelli 2000). Highland inselbergs are known as campos de altitude (Brazilian Highlands, sensu Safford 1999) and can be found in the mountain ranges of Serra da Mantiqueira and Serra do Mar, as well as in their subranges and disjunctions, such as Serra do Itatiaia, Serra dos Órgãos, Serra do Brigadeiro and Serra do Caparaó (Fig. 1, A-B). On the other hand, lowland inselbergs are a group of dome-shaped monolithic outcrops with a sugarloaf morphology (Ab’Sáber 1967, Fig. 1, C-D), the most iconic example of which is Sugarloaf Mountain (Pão de Açúcar) in Rio de Janeiro. The core area encompassing the states of Rio de Janeiro, Espírito Santo, southern Bahia and the adjacent region in Minas Gerais, were recently denominated Sugarloaf Land due to the concentration of lowland inselbergs that harbour high levels of plant species richness and elevated rates of endemism (de Paula et al. 2016). However, estimates of diversity in this area have been based solely on local inventories or on 4 de Paula L et al surveys of specific taxonomic plant groups (e.g. Porembski et al. 1998, Meirelles et al. 1999, de Paula et al. 2016, de Paula et al. 2017a). surveys of specific taxonomic plant groups (e.g. Porembski et al. 1998, Meirelles et a 1999, de Paula et al. 2016, de Paula et al. 2017a). Figure 1. Examples of highland and lowland inselbergs occurring in SE Brazil. The first row represents highland inselbergs; A. Serra do Caparaó, Minas Gerais State; B. Serra do Itatiaia, Rio de Janeiro State. The second row represents lowland inselbergs, also known as sugarloaves; C. Sugarloaf Mountain, Rio de Janeiro; D. Pedra da Boca, Minas Gerais. Photos by L.F.A. de Paula, except for A. by N.F.O. Mota. Figure 1. Examples of highland and lowland inselbergs occurring in SE Brazil. The first row represents highland inselbergs; A. Serra do Caparaó, Minas Gerais State; B. Serra do Itatiaia, Rio de Janeiro State. The second row represents lowland inselbergs, also known as sugarloaves; C. Sugarloaf Mountain, Rio de Janeiro; D. Pedra da Boca, Minas Gerais. Photos by L.F.A. de Paula, except for A. by N.F.O. Mota. Examples of highland and lowland inselbergs occurring in SE Brazil. Project description The first row represents highland inselbergs; A. Serra do Caparaó, Minas Gerais State; B. Serra do Itatiaia, Rio de Janeiro State. The second row represents lowland inselbergs, also known as sugarloaves; C. Sugarloaf Mountain, Rio de Janeiro; D. Pedra da Boca, Minas Gerais. Photos by L.F.A. de Paula, except for A. by N.F.O. Mota. Highland and lowland inselbergs have distinct floristic composition (Safford 1999, Safford and Martinelli 2000). The present checklist focuses on the lowland inselbergs from Sugarloaf Land (de Paula et al. 2016), which are defined as dome-shaped with isolated peaks at an elevation of less than 1000 m above sea level (a.s.l.). Highland inselbergs were not considered in the present study, nor were transitional areas where most inselbergs are located above 1000 m a.s.l., but not considered campos de altitude, such as Pedra Azul, Forno Grande, Alto Misterioso (Espírito Santo State) and Pico da Caledônia (Rio de Janeiro State). We also excluded rocky shores (costões rochosos), inselbergs located in domains other than the Atlantic Forest biome and other types of rock outcrops, such as campos rupestres (sensu Silveira et al. 2016), karstic outcrops (sensu Bystriakova et al. 2019) and cangas (sensu Carmo et al. 2018). Study extent:  Vascular plant dataset Study extent:  Vascular plant dataset Species list compilation: We obtained a list of Brazilian angiosperms from Brazilian Flora Group (BFG 2015) and a list of Brazilian ferns and lycophytes from Prado et al. (2015). To Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... 5 filter the species unique to Sugarloaf Land, we created a protocol (procedures 1 and 2) to clean the datasets. In procedure 1, four steps were conducted separately for the angiosperm (Fig. 2) and fern and lycophyte datasets (Fig. 3). In the first step, we filtered the original datasets (32,086 angiosperms and 1,253 ferns and lycophytes) for species that occur in the Atlantic Forest domain, resulting in 15,001 angiosperms and 883 ferns and lycophytes. In the second step, we searched the resulting list for species identified as occurring on rock outcrops through the “vegetation type” field present in the original datasets, resulting in 1,023 angiosperms and 47 ferns and lycophytes. In the third step, we filtered for species that occur in Bahia, Espírito Santo, Minas Gerais and Rio de Janeiro States, corresponding to Sugarloaf Land, resulting in 884 angiosperms and 44 ferns and lycophytes. We conducted the first three steps using the “filter” tool in Microsoft Excel v. 14.5 (Microsoft Office 2010 Proofing Tools). Figure 2. Procedure 1: stages of data filtering to obtain the angiosperm species list for lowland inselbergs, SE Brazil, based on the list available from BFG (2015). The left column shows species that remained during the construction and validation of the list and the right column represents species removed from the list; S1-S4: indicates steps 1- 4 (see text for more details). Procedure 2: stages of data addition to obtain the final angiosperm species list for lowland inselbergs, SE Brazil. Figure 3. Figure 3. Procedure 1: stages of data filtering to obtain the fern and lycophyte species list from lowland inselbergs, SE Brazil, based on the list available from Prado et al. (2015). The left column represents species that remained during the construction and validation of the list and the right column represents species removed from the list; S1-S4: indicates steps 1- 4 (see text for more details). Procedure 2: stages of data addition to obtain the final fern and lycophyte species list for lowland inselbergs, SE Brazil. gu e 3. Procedure 1: stages of data filtering to obtain the fern and lycophyte species list from lowland inselbergs, SE Brazil, based on the list available from Prado et al. (2015). The left column represents species that remained during the construction and validation of the list and the right column represents species removed from the list; S1-S4: indicates steps 1- 4 (see text for more details). Procedure 2: stages of data addition to obtain the final fern and lycophyte species list for lowland inselbergs, SE Brazil. g Procedure 1: stages of data filtering to obtain the fern and lycophyte species list from lowland inselbergs, SE Brazil, based on the list available from Prado et al. (2015). The left column represents species that remained during the construction and validation of the list and the right column represents species removed from the list; S1-S4: indicates steps 1- 4 (see text for more details). Procedure 2: stages of data addition to obtain the final fern and lycophyte species list for lowland inselbergs, SE Brazil. Finally, in the fourth step, we verified the remaining species as those that occur either on lowland inselbergs or other types of rock outcrops, excluding those that occur on highland inselbergs, i.e. above 1000 m a.s.l. (campos de altitude), cangas, campo rupestre and karstic rock outcrops. This verification was based on our own field observations and records of the species in online databases (Jabot-JBRJ 2020, www.jbrj.gov.br/jabot; Reflora - Virtual Herbarium 2020, http://reflora.jbrj.gov.br/reflora/herbarioVirtual/; INCT 2020, http://inct.splink.org.br). We conducted several searches over the period from May 2016 to August 2019 using the keywords “granito”, “granítica”, “granítico”, “gneiss”, “gnáissicos”, “gnaisses”, “inselberg”, “inselbergue”, “incelberg”, “incelbergue”, “pão de açúcar”. At the end of procedure 1, we compiled a list of 208 angiosperms and 16 ferns and lycophytes that are documented as occurring on lowland inselbergs. In procedure 2, we built new datasets for angiosperms (Fig. Figure 2. Figure 2. Procedure 1: stages of data filtering to obtain the angiosperm species list for lowland inselbergs, SE Brazil, based on the list available from BFG (2015). The left column shows species that remained during the construction and validation of the list and the right column represents species removed from the list; S1-S4: indicates steps 1- 4 (see text for more details). Procedure 2: stages of data addition to obtain the final angiosperm species list for lowland inselbergs, SE Brazil. Figure 2. Procedure 1: stages of data filtering to obtain the angiosperm species list for lowland inselbergs, SE Brazil, based on the list available from BFG (2015). The left column shows species that remained during the construction and validation of the list and the right column represents species removed from the list; S1-S4: indicates steps 1- 4 (see text for more details). Procedure 2: stages of data addition to obtain the final angiosperm species list for lowland inselbergs, SE Brazil. Figure 2. Procedure 1: stages of data filtering to obtain the angiosperm species list for lowland inselbergs, SE Brazil, based on the list available from BFG (2015). The left column shows species that remained during the construction and validation of the list and the right column represents species removed from the list; S1-S4: indicates steps 1- 4 (see text for more details). Procedure 2: stages of data addition to obtain the final angiosperm species list for lowland inselbergs, SE Brazil. de Paula L et al 6 Figure 3. Procedure 1: stages of data filtering to obtain the fern and lycophyte species list from lowland inselbergs, SE Brazil, based on the list available from Prado et al. (2015). The left column represents species that remained during the construction and validation of the list and the right column represents species removed from the list; S1-S4: indicates steps 1- 4 (see text for more details). Procedure 2: stages of data addition to obtain the final fern and lycophyte species list for lowland inselbergs, SE Brazil. 6 de Paula L et al Figure 3. 2) and ferns and lycophytes (Fig. 3). We combined the species identified in procedure 1 with those sampled by the Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... 7 7 authors, i.e. species that, to date, have not been documented for lowland inselbergs or were removed during procedure 1 due to incomplete or incorrect information in the original datasets. We also added species based on available literature, such as floristic inventories, original species’ descriptions and ecological studies. The final checklist is composed of native and non-native plants and includes only vouchers identified to the species level, based on the List of Species of the Brazilian Flora (Flora do Brasil under construction 2020). Correction and updating of the names were performed using the function get.taxa in the flora package (Carvalho 2017) for the R software environment (R Core Team 2016), which compares the names in our list with those in the Brazilian Flora online (Flora do Brasil under construction 2020). Families and genera follow Flora do Brasil under construction (2020) and are listed in alphabetical order. The herbaria acronyms are according to Thiers (2020). Vouchers were carefully chosen from specimens collected on vegetation islands and scrub from lowland inselbergs. We added link for the vouchers with available images in Jabot-JBRJ (2020) and Reflora - Virtual Herbarium 2020, which can be continuously updated with regards to taxonomic alterations. A map was built, based on the available coordinates of the vouchers assigned in the lists for angiosperms and ferns and lycophytes in order to ascribe the SLL region. All coordinates were checked to ensure that they occurred on lowland inselbergs; coordinates of municipalities related to the species vouchers were excluded from our sampling. The map was generated in R (R Core Team 2016), using the 'rgdal' and 'concaveman' packages. The first allows the manipulation of shapefiles, the second allows the generation of concave polygons (concave hulls). The Brazilian biomes shapefile came from the Brazilian Ministry of the Environment. Habitat types and life forms: The sampling was restricted to vascular plants that occur in vegetation islands (Porembski et al. 2000), i.e. isolated vegetation patches of various shapes and sizes, bounded by bare rock or directly seated on bare rock (Caiafa and Silva 2005, de Paula et al. 2017, Fig. 4). Another vegetation type, “thicket” or “scrub” (Rizzini 1997, Fig. Geographic coverage Description: The geographic coverage encompasses lowland inselbergs in the States of Rio de Janeiro, Espírito Santo, southern Bahia and eastern Minas Gerais, i.e. Sugarloaf Land (de Paula et al. 2016). Figure 4. Schematic representation of inselberg vegetation. Vegetation islands are represented by patches 1-5 of varying shapes and sizes, which are bounded by bare rock or directly seated on bare rock (Porembski et al. 2000). Scrub vegetation (Rizzini 1997) appears in the transition zone between the outcrop and the forest that borders the inselberg. The matrix is characterised by the surrounding vegetation, in this case represented by the forest physiognomies belonging to the Atlantic Forest domain. The schematic representation was adapted from de Paula et al. (2015). Figure 3. 4; from hereon, treated as scrub), which occurs in the transition zone between the outcrop and the forest that borders the inselberg, is composed mainly of trees and shrubs from the Atlantic Forest matrix surrounding the inselbergs, with the occasional incidence of species from the vegetation islands. The scrub also includes endemic inselberg species; however, in order to standardise our sampling and filtering in the virtual databases and because of misleading information in the records, we did not include species exclusive to the scrub habitat. Nevertheless, we do include species from vegetation islands that occur occasionally in the scrub, ascribing them to both habitats. We obtained information on life forms for every species, based on the Brazilian Flora online (Flora do Brasil under construction 2020) and classified them into six categories: herbs, lianas/vines, palm trees, shrubs, sub-shrubs and trees. Threatened species: To assign conservation status, we used information from the Centro Nacional de Conservação da Flora (http://www.cncflora.jbrj.gov.br/portal), which provides a continuously-updated list of threatened plant species in the country (MMA 2014). de Paula L et al 8 Figure 4. Schematic representation of inselberg vegetation. Vegetation islands are represented by patches 1-5 of varying shapes and sizes, which are bounded by bare rock or directly seated on bare rock (Porembski et al. 2000). Scrub vegetation (Rizzini 1997) appears in the transition zone between the outcrop and the forest that borders the inselberg. The matrix is characterised by the surrounding vegetation, in this case represented by the forest physiognomies belonging to the Atlantic Forest domain. The schematic representation was adapted from de Paula et al. (2015). Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... 9 9 We identified a total of 505 species of angiosperms (493 species are native and 12 are naturalised) belonging to 58 families and 192 genera (Suppl. material 1; Fig. 5). The richest families are Bromeliaceae (102 spp.), Orchidaceae (63 spp.), Asteraceae (30 spp.), Melastomataceae (25 spp.), Begoniaceae (19 spp.), Cyperaceae (19 spp.), Apocynaceae (18 spp.), Cactaceae (17 spp.), Euphorbiaceae (17 spp.), Poaceae (17 spp.), Fabaceae (15 spp.), Velloziaceae (12 spp.) and Araceae (10 spp.) (Fig. 6). Sixteen families are represented by only one species. The richest genera are Alcantarea (22 spp.), Orthophytum (20 spp.), Begonia (19 spp.), Pitcairnia (13 spp.), Pleroma (13 spp.), Stigmatodon (13 spp.), Tillandsia (13 spp.), Pseudolaelia (10 spp.), Anthurium (9 spp.), Dioscorea (7 spp.), Epidendrum (7 spp.), Mandevilla (7 spp.), Barbacenia (6 spp.), Vellozia (6 spp.) and Vriesea (6 spp.) (Fig. 7). A total of 96 genera are represented by only one species. We identified a total of 505 species of angiosperms (493 species are native and 12 are naturalised) belonging to 58 families and 192 genera (Suppl. material 1; Fig. 5). The richest families are Bromeliaceae (102 spp.), Orchidaceae (63 spp.), Asteraceae (30 spp.), Melastomataceae (25 spp.), Begoniaceae (19 spp.), Cyperaceae (19 spp.), Apocynaceae (18 spp.), Cactaceae (17 spp.), Euphorbiaceae (17 spp.), Poaceae (17 spp.), Fabaceae (15 spp.), Velloziaceae (12 spp.) and Araceae (10 spp.) (Fig. 6). Sixteen families are represented by only one species. The richest genera are Alcantarea (22 spp.), Orthophytum (20 spp.), Begonia (19 spp.), Pitcairnia (13 spp.), Pleroma (13 spp.), Stigmatodon (13 spp.), Tillandsia (13 spp.), Pseudolaelia (10 spp.), Anthurium (9 spp.), Dioscorea (7 spp.), Epidendrum (7 spp.), Mandevilla (7 spp.), Barbacenia (6 spp.), Vellozia (6 spp.) and Vriesea (6 spp.) (Fig. 7). A total of 96 genera are represented by only one species. Figure 5. Species belonging to the most representative angiosperm families from vegetation islands occurring on lowland inselbergs in the Atlantic Forest, SE Brazil. A. Encholirium gracile L.B.Sm. (Bromeliaceae); B. Encyclia spiritusanctensis L.C.Menezes (Orchidaceae); C. Barbacenia tomentosa Mart. (Velloziaceae); D. Begonia aguiabrancensis L.Kollmann (Begoniaceae); E. Vellozia pulchra L.B.Sm. (Velloziaceae); F. Coleocephalocereus fluminensis (Miq.) Backeb. (Cactaceae); G. Pleroma marinanum P.J.F. Guim. & Fraga (Melastomataceae); H. Cnidoscolus lombardii Fern.Casas (Euphorbiaceae); I. Mandevilla grazielae M.F.Sales et al. (Apocynaceae). Photos A, B, D, E, I by L.F.A. de Paula; C, F, G, H by L.O. Azevedo. Figure 5. Taxonomic coverage Description: Our final list consisted of a total of 548 species of vascular plants (505 angiosperms; 43 ferns and lycophytes), belonging to 69 families and 212 genera, occurring on lowland inselbergs in the Atlantic Forest in SE Brazil, i.e. Sugarloaf Land. From procedure 1, based originally on BFG (2015)and Prado et al. (2015), we recorded 208 angiosperms and 16 ferns and lycophytes. From procedure 2, which took into account species sampled by the authors (that were not compiled in BFG 2015 and Prado et al. 2015) and those identified in previous published articles, we recorded 297 angiosperms and 27 ferns and lycophytes. Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... Species belonging to the most representative angiosperm families from vegetation islands occurring on lowland inselbergs in the Atlantic Forest, SE Brazil. A. Encholirium gracile L.B.Sm. (Bromeliaceae); B. Encyclia spiritusanctensis L.C.Menezes (Orchidaceae); C. Barbacenia tomentosa Mart. (Velloziaceae); D. Begonia aguiabrancensis L.Kollmann (Begoniaceae); E. Vellozia pulchra L.B.Sm. (Velloziaceae); F. Coleocephalocereus fluminensis (Miq.) Backeb. (Cactaceae); G. Pleroma marinanum P.J.F. Guim. & Fraga (Melastomataceae); H. Cnidoscolus lombardii Fern.Casas (Euphorbiaceae); I. Mandevilla grazielae M.F.Sales et al. (Apocynaceae). Photos A, B, D, E, I by L.F.A. de Paula; C, F, G, H by L.O. Azevedo. 10 10 de Paula L et al Figure 6. Richest angiosperm families from lowland inselbergs in the Atlantic Forest, SE Brazil. Figure 7. Richest angiosperm genera from lowland inselbergs in the Atlantic Forest, SE Brazil. Figure 6. Richest angiosperm families from lowland inselbergs in the Atlantic Forest, SE Brazil. Figure 7. Richest angiosperm genera from lowland inselbergs in the Atlantic Forest, SE Brazil. Richest angiosperm families from lowland inselbergs in the Atlantic Forest, SE Braz Figure 7. Richest angiosperm genera from lowland inselbergs in the Atlantic Forest, SE Brazil. Richest angiosperm genera from lowland inselbergs in the Atlantic Forest, SE Brazil Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... 11 We recorded a total of 43 species of ferns and lycophytes (all species are native) belonging to 11 families and 20 genera (Suppl. material 2; Fig. 8). The richest families are Anemiaceae (13 spp.), Polypodiaceae (11 spp.), Pteridaceae (6 spp.), Selaginellaceae (3 spp.), Lomariopsidaceae (3 spp.) and Gleicheniaceae (2 spp.) (Fig. 9). Five families are represented by only one species. The richest genera are Anemia (13 spp.), Serpocaulon (4 spp.), Microgramma (3 spp.), Nephrolepis (3 spp.), Selaginella (3 spp.), Doryopteris (2 spp.) and Cheilanthes (2 spp.) (Fig. 10). There were 13 genera presenting only one species. p Figure 8. Species belonging to the most representative fern and lycophyte families from vegetation islands occurring on lowland inselbergs in the Atlantic Forest, SE Brazil. A. Selaginella convoluta (Arn.) Spring (Selaginellaceae); B. S. sellowii Hieron. (Selaginellaceae); C. Cheilanthes geraniifolia (Weath.) R.M.Tryon & A.F.Tryon (Pteridaceae); D. Doryopteris collina (Raddi) J.Sm. (Pteridaceae). Figure 8. Species belonging to the most representative fern and lycophyte families from vegetation islands occurring on lowland inselbergs in the Atlantic Forest, SE Brazil. A. Selaginella convoluta (Arn.) Spring (Selaginellaceae); B. S. sellowii Hieron. (Selaginellaceae); C. Cheilanthes geraniifolia (Weath.) R.M.Tryon & A.F.Tryon (Pteridaceae); D. Doryopteris collina (Raddi) J.Sm. (Pteridaceae). Species belonging to the most representative fern and lycophyte families from vegetation islands occurring on lowland inselbergs in the Atlantic Forest, SE Brazil. A. Selaginella convoluta (Arn.) Spring (Selaginellaceae); B. S. sellowii Hieron. (Selaginellaceae); C. Cheilanthes geraniifolia (Weath.) R.M.Tryon & A.F.Tryon (Pteridaceae); D. Doryopteris collina (Raddi) J.Sm. (Pteridaceae). 12 12 de Paula L et al Life forms and habitat types W d t i d th t 53% (268 ) f i h b f ll d b h b (15 Figure 9. Richest fern and lycophyte families from lowland inselbergs in the Atlantic Forest, SE Brazil. Figure 10. Richest fern and lycophyte genera from lowland inselbergs in the Atlantic Forest, SE Brazil. Figure 9. Richest fern and lycophyte families from lowland inselbergs in the Atlantic Forest, SE Braz Figure 9. Richest fern and lycophyte families from lowland inselbergs in the Atlantic Forest, SE Brazil. Figure 10. Richest fern and lycophyte genera from lowland inselbergs in the Atlantic Forest, SE Brazil. hest fern and lycophyte families from lowland inselbergs in the Atlantic Forest, SE Brazil. Figure 10. Richest fern and lycophyte genera from lowland inselbergs in the Atlantic Forest, SE Brazi Life forms and habitat types We determined that 53% (268 spp.) of angiosperms are herbs, followed by shrubs (15%; 75 spp.), subshrubs (15%; 78 spp.), lianas (8%; 39 spp.), trees (8%; 42 spp.) and palm trees (1%; 3 spp.) (Fig. 11). All species of ferns and lycophytes are herbs (Fig. 11). Most of the species (73% of the angiosperms and 58% of the ferns and lycophytes) occurred in Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... 13 vegetation islands, but there were species which occurred in both vegetation islands and scrub (27% of angiosperms and 42% of ferns and lycophytes) (Fig. 12). scrub (27% of angiosperms and 42% of ferns and lycophytes) (Fig. 12). Figure 11. Life forms of the vascular plant species occurring on lowland inselbergs in the Atlantic Forest, SE Brazil. Figure 12. Percentage of vascular plant species occurring in different habitat types on lowland inselbergs in the Atlantic Forest, SE Brazil. VI = vegetation islands; VI_S = vegetation islands and scrub. Figure 11. Life forms of the vascular plant species occurring on lowland inselbergs in the Atlantic Fores SE Brazil. Percentage of vascular plant species occurring in different habitat types on lowland inselbergs in the Atlantic Forest, SE Brazil. VI = vegetation islands; VI_S = vegetation islands and scrub. Percentage of vascular plant species occurring in different habitat types on lowland inselberg in the Atlantic Forest, SE Brazil. VI = vegetation islands; VI_S = vegetation islands and scrub. Vegetation type, vouchers, conservation status A total of 59% of the angiosperms and 63% of the ferns and lycophytes on our final list were not described as occurring on “rock outcrops” in BFG (2015) and Prado et al. (2015), respectively. Therefore, this study helps to better define information in the "vegetation type" field in Flora do Brasil under construction (2020). Furthermore, 49% (249 spp.) of the angiosperms and 72% (31 spp.) of the ferns and lycophytes were collected by the authors (Fig. 13), the remaining species being vouchered indirectly from other sources. As a first de Paula L et al 14 step to generate a Virtual Herbarium of the vascular plants of Sugarloaf Land, we added links with available images for the vouchers for 75% of the total number of species (375 angiosperms and 37 ferns and lycophytes). The advantage of the link, besides enabling the reader to see the image of the voucher with the respective original collection label, is that taxonomic changes can be revised in the databases, which allows the list to be continuously updated. In the official lists of endangered species, 115 angiosperms and five ferns and lycophytes are mentioned. In the angiosperm list, 9 species are vulnerable (VU), 30 are endangered (EN) and 12 are critically endangered (CR), while in the fern and lycophyte list, two species are vulnerable; the remaining species are in least concern categories (see Suppl. materials 1, 2). However, the conservation status of most of the angiosperm (77%), fern and lycophyte (88%) species is unknown. Figure 13. Percentages of the vascular plant species included in the checklist that were collected by the authors (C) or vouchered indirectly from other sources (OS). Figure 13. Percentages of the vascular plant species included in the checklist that were collected by th authors (C) or vouchered indirectly from other sources (OS). Percentages of the vascular plant species included in the checklist that were collected by the authors (C) or vouchered indirectly from other sources (OS). Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... 15 15 Figure 14. General overview of Sugarloaf Land (SLL) region. The concave polygon that indicates SLL was built, based on the available coordinates of the vouchers assigned in the vascular plant list for lowland inselbergs occurring in the Atlantic Forest, SE Brazil. Fi 14 Figure 14. General overview of Sugarloaf Land (SLL) region. The concave polygon that indicates SLL was built, based on the available coordinates of the vouchers assigned in the vascular plant list for lowland inselbergs occurring in the Atlantic Forest, SE Brazil. Usage rights Use license:  Creative Commons Public Domain Waiver (CC-Zero) Sugarloaf Land region SLL region was originally baptised, based on projections generated through modelling techniques only for the Bromeliaceae family (de Paula et al. 2016) and here we aimed to cover the occurrence of all the vascular plant species occurring on lowland inselbergs. The convex envelope representing the SLL region is displayed in Fig. 14 and contains the records for lowland inselbergs where we had access to the coordinates. It is important to note that lowland inselbergs might exist outside the area ascribed here as SLL, due to the following facts: i) many lowland inselbergs have never been sampled; ii) some vouchers did not have available coordinates; these factors culminate in the absence of possible lowland inselberg areas in our map. However, it gives a better estimation and a finer overview of the core area named as SLL. Data resources Data package title:  Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg plant diversity - Supplementary Material Resource link:  https://ckan.jbrj.gov.br/dataset/2020_bdj_inselberg Number of data sets:  2 Data set name: List of angiosperms occurring on lowland inselbergs in the Atlantic Forest, SE Brazil Download URL:  https://ckan.jbrj.gov.br/dataset/2020_bdj_inselberg Data package title:  Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg plant diversity - Supplementary Material Resource link:  https://ckan.jbrj.gov.br/dataset/2020_bdj_inselberg Data set name: List of angiosperms occurring on lowland inselbergs in the Atlant Forest, SE Brazil Download URL:  https://ckan.jbrj.gov.br/dataset/2020_bdj_inselberg de Paula L et al de Paula L et al 16 de Paula L et al Data format: CSV and XLSX Description: List containing 505 angiosperm species occurring on lowland inselbergs in the Atlantic Forest, SE Brazil, highlighting species included in official lists of endangered flora, life forms, habitat type of occurrence, origin, voucher, link for the herbarium image of the voucher. Column label Column description Family The full scientific name of the family in which the taxon is classified Species The full scientific name Author Authorship of the scientific name Origin If the species is native or non-native, followed Flora do Brasil 2020 (under construction; http:// floradobrasil.jbrj.gov.br) Threatened category Followed Centro Nacional de Conservação da Flora (http://www.cncflora.jbrj.gov.br/portal); DD = Data deficient, NT = Near Threatened, VU = Vulnerable, CR = Critically Endangered, EN = Endangered, LC = Least Concern, NE = Not evaluated Life form Followed Flora do Brasil 2020 (under construction; http://floradobrasil.jbrj.gov.br); life forms are classified into six categories: herbs, lianas/vines, palm trees, shrubs, sub-shrubs and trees Source Indicates where we achieved the information that the species is occurring on lowland inselbergs Habitat Indicates if the species occurred in vegetation islands (VI; ellipsoid vegetation patches surrounded by bare rock) and scrub (S; arboreal-shrub vegetation bordering inselberg) Reference or voucher Indicates vouchers (collector and number) with the respective acronym of the herbarium according to Thiers (http://sweetgum.nybg.org/ih/, continuously updated) when the source is C or EC; in case the source is A, it was provided by the reference of the article (see references at the end of the table) Link for the voucher Provides the link for the online image of the respective voucher; links are from JABOT-JBRJ and Reflora-Herbário Virtual Data set name: List of ferns and lycophytes occurring on lowland inselbergs in the Atlantic Forest, SE Brazil Data set name: List of ferns and lycophytes occurring on lowland inselbergs in the Atlantic Forest, SE Brazil Data set name: List of ferns and lycophytes occurring on lowland inselbergs in the Atlantic Forest, SE Brazil Download URL:  https://ckan.jbrj.gov.br/dataset/2020_bdj_inselberg Description: List containing 43 fern and lycophyte species occurring on lowland inselbergs in the Atlantic Forest, SE Brazil, highlighting species included in official lists of endangered flora, life forms, habitat type of occurrence, origin, voucher, link for the herbarium image of the voucher. Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... Column label Column description Family The full scientific name of the family in which the taxon is classified Species The full scientific name Author Authorship of the scientific name Origin If the species is native or non-native, followed Flora do Brasil 2020 (under construction; http:// floradobrasil.jbrj.gov.br) Threatened category Followed Centro Nacional de Conservação da Flora (http://www.cncflora.jbrj.gov.br/portal); DD = Data deficient, NT = Near Threatened, VU = Vulnerable, CR = Critically Endangered, EN = Endangered, LC = Least Concern, NE = Not evaluated Life form Followed Flora do Brasil 2020 (under construction; http://floradobrasil.jbrj.gov.br); life forms are classified into six categories: herbs, lianas/vines, palm trees, shrubs, sub-shrubs and trees Source Indicates where we achieved the information that the species is occurring on lowland inselbergs Habitat Indicates if the species occurred in vegetation islands (VI; ellipsoid vegetation patches surrounded by bare rock) and scrub (S; arboreal-shrub vegetation bordering inselberg) Reference or voucher Indicates vouchers (collector and number) with the respective acronym of the herbarium according to Thiers (http://sweetgum.nybg.org/ih/, continuously updated) when the source is C or EC; in case the source is A, it was provided by the reference of the article (see references at the end of the table) Link for the voucher Provides the link for the online image of the respective voucher; links are from JABOT-JBRJ and Reflora-Herbário Virtual Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... 17 17 Checklist, ecological aspects and implications for conservation The beta diversity of lowland inselbergs in Brazil is outstanding (Meirelles et al. 1999, Safford and Martinelli 2000, de Paula et al. 2016, de Paula et al. 2019b), with each outcrop containing exclusive flora, even though they may be located only a few kilometres apart. The high species turnover across individual inselbergs is a common pattern worldwide, for instance, it has been shown for inselberg mosaics in Australia (Yates et al. 2019) and Northern South America (Sarthou et al. 2017). Moreover, studies on neotropical inselberg- adapted species have shown high population differentiation, high genetic diversity levels and strong phylogeographic structure in this naturally-fragmented environment (e.g. Barbará et al. 2007, Palma-Silva et al. 2011, Hmeljevski et al. 2015, Hmeljevski et al. 2017, de Paula et al. 2017b, Nazareno et al. 2020). Species with patchy distribution usually experience reduced gene flow, significant genetic drift and high levels of population divergence, supporting the view of inselbergs as centres of species diversity and endemism. de Paula L et al 18 Furthermore, a common phenomenon on these outcrops is the occurrence of polymorphic species (Cronk 1998, Mello-Silva 2004). Many inselberg taxa have previously been described as displaying extensive intraspecific variation amongst and within populations, especially monocot taxa, such as Anthurium, Philodendron (Araceae), Alcantarea, Encholirium, Pitcairnia (Bromeliaceae), Pseudolaelia (Orchidaceae), Barbacenia and Vellozia (Velloziaceae), but also eudicot taxa, such as Pleroma (Melastomataceae) and Coleocephalocereus (Cactaceae) (Mello-Silva 2004, Taylor and Zappi 2004, Hmeljevski et al. 2017, de Paula et al. 2017a, Menini Neto et al. 2019). The spatial and ecological isolation of these outcrops as a result of their immersion in the surrounding rainforest is thought to be responsible for the high levels of genetic differentiation amongst populations, including Alcantarea (Barbará et al. 2007), Encholirium (Hmeljevski et al. 2015, Hmeljevski et al. 2017) and Pitcairnia (Palma-Silva et al. 2011), as well as for the resulting morphological variability commonly reported in adaptive radiation events (Palma-Silva et al. 2011). In developing the present checklist, we could not provide information on species complexes, but note that they should be taken into consideration in conservation analyses since outcrops harbour specific morphotypes. Attention to these processes is also important for solving taxonomic problems and understanding processes of species evolution within terrestrial islands. Checklist, ecological aspects and implications for conservation The high levels of species diversity on inselbergs is usually linked to the fact that species capable of surviving in such harsh environmental conditions display diverse forms of adaptation (Biedinger et al. 2000, de Paula et al. 2015, de Paula et al. 2019a). With regard to water scarcity, distinct plant groups in SE Brazil have adopted several strategies to mitigate such stresses. The succulent leaves of bromeliads of the genus Encholirium and orchids of the genus Epidendrum, for example, enable plants to accumulate water in their tissue. Other bromeliads, such as Alcantarea spp. and Vriesea spp., can store water in reservoirs due to their rosette-shaped leaf arrangement, which also attracts a variety of fauna (Lacerda et al. 2009). Cacti, in turn, not only accumulate water, but also have spines (modified leaves) and trichome-like thorns/spines at the stem base, which enables them to avoid overheating when lying directly on the rock, an adaption typical of Coleocephalocereus species. Geophytes that store water in underground bulbs are also very common, such as members of the Apocynaceae family (e.g. Mandevilla spp.). In contrast, desiccation tolerance is an effective strategy used by some inselberg species to cope with drought conditions, for instance, present in Velloziaceae members (e.g. Barbacenia spp. and Vellozia spp.), but also in fern and lycopyte species (e.g. Cheilanthes spp., Doryopteris spp., Selaginella spp.). This strategy is characterised by the ability of plants to survive cycles of dehydration and rehydration without affecting viability (Porembski and Barthlott 2000). During the dry season, desiccation-tolerant species enter a dehydrated state, losing a considerable amount of water in their tissue. With the occurrence of rain, they quickly rehydrate, in many cases without any structural or physiological damage (Vicré et al. 2004). Due to this unique feature, they are called resurrection plants. Understanding the heterogeneity of ecological strategies found amongst inselberg species can be useful in developing conservation strategies, for example, for restoration after disturbance, especially in the context of global environmental change (Drenovsky et al. 2012, de Paula et al. 2015, Ottaviani et al. 2016). Since species Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... 19 have different levels of tolerance to abiotic stresses, conservationists can select species based on their traits, which includes attributes associated with resource acquisition and growth and their functional role (Tecco et al. 2010, de Paula et al. 2015). Checklist, ecological aspects and implications for conservation Due to difficulties in accessing many inselbergs, researchers concerned with biodiversity have overlooked the significant potential of these outcrops. The high levels of beta diversity and genetically-differentiated populations on outcrops in SE Brazil underscore the fact that there are insufficient numbers of inselbergs protected within conservation units. Furthermore, lowland inselbergs are most often located in small reserves within urban areas, such as the Monumento Natural dos Morros do Pão de Açúcar e da Urca, the Parque Nacional da Tijuca, the Parque Natural Municipal Paisagem Carioca Rio de Janeiro and the Parque Estadual Serra da Tiririca, all of which are located within the metropolitan area of Rio de Janeiro. In these reserves, inselbergs can be visited by tourists, which can have a significant impact on the natural vegetation. There are also some lowland inselbergs located in conservation units which permit a certain amount of human occupation, for example, the Pedra do Elefante in Espírito Santo State (Pena and Alves-Araújo 2017). The Monumento Natural Pontões Capixabas, in the heart of the Sugarloaf Land in Espírito Santo, is the only conservation area in Brazil that encompasses a considerable number of lowland inselbergs. However, its effectiveness is threatened by the absence of a reasonable management plan (http://www.icmbio.gov.br/portal/ planosmanejo) that could help to balance the competing interests of local farmers with the preservation of its unique biota. In Brazil, threats to inselberg biodiversity is increasing dramatically due to quarrying, mining, grazing, goat herding, fire, biological invasions, urban expansion and unsustainable removal of attractive floral species (Martinelli 2007, Porembski et al. 2016, Fig. 15). In Sugarloaf Land, not only is the extraction of natural resources far too vast to be sustainable (Ferreira et al. 2014), but the area is also experiencing rapid fragmentation of the Atlantic Forest biome (Tabarelli et al. 2004), both of which increase the risks to vegetation refugia in inselbergs. In several instances, rocks have taken the place of wood as building and finishing material and the destructive cycle of forest logging (Dean 1995) has been replaced by the extraction of geological resources, once again fuelling an uncontrolled and unsustainable industry. The lack of conservation units that harbour inselbergs demonstrates the need for inventories of those that occur on private lands. Meanwhile, taxonomically-verified checklists are not only necessary for ecological, genetic, biogeographic and evolutionary studies (Cardoso et al. Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... 21 Acknowledgements We are thankful to the staff from RB and MBML herbaria for helping in plant cataloguing and dataset management. We are grateful to F. Alves, L.A. Estevão, M.M. Moreira, R. Ocelli and R. Dias for helping in database management and graphical work. We also thank the taxonomists who kindly helped with plant identification: A. Chautems (Gesneriaceae), D. Zappi (Cactaceae), L. Versieux and R. Louzada (Bromeliaceae), P.L. Viana (Poaceae), R. Trevisan (Cyperaceae), R. Goldenberg and F. Meyer (Melastomataceae). L.F.A. de Paula was supported by a research grant from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico; 290155/2014-2) and DAAD (Deutscher Akademischer Austauschdienst). We also thank CNPq for the research grants provided to R.C. Forzza (303420/2016-2) R. Mello-Silva (research fellow) and P. Labiak (311352/2019-7). R.C. Forzza thanks the grants from FAPERJ (processes n° E-26/202.778/2018) through Programa Cientistas do Nosso Estado. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) – Finance Code 001. We finally thank Dr. Evelyn R. Nimmo for editing the English of the manuscript. Checklist, ecological aspects and implications for conservation 2017), but also to provide information for Brazilian policies of biological conservation that include rock outcrop ecosystems. Correct estimates of the flora provide reliable data and information to promote the activities necessary to preserve these extremely threatened environments. Finally, as populations on individual inselbergs are genetically distinct and beta diversity between outcrops is extremely high, effective conservation strategies will face serious challenges to protect multiple inselbergs across the whole Sugarloaf Land region. de Paula L et al 20 Figure 15. Main threats to inselberg biodiversity in SE Brazil. A, B. Mining in Espírito Burned populations of Alcantarea extensa (Bromeliaceae) and Vellozia plic respectively, due to anthropogenically-caused fire in Minas Gerais State; E, Melinis repens) on inselbergs in Minas Gerais State; G, H. Grazing on inse and on the top of an inselberg, respectively, in Minas Gerais State; I, J. Urb inselbergs in the municipalities of Rio de Janeiro (Rio de Janeiro State) a Santo State), respectively. Photos by L.F.A. de Paula, except for photo I by J Figure 15. Main threats to inselberg biodiversity in SE Brazil. A, B. Mining in Espírito Santo State; C, D. Burned populations of Alcantarea extensa (Bromeliaceae) and Vellozia plicata (Velloziaceae), respectively, due to anthropogenically-caused fire in Minas Gerais State; E, F. Invasive grass ( Melinis repens) on inselbergs in Minas Gerais State; G, H. Grazing on inselberg surroundings and on the top of an inselberg, respectively, in Minas Gerais State; I, J. Urban expansion on to inselbergs in the municipalities of Rio de Janeiro (Rio de Janeiro State) and Vitória (Espírito Santo State), respectively. Photos by L.F.A. de Paula, except for photo I by J.M.A. Braga. Figure 15. Main threats to inselberg biodiversity in SE Brazil. A, B. Mining in Espírito Santo State; C, D. Burned populations of Alcantarea extensa (Bromeliaceae) and Vellozia plicata (Velloziaceae), respectively, due to anthropogenically-caused fire in Minas Gerais State; E, F. Invasive grass ( Melinis repens) on inselbergs in Minas Gerais State; G, H. Grazing on inselberg surroundings and on the top of an inselberg, respectively, in Minas Gerais State; I, J. Urban expansion on to inselbergs in the municipalities of Rio de Janeiro (Rio de Janeiro State) and Vitória (Espírito Santo State), respectively. Photos by L.F.A. de Paula, except for photo I by J.M.A. Braga. Sugarloaf Land in south-eastern Brazil: a tropical hotspot of lowland inselberg ... References https://doi.org/10.1073/pnas.1706756114 • Carmo F, Mota R, Kamino L, Jacobi C (2018) Check-list of vascular plant communities on ironstone ranges of south-eastern Brazil: dataset for conservation. 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Kew, Royal Botanic Gardens, London. • Tecco PA, Díaz S, Cabido M, Urcelay C (2010) Functional traits of alien plants across contrasting climatic and land-use regimes: do aliens join the locals or try harder than them? Journal of Ecology 98: 17‑27. https://doi.org/10.1111/j.1365-2745.2009.01592.x • Thiers B (2020) A global directory of public herbaria and associated staff. New York Botanical Garden's Virtual Herbarium. http://sweetgum.nybg.org/ih/ • Valadares RT, Kollmann LJ, Sakuragui CM (2019) A new species of Anthurium (Araceae) with cordate leaves from southeastern Brazil. Novon: A Journal for Botanical Nomenclature 27: 3‑7. References Journal of Biogeography 26: 693‑712. https:// d i /10 1046/j 1365 2699 1999 00313 doi.org/10.1046/j.1365-2699.1999.00313.x https://doi.org/10.3417/2018270 , , g ( ) p (Araceae) with cordate leaves from southeastern Brazil. Novon: A Journal for Botanical Nomenclature 27: 3‑7. https://doi.org/10.3417/2018270 Viana PL, de Paula LF (2013) Axonopus graniticola, a new species of A. ser. Suffulti (Poaceae, Panicoideae, Paspaleae) from Minas Gerais, Brazil. PhytoKeys 21: 7‑16. https://doi org/10 3897/phytokeys 21 4157 • Viana PL, de Paula LF (2013) Axonopus graniticola, a new species of A. ser. Suffulti (Poaceae, Panicoideae, Paspaleae) from Minas Gerais, Brazil. PhytoKeys 21: 7‑16. https://doi.org/10.3897/phytokeys.21.4157 • Vicré M, Farrant JM, Driouich A (2004) Insights into the cellular mechanisms of desiccation tolerance among angiosperm resurrection plant species. Plant, Cell & Environment 27: 1329‑1340. https://doi.org/10.1111/j.1365-3040.2004.01212.x • Vicré M, Farrant JM, Driouich A (2004) Insights into the cellular mechanisms of desiccation tolerance among angiosperm resurrection plant species. Plant, Cell & Environment 27: 1329‑1340. https://doi.org/10.1111/j.1365-3040.2004.01212.x 26 de Paula L et al • Vieira BC, Salgado AA, Santos LJ (2015) Landscapes and Landforms of Brazil - World Geomorphological Landscapes. Springer, Dordrecht. • Yates CJ, Robinson T, Wardell‐Johnson GW, Keppel G, Hopper SD, Schut AG, Byrne M (2019) High species diversity and turnover in granite inselberg floras highlight the need for a conservation strategy protecting many outcrops. Ecology and Evolution 9: 7660‑7675. https://doi.org/10.1002/ece3.5318 Geomorphological Landscapes. Springer, Dordrecht. • Yates CJ, Robinson T, Wardell‐Johnson GW, Keppel G, Hopper SD, Schut AG, Byrne M (2019) High species diversity and turnover in granite inselberg floras highlight the need for a conservation strategy protecting many outcrops. Ecology and Evolution 9: 7660‑7675. https://doi.org/10.1002/ece3.5318 Forest, SE Brazil Authors:  de Paula, LFA; Azevedo, LO; Mauad, LP; Cardoso, LJT; Braga, JMA; Kollmann, LJC; Fraga, CN; Menini Neto, L; Labiak, PH; Mello-Silva, R; Porembski, S; Forzza, RC Data type:  List of species Brief description:  List containing 505 angiosperm species occurring on lowland inselbergs in the Atlantic Forest, SE Brazil, highlighting species included in official lists of endangered flora, life forms, habitat type of occurrence, origin, voucher, link for the herbarium image of the voucher. Download file (81.50 kb) Authors:  de Paula, LFA; Azevedo, LO; Mauad, LP; Cardoso, LJT; Braga, JMA; Kollmann, LJC; Fraga, CN; Menini Neto, L; Labiak, PH; Mello-Silva, R; Porembski, S; Forzza, RC Data type:  List of species Brief description:  List containing 505 angiosperm species occurring on lowland inselbergs in the Atlantic Forest, SE Brazil, highlighting species included in official lists of endangered flora, life forms, habitat type of occurrence, origin, voucher, link for the herbarium image of the voucher. Download file (81.50 kb) Suppl. material 1: List of angiosperms occurring on lowland inselbergs in the Atlantic Forest, SE Brazil Forest, SE Brazil Atlantic Forest, SE Brazil Authors:  de Paula, LFA; Azevedo, LO; Mauad, LP; Cardoso, LJT; Braga, JMA; Kollmann, LJC; Fraga, CN; Menini Neto, L; Labiak, PH; Mello-Silva, R; Porembski, S; Forzza, RC Data type:  List of species Brief description:  List containing 43 fern and lycophyte species occurring on lowland inselbergs in the Atlantic Forest, SE Brazil, highlighting species included in official lists of endangered flora, life forms, habitat type of occurrence, origin, voucher, link for the herbarium image of the voucher. Download file (8.60 kb) Authors:  de Paula, LFA; Azevedo, LO; Mauad, LP; Cardoso, LJT; Braga, JMA; Kollmann, LJC; Fraga, CN; Menini Neto, L; Labiak, PH; Mello-Silva, R; Porembski, S; Forzza, RC Data type:  List of species Brief description:  List containing 43 fern and lycophyte species occurring on lowland inselbergs in the Atlantic Forest, SE Brazil, highlighting species included in official lists of endangered flora, life forms, habitat type of occurrence, origin, voucher, link for the herbarium image of the voucher. Download file (8.60 kb)
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Seroprevalence and Modifiable Risk Factors for Toxocara spp. in Brazilian Schoolchildren
PLoS neglected tropical diseases
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Introduction commonly results from contact with contaminated soil and rarely from the ingestion of undercooked meat containing Toxocara larvae. Children are notably susceptible to infection because they are exposed to the eggs during recreational activities in sandboxes and playgrounds contaminated with dog and cat feces [2],[8],[9]. The clinical manifestations of toxocariasis are related to the location and degree of damage to host tissues caused by the inflammatory response to larval migration. These manifestations can vary from asymptomatic infection to the most severe: visceral toxocariasis (VT), characterized by larval migration through major organs including the liver, lungs and, more rarely, the central nervous system (CNS) [8],[10],[11]; ocular toxocariasis (OT), which occurs when the larvae of the parasite affect the human eye, causing severe inflammation and potentially producing partial or total loss of vision [12]; and less severe syndromes, characterized by nonspecific signs and symptoms, which have been primarily described in children. These syndromes include covert toxocariasis [13] and common toxoca- riasis [14] in adults. commonly results from contact with contaminated soil and rarely from the ingestion of undercooked meat containing Toxocara larvae. Children are notably susceptible to infection because they are exposed to the eggs during recreational activities in sandboxes and playgrounds contaminated with dog and cat feces [2],[8],[9]. Human toxocariasis is a helminthic zoonosis caused by the larvae of the ascarid worms of Toxocara spp. In the early 1950s, Toxocara canis was recognized as a human pathogen [1], and the term ‘‘visceral larva migrans’’ was so widely used that human toxocariasis is also known as visceral larva migrans syndrome. Two species of roundworms, Toxocara canis and Toxocara cati, are recognized as causative agents of human toxocariasis [2],[3],[4],[5]. The adults of both of these species parasitize the small intestines of their definitive hosts, which are canids and felids, respectively [6]. The clinical manifestations of toxocariasis are related to the location and degree of damage to host tissues caused by the inflammatory response to larval migration. Received Jone 30, 2013; Accepted March 10, 2014; Published May 29, 2014 Received Jone 30, 2013; Accepted March 10, 2014; Published May 29, 2014 Copyright:  2014 Cassenote et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: AJFC was given scholarship from Brazilian government by CAPES (Coordination for the Improvement of Higher Education Personnel). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Funding: AJFC was given scholarship from Brazilian government by CAPES (Coordination for the Improvement of Higher Education Personnel). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cassenote@usp.br * E-mail: cassenote@usp.br Alex J. F. Cassenote1*, Alba R. de Abreu Lima2, Jose´ M. Pinto Neto3, Guita Rubin 1 Postgraduate Program in Infectious and Parasitic Diseases, Faculty of Medicine, University of Sa˜o Paulo, Cerqueira Ce´sar, Sa˜o Paulo, Brazil, 2 Faculty of Medicine of Sa˜o Jose´ do Rio Preto, Department of Molecular Biology, Vila Sa˜o Jose´, Sa˜o Jose´ do Rio Preto, Brazil, 3 Faculty of Health Sciences, University Camilo Castelo Branco, Sa˜o Paulo, Brazil, 4 Institute of Tropical Medicine of Sa˜o Paulo, University of Sa˜o Paulo, Cerqueira Ce´sar, Sa˜o Paulo, Brazil PLOS Neglected Tropical Diseases | www.plosntds.org Abstract Background: Toxocariasis is a worldwide helminthic zoonosis caused by infection with the larvae of the ascarid worms that comprise the Toxocara spp. Children are particularly prone to infection because they are exposed to the eggs in sandboxes and playgrounds contaminated with dog and cat feces. Certain behaviors, such as a geophagy habit, poor personal hygiene, a lack of parental supervision, close contact with young dogs, and ingestion of raw meat, as well as gender, age, and socioeconomic status, affect the prevalence of the disease. However, previous studies of the risk factors for toxocariasis have generally produced inconsistent results. An epidemiological cross-sectional study was conducted to evaluate the seroprevalence of IgG anti-Toxocara spp. antibodies and associated factors in schoolchildren from a region in the southeast of Brazil. Methodology/Principal Findings: A total of 252 schoolchildren aged 1 to 12 years (120 males and 132 females) were assessed. An enzyme-linked immunosorbent assay based on Toxocara canis larval excretory-secretory antigens was used to determine outcomes. A questionnaire was used to collect information on children, family, and home characteristics. Clinical and laboratory data completed the dataset investigated in this study. Seroprevalence was 15.5% (95%CI 11.5–19.8). Geophagy (aPR 2.38 [95%CI 1.36–4.18], p-value 0.029) and the habit of hand washing before meals (aPR 0.04 [95%CI 0.01– 0.11], p-value #0.001) were factors associated with increased and decreased seroprevalence, respectively. The income factor and its related variables lost statistical significance after adjustment with a multiple Poisson regression model. Conclusions/Significance: The current study confirms that toxocariasis is a public health problem in the evaluated area; modifiable factors such as soil contact and personal hygiene appear to have a greater influence on the acquisition of infection than sociodemographic attributes, thus representing direct targets for disease prevention and control. Citation: Cassenote AJF, de Abreu Lima AR, Pinto Neto JM, Rubinsky-Elefant G (2014) Seroprevalence and Modifiable Risk Factors for Toxocara spp. in Brazilian Schoolchildren. PLoS Negl Trop Dis 8(5): e2830. doi:10.1371/journal.pntd.0002830 Editor: Emily Jenkins, University of Saskatchewan, Canada Citation: Cassenote AJF, de Abreu Lima AR, Pinto Neto JM, Rubinsky-Elefant G (2014) Seroprevalence and Modifiable Risk Factors for Toxocara spp. in Brazilian Schoolchildren. PLoS Negl Trop Dis 8(5): e2830. doi:10.1371/journal.pntd.0002830 Editor: Emily Jenkins, University of Saskatchewan, Canada Seroprevalence and Modifiable Risk Factors for Toxocara spp. in Brazilian Schoolchildren assenote1*, Alba R. de Abreu Lima2, Jose´ M. Pinto Neto3, Guita Rubinsky-Elefant4 Author Summary Author Summary Human toxocariasis is a neglected parasitic infection occurring in Brazil and worldwide. The combination of the close proximity of canines and felines to humans, environmental contamination by infectious forms of the parasite and neglect by public health officials provides a favorable situation for the spread of this zoonosis, which primarily affects pediatric populations. An epidemiological study in an area of known parasitic soil contamination was conducted to evaluate the impact of this disease and the factors that influence its occurrence. A sample of 1- to 12- year-old children was obtained with random selection of schools, classrooms and schoolchildren. A questionnaire was used to collect socioeconomic and behavioral infor- mation, laboratory tests determined the presence of antibodies against Toxocara spp. parasites and a medical evaluation noted clinical features of the disease. The study found that antibodies is present in more than 15% of subjects and can be in as many as 30% depending on individual characteristics. The analysis also indicated that behaviors that increase soil ingestion, such as nail biting, eating dirt or poor personal hygiene, contribute to increased frequency of the disease and may be even more important than the socioeconomic status of individuals. These behaviors therefore represent real and simple targets for the control of this neglected tropical disease. A complex sample (multi-stage cluster and strata sampling) [21],[22] was prepared. In the first stage, the number of schools with students within the age range being studied was identified (n = 31 schools), and then, schools were selected with a probability proportional to the type of school (public, philanthropic, or private). In total, 2 public schools, 2 philanthropic schools and 1 private school were selected. In the second stage, simple random sampling was used to select groups, the number of classrooms with students within the age range under study was identified (n = 40 classrooms), and 25 classrooms were selected (5 classrooms for each school) with a probability proportional to the eligible age ranges. Three operational strata were used: 1 to 4, 5 to 8, and 9 to 12 years old. The representativeness of the age groups was thus conserved intra-classroom. Author Summary The children outside the specific age group within a classroom were excluded from the final stage, in which children were selected within each classroom by simple random sampling with replacement (n = 10 children per classroom for original sample members and n = 3 children per classroom for replacement sample members). Additionally, 2 children were randomly selected from the 25 classrooms to fulfill the prespecified sample size of 252. In this stage, the sampling also preserved 2 income strata (with similar population proportions): less than or equal to 2 minimum wages were included in stratum stA, and incomes higher than 2 minimum wages were included in stratum stB. The literature reports that behaviors such as geophagy, poor personal hygiene and a lack of parental supervision, close contact with young dogs and ingestion of raw meat as well as gender, age, and socioeconomic status affect the frequency of the disease [7]. Nevertheless, the results of various studies of the risk factors for toxocariasis have generally been inconsistent [15],[16]. The replacement sample members were identified as alternates and placed on a waiting list; they were included in the study if original sample members refused to participate. The process of identifying the classrooms and the students was based on an updated list of the students in each participating school, with birth dates previously provided by the 5 schools that participated in the study. This list permitted all procedures to be performed randomly. Between 2007 and 2008, soil contamination by eggs of soil- transmitted helminths was evaluated in the same area investigated in the current study [17]. The authors observed a high rate of soil contamination by parasites with zoonotic potential (30.2%), including Toxocara spp. (79.3%), Trichuris spp. (13.8%) and Ancylostoma spp. (6.9%) in the evaluated samples from sandboxes and playgrounds located in public squares, an observation that prompted a serological evaluation of children in the region. The aim of this paper is to present the methodology and preliminary results of a cross-sectional study conducted to estimate the seroprevalence of IgG anti-Toxocara spp. and associated factors in schoolchildren aged 1 to 12 years from a region in southeast Brazil. Data collection, variables, outcome definitions and ethical considerations After receiving a formal request and information on the importance, objectives and methodology of this study, the school board of each selected school gave permission for the study to be conducted. Selected children and their families were approached by interviewers to validate the prospective subjects’ consent to participate in this research, and over the next week, the interviewers visited the families to collect data and schedule a medical visit for the child at the Support Center for Infectious and Parasitic Diseases in Fernando´polis. Introduction These manifestations can vary from asymptomatic infection to the most severe: visceral toxocariasis (VT), characterized by larval migration through major organs including the liver, lungs and, more rarely, the central nervous system (CNS) [8],[10],[11]; ocular toxocariasis (OT), which occurs when the larvae of the parasite affect the human eye, causing severe inflammation and potentially producing partial or total loss of vision [12]; and less severe syndromes, characterized by nonspecific signs and symptoms, which have been primarily described in children. These syndromes include covert toxocariasis [13] and common toxoca- riasis [14] in adults. Toxocara spp. have a worldwide distribution and tend to be more prevalent in tropical regions, including industrialized countries, where they are considered the cause of the most frequent form of helminthiasis. Toxocariasis has been described as the most common human parasitic worm infection in developed countries [2],[7],[8]. Toxocara spp. have a worldwide distribution and tend to be more prevalent in tropical regions, including industrialized countries, where they are considered the cause of the most frequent form of helminthiasis. Toxocariasis has been described as the most common human parasitic worm infection in developed countries [2],[7],[8]. Humans are infected via the accidental ingestion of embryo- nated eggs containing Toxocara spp. larvae. This ingestion most Humans are infected via the accidental ingestion of embryo- nated eggs containing Toxocara spp. larvae. This ingestion most May 2014 | Volume 8 | Issue 5 | e2830 May 2014 | Volume 8 | Issue 5 | e2830 1 PLOS Neglected Tropical Diseases | www.plosntds.org PLOS Neglected Tropical Diseases | www.plosntds.org Toxocarariasis Risk Factors in Brazilian Children The sample size (n = 252) was based on the following parameters: 95% confidence level and a 5% margin of error, a statistical power of 80%, design effect of 1.5, and an estimated prevalence of 12.4% (average proportion of two sampling strata), as observed by Campos Junior et al. [20]. Scenario, study and sample design Between 2007 and 2010, a cross-sectional study was conducted in urban areas of Fernando´polis (S.20u17’02’’ W.50u14’47’’), a city with 59,580 inhabitants located in the northwestern Sa˜o Paulo State in the southeast of Brazil (Figure 1). Fernando´polis has a high Human Development Index (a comprehensive statistic that incorporates population life expectancy, education and income) of 0.83, whereas the HDI value for Brazil is 0.71 [18]. p Some of the variables investigated in blocks were as follows: 1) characteristics of children (collected from family interview questionnaire): gender, age, type of school (public, philanthropic, or private), geophagy, onychophagy and hand-washing habits; 2) family and home characteristics (collected from family interview questionnaire): SUS users only (SUS - Brazilian Unified Health System) and income (wage of head of household); 3) clinical characteristics (collected from medical records): history or presence of hepatomegaly, splenomegaly, adenomegaly, cutaneous manifestations (urticarial, pruritus, or eczema), lung manifestations (wheezing, bronchitis, or cough), pneumonia, and seizures; 4) laboratory characteristics (collected from laboratory reports): complete blood count (CBC; absolute and relative eosinophils, and other cells), intestinal parasites (larvae and eggs in The population of individuals aged 1 to 12 years in the city was estimated to total approximately 22,000 children studying in 31 schools, 18 of which were public (funded by the state or municipal government, with no cost to students), 8 philanthropic (funded in whole or in part by churches, with no cost to students), and 5 private schools (self-funded by charging their student body) [19]. May 2014 | Volume 8 | Issue 5 | e2830 PLOS Neglected Tropical Diseases | www.plosntds.org 2 Toxocarariasis Risk Factors in Brazilian Children Figure 1. Geographic location of the study area. Sa˜o Paulo is located in southeast Brazil, and Fernando´polis is situated in the northwest of Sa˜o Paulo State. doi:10.1371/journal.pntd.0002830.g001 Figure 1. Geographic location of the study area. Sa˜o Paulo is located in southeast Brazil, and Fernando´polis is situated in the northwest of Sa˜o Paulo State. doi:10 1371/journal pntd 0002830 g001 fecal samples), and anti-Toxocara spp. IgG antibodies evaluated with an enzyme-linked immunosorbent assay (ELISA). CBC). As the main objective of the study was to evaluate the risk factors for contact with the parasite, greater emphasis was placed on the factors associated with the first outcome. Scenario, study and sample design Data from different sources were collected by a team of 8 interviewers who were previously trained in pilot studies over a 1- week interval at 2 schools that were not included in the final sample. The kappa coefficient was used to verify agreement between the 2 questionnaire applications. The agreement between the questionnaire applications was considered very good (kappa coefficient = 0.94). Refusal at the blood collection or interview stage was observed in 3.17% (8) of individuals without prejudice to the study because the alternate participants were then included in the study. The study was reviewed and approved by the Research Ethics Committee of the Clinics Hospital, Sa˜o Paulo University Medical School (Protocol Number #0518-07), in accordance with Brazil- ian and international laws. Legal guardians were informed about the study ethical criteria and procedures by letter and by interview with the research assistant; they all signed the informed consent for the children that participated in this study. PLOS Neglected Tropical Diseases | www.plosntds.org Antigen preparation, serum processing and antibody detection A preparation of infective Toxocara canis larval excretory- secretory antigens (TES) was obtained as described by Rubinsk- Elefant et al. [27], with several modifications briefly described as follows. Eggs obtained from the uteri of female worms were incubated in 2% formalin for approximately 1 month at 28uC. Formalin was removed after exhaustive washing with physiological sodium chloride solution (0.85% NaCl), and the embryonated eggs were artificially hatched in serum-free Eagle’s medium. Larvae were recovered by transferring the suspension on a loose cotton wool plug to a Baermann apparatus. After 18 h, larvae were collected from the bottom of the apparatus, and cultures were incubated at 37uC. The supernatants that contained the antigens were removed and replaced with fresh Eagle’s medium at weekly intervals. Different batches were pooled, concentrated in an Amicon apparatus, dialyzed against distilled water, centrifuged (18,500 g) at 4uC for 60 min, and filtered with a 0.22 mM Millipore membrane (Millipore, Danvers, MA, USA). The protein content was determined with the Lowry protein assay [28]. Antibodies cross-reacting to Ascaris spp. were removed from the sera by preincubating with an adult worm extract (AWE) of Ascaris suum [27][29]. In the enzyme-linked immunosorbent assay (ELISA), all sera were preincubated with a solution (25 mg/mL) of AWE in 0.01 M phosphate buffered saline (PBS, pH 7.2) containing 0.05% Tween-20 (PBS-T) for 30 min at 37uC. Ninety- six-well microtitration polystyrene plates (Corning, Costar, New York, NY) were coated (100 mL/well) with TES antigen solution (1.9 mg/mL in a 0.1 M carbonate–bicarbonate buffer, pH 9.6), incubated for 2 h at 37uC, and then incubated for 18 h at 4uC in a humidified chamber. After the wells were washed 3 times with 0.01 M (PBS, pH 7.2) containing 0.05% Tween-20 (PBS-T), the plates were blocked (200 mL/well) with 1% bovine-serum albumin in PBS-T (BSA, Sigma, St. Louis, MO, USA) for 1 h at 37uC. After 3 washing cycles with PBS-T, the plates were incubated (100 mL/well) with serum samples (dilution: 1/320) for 40 min at 37uC. After incubation with serum samples, the plates were washed 3 times and incubated (100 mL/well) with horseradish peroxidase-conjugated goat anti-human IgG (Sigma) at a 1:10,000 dilution in PBS-T for 40 min at 37uC. The plates were washed 3 times and incubated (100 mL/well) with ortho-phenylenediamine (0.4 mg/mL, OPD-Fast, Sigma, Dorset, United Kingdom) and H2O2-urea (0.4 mg/mL) in 0.05 M citrate buffer for 15 min at 37uC. Database and statistical analysis Data were entered by 2 research assistants on a specific EpiData 3.1 form (The EpiData Association, Odense, Denmark) using the double entry method with subsequent validation. The agreement observed was high (kappa = 0.92), and inconsistencies were resolved to complete agreement (kappa = 1.00). The database was exported to the Statistical Package for the Social Sciences (SPSS) 20 for Windows (International Business Machines Corp, New York, USA), to STATA 12 (StataCorp LP, Texas, USA), and R version 3.0.2 (http://www.r-project.org/) for statistical treat- ments. Antigen preparation, serum processing and antibody detection The reaction was stopped (50 mL/well) with 2 M H2SO4. The assay was monitored by including negative and positive serum The selected variables were initially studied in conjunction with confidence intervals (CI) estimated from 1,000 bootstrap samples [30]. The design effect (deff) was estimated to study the variance between and within the clusters (classrooms) in relation to the income sampling stratum for all the presented variables [31],[32],[33],[34]. A post-hoc proportion Z-test was conducted between sample and population categories [35]. The variables analyzed were gender, age, income (wage of head of household), and school type. A univariate analysis was performed with the Pearson chi- squared test (x2), while a chi-square test for linear trends or Fisher’s exact test were initially used to examine the association between positivity in the IgG anti-Toxocara spp. test and the analyzed factors [36]. A crude prevalence ratio (cPR) was applied to assess the impact of individual factors on outcomes [37]. A multiple Poisson regression with robust variance was used to estimate the adjusted prevalence ratio (aPR), and a 95% confidence interval (95% CI) was also applied [38],[39],[40], as recommended for high-prevalence outcomes [41],[42] in con- junction with consideration of deff [43],[44]. The variables significantly associated in the univariate model (p-value ,0.05) remained in the final models (all entered simultaneously). To improve the final model, the predictor variables were tested for collinearity with variance inflation factor (VIF) and for the presence of influential values. The accuracy of the model was evaluated using a cross-validation system [45],[46]. The signifi- cance level was set at p-value ,0.05. Toxocarariasis Risk Factors in Brazilian Children evaluated via the microscopic examination of stained blood smears. Eosinophil levels (to characterize relative eosinophilia) were assessed as the percentage of total leucocytes represented by eosinophils [23]. samples in addition to a blank (no serum sample). Absorbance at 492 nm was determined in an automatic microplate reader (Titertek Multiskan MCC/340, Lab-system, Helsinki, Finland). A cut-off absorbance value was defined as the mean absorbance reading for 96 negative control sera plus 3 standard deviations. Antibody levels were expressed as reactivity indices (RIs), which were calculated as the ratio between the absorbance values of each test sample and the cut-off value; positive samples had RIs greater than 1. Serum samples were separated from the blood in EDTA-free tubes by centrifugation and stored at 220uC prior to use. The samples were sent to the Seroepidemiology and Immunobiology Laboratory of the Sa˜o Paulo Tropical Medicine Institute for the IgG anti-Toxocara spp. ELISA test. A series of stool samples for parasitological examinations were collected from the schoolchildren on 3 different days in the same container with the liquid preservative MIF (merthiolate-iodine- formaldehyde). The following parasitological methods to detect the larvae and eggs of parasites were used in the laboratory analyses: the flotation technique in a saturated sodium chloride solution with a density of 1.20 g/mL [24], the spontaneous fecal sedimentation technique [25], and the formol-ethyl acetate concentration technique [26]. Blood collection, complete blood count (CBC) and parasitological examination of stools For this specific study, 2 binary (yes/no) outcomes were considered: (1) positivity by ELISA test for anti-Toxocara spp. antibodies, described in the following methodology sections, and (2) physician diagnosis that was performed by an infectious and parasitic diseases expert based on the clinical history of charac- teristic signs and symptoms (described in item 3 of this section) and the assessment of laboratory findings, the most relevant of which are the serology (ELISA anti-Toxocara spp.) and eosinophilia (from Blood samples were collected by the Clinical Laboratory School from the Fernando´polis Educational Foundation into Vacutai- nerTM tubes (BD, Franklin Lakes, NJ, USA) with and without EDTA. The blood in the EDTA tubes was used for CBC counts and differentiation. Red and white cell counts were obtained with a Coulter T890 automated hematology analyzer (Beckman Coulter, Brea, CA, USA), and differential leucocyte counts were May 2014 | Volume 8 | Issue 5 | e2830 PLOS Neglected Tropical Diseases | www.plosntds.org 3 Toxocarariasis Risk Factors in Brazilian Children Results The general prevalence of IgG anti-Toxocara spp. antibodies was 15.5%, with equal variability between and within clusters considering income strata (deff 1.0). The descriptive analyses of the evaluated attributes, socio-demographic characteristics (in- cluding sample and population comparisons), behaviors that increase soil ingestion, and clinical laboratory characteristics including the prevalence of IgG anti-Toxocara spp. antibodies are shown in Table 1. The variable gender showed an equal distribution in the sample, already age showed high percentages for the categories 4 |- 6 years and 6 |- 9 years. Income was concentrated in the first category, , 1 minimum wage, and in the last category, $5 minimum wages. The public and philanthropic categories for the type of school PLOS Neglected Tropical Diseases | www.plosntds.org May 2014 | Volume 8 | Issue 5 | e2830 4 Toxocarariasis Risk Factors in Brazilian Children Table 1. Proportions with a 95% confidence interval (95% CI) pertaining to the characteristics of 252 schoolchildren aged 1 to 12 years from urban areas of Fernando´polis, in the northwest of Sa˜o Paulo State, Brazil. Absolute frequency % (95% IC {) DEFFˇ Population (%)` p-value`` Socio-demographic characteristics Gender Female 132 52.4 (46–57.9) 1.0 50.7 0.821 Male 120 47.6 (42.1–54) 1.0 49.3 0.179 $9 6 |- 9 47 18.7 (13.9–23.0) 1.6 21.9 0.771 4 |- 6 69 27.4 (22.2–32.5) 1.9 25.9 0.178 , 4 88 34.9 (29.4–41.3) 1.5 31.3 0.223 Overall 48 19.0 (13.9–23.8) 1.7 20.9 0.867 Income (head of household) $5 67 26.6 (21.4–32.1) 1.6 25.4 0.708 4 |- 5 37 14.7 (10.3–19.4) 1.7 9.9 0.802 3 |- 4 15 6.0 (3.6–9.1) 1.5 7.4 0.810 2 |- 3 13 5.2 (2.4–7.9) 1.1 13.9 0.445 1 |- 2 70 27.8 (22.6–33.3) 1.1 20.2 0.103 ,1 50 19.8 (15.1–25.0) 0.8 23.2 0.353 Type of School Private 103 40.9 (34.9–46.8) 1.9 35.5 0.094 Public and philanthropic 149 59.1 (53.2–65.1) 1.9 64.5 0.081 SUS users only{{ Yes 99 39.3 (32.9–45.6) 1.8 - - No 153 60.7 (54.4–67.1) 1.8 - - Behaviors that increase soil ingestion Geophagy £ Yes 85 33.7 (28.2–40.1) 0.9 - - No 167 66.3 (59.9–71.8) 0.9 - - Onychophagy £ Yes 96 38.1 (32.1–43.7) 1.8 - - No 156 61.9 (56.3–67.9) 1.8 - - Hand washing habitm Yes 193 76.6 (71.4–81.3) 0.9 - - No 59 23.4 (18.7–28.6) 0.9 - - Clinical laboratory characteristics ELISA anti-Toxocara spp. Results Age was associated only considering a linear trend effect but was not significant as an estimator of risk. Income in the categories of $5 minimum wages, 4 |- 5 minimum wages, private school, and the habit of hand washing were factors that contributed to low prevalence for this outcome. After adjustment in the Poisson model, however, most analyzed variables lost their statistical significance and remained associated only with the habit of hand washing and geophagy (Table 3). Accordingly, a deff value of 1 indicates that the variance for the estimate under cluster sampling is the same as the variance under simple random sampling. In contrast, a deff value greater than 1 indicates that the effective sample size is less than the number of sampled persons but greater than the number of clusters; moreover, there is a loss of precision and a reduction in the effective sample size because individuals are chosen within clusters rather than sampled randomly throughout the population [22],[34]. In this study, a deff of 1.5 was used in the sample size calculation step to prevent a loss of precision, but an initial analysis demonstrated that the variables of age, income, type of school, SUS users only, onychophagy and physician diagnosis were associated with higher deff values. For this reason, the univariate and multivariate analyses incorporated the design effect to ensure more accurate inferences [43],[44]. Intestinal parasite infections or commensal species were detected in 33 (14.9%) children with prevalences of 9.7% for protozoa and 5.8% for helminths. The predominant species were Entamoeba coli (4.5%), Giardia spp. (4.1%) and Strongyloides stercoralis (3.1%). No significant association was observed between intestinal parasites and anti-Toxocara spp. antibody positivity (Table 4). In addition to the enabling environment for toxocariasis transmission observed in a previous study [17], the municipality under study has a large dog population. The most recent official data, based on a rabies vaccination campaign, reported approx- imately 18,000 dogs (an average based on the years 2008–2010) [55], corresponding to a ratio greater than 3 people for every dog. This phenomenon is difficult to measure and may contribute to the epidemiological context for toxocariasis in Fernando´polis. Results Crude prevalence ratio with a 95% confidence interval (95% CI) based on the sampling strata of schoolchildren aged 1 to 12 years from urban areas of Fernando´polis, in the northwest of Sa˜o Paulo State, Brazil. Table 2. Crude prevalence ratio with a 95% confidence interval (95% CI) based on the sampling strata of schoolchildren aged 1 to 12 years from urban areas of Fernando´polis, in the northwest of Sa˜o Paulo State, Brazil. {{Fisher exact test ¥Based on clinical presentation (signs and symptoms) and laboratory results (serology and eosinophilia). doi:10.1371/journal.pntd.0002830.t002 Toxocara seroprevalence (based on an ELISA test) in children has been reported to range from 7.3% to 62.3% [50],[51],[52],[53],[54]. were combined due to the similarity of individuals and the representativeness of the sample. The category of exclusive users of SUS completed the socio-demographic characteristics block. For the socio-demographic variables (excluding SUS users only), all the tested attributes were found to be comparable to the statistical distribution in the population. The data on behaviors that increase soil ingestion showed that participants without geophagy, without onychophagy, and with the habit of hand washing dominated the sample (Table 1). The sample investigated in this study showed good inferential quality for several reasons. First, the application of the resampling method helped to obtain narrower CIs to compare the sample with the population, and all CIs included the corresponding population parameter. Furthermore, no statistical significance was observed in the classic Z-test for proportions, also applied for this purpose. Thus, the sample showed similarities to several charac- teristics of the original population (compare the CI sample with the population proportion and the p-values in Table 1). Prevalence according to the sampling strata was studied for two possible outcomes, IgG anti-Toxocara spp. antibodies and physician diagnosis, with statistical significance (p-value # 0.001) observed for both variables and a greater impact on stA in both cases (Table 2). The impact of the complex sample design upon variance estimates is measured by the deff. It is defined as the ratio of the variance of a statistic that accounts for the complex sample design to the variance of the same statistic based on a hypothetical simple random sample of the same size. The unadjusted analysis showed that factors associated with IgG anti-Toxocara spp. antibodies that contributed to higher prevalence were SUS users only, geophagy, and onychophagy. Relative eosinophilia was also suggested as an associated factor. PLOS Neglected Tropical Diseases | www.plosntds.org Results antibodies Positive 39 15.5 (11.5–19.8) 1.0 - - Negative 213 84.5 (80.2–88.5) 1.0 - - Relative eosinophilia Yes 53 21.0 (15.9–26.2) 1.4 - - No 199 79.0 (73.8–84.1) 1.4 - - Physician diagnosis ¥ Yes 20 7.9 (4.8–11.5) 1.6 - - No 232 92.1 (88.5–95.2) 1.6 - - ˇDesign effect considering income strata. {The % refers to the distribution in the studied sample. The confidence interval (95% CI) was estimated based on 1,000 bootstrap samples. {{Refers to the families who have no health insurance at their disposal and are solely and exclusively dependent on the Brazilian Unified Health System. £Note these habits at least 1 time in the past month. mNote the habit of hand washing before meals (at least 3 times a day). ¥Based on clinical presentation (signs and symptoms) and laboratory results (serology and eosinophilia). `Relative frequency in the Fernando´polis population. ``Proportion Z-test comparing sample vs. population. -There are no population data available. Table 1. Proportions with a 95% confidence interval (95% CI) pertaining to the characteristics of 252 schoolchildren aged 1 to 12 years from urban areas of Fernando´polis, in the northwest of Sa˜o Paulo State, Brazil. Table 1. Proportions with a 95% confidence interval (95% CI) pertaining to the characteristics of 252 schoolchildren aged 1 to 12 years from urban areas of Fernando´polis, in the northwest of Sa˜o Paulo State, Brazil. May 2014 | Volume 8 | Issue 5 | e2830 PLOS Neglected Tropical Diseases | www.plosntds.org Toxocarariasis Risk Factors in Brazilian Children Table 2. Crude prevalence ratio with a 95% confidence interval (95% CI) based on the sampling strata of schoolchildren aged 1 to 12 years from urban areas of Fernando´polis, in the northwest of Sa˜o Paulo State, Brazil. Positive % cPR (95% CI) p-value ELISA anti-Toxocara spp. antibodies stA 34 28.3 7.48 (3.24–18.50) stB 5 3.8 1.00 ,0.001{ Overall 39 15.5 Physician diagnosis ¥ stA 17 14.2 6.23 (1.87–20.74) stB 3 2.3 1.00 0.001{{ Overall 20 7.9 st – stratum. cPR - crude prevalence ratio {Chi-squared test {{Fisher exact test ¥Based on clinical presentation (signs and symptoms) and laboratory results (serology and eosinophilia). doi:10.1371/journal.pntd.0002830.t002 Table 2. Crude prevalence ratio with a 95% confidence interval (95% CI) based on the sampling strata of schoolchildren aged 1 to 12 years from urban areas of Fernando´polis, in the northwest of Sa˜o Paulo State, Brazil. Table 2. Discussion The seroprevalence of Toxocara spp. in schoolchildren aged 1 to 12 years from urban areas of Fernando´polis, in northwestern Sa˜o Paulo State, was examined with an anti-Toxocara spp. ELISA test and estimated as 15.5% (95% CI 11.5–19.8). In previous studies of human toxocariasis in Brazilian children based on use of the same method, higher rates were reported: 51.6% [47] and 36.8% [48]. Other studies, such as Manini et al. [49], demonstrated rates (17.8%) similar to that found in the current study. Worldwide, In general, variation in the rate of seropositivity can occur due to the presence of dog and cat populations with high prevalences May 2014 | Volume 8 | Issue 5 | e2830 PLOS Neglected Tropical Diseases | www.plosntds.org 6 Toxocarariasis Risk Factors in Brazilian Children Toxocarariasis Risk Factors in Brazilian Children Table 3. Crude and adjusted prevalence ratio with a 95% confidence interval (95% CI) based on independent variables for schoolchildren aged 1 to 12 years from urban areas of Fernando´polis, in the northwest of Sa˜o Paulo State, Brazil. Discussion However, certain factors do not represent the life cycle of the parasite or the natural history of the disease but rather the research design and the outcome of the diagnostic method. First, the sampling design should consider the principles of randomness, uniformity and stratification of individuals relative to the group composition to ensure that the exposure factors and outcome found for the sample will be similar to the population. Considerable variation among laboratory methods, technical modifications in the production of TES, and disagreement over the cutoff definition must also be considered [27]. These factors can generate results that do not necessarily reflect the prevalence in the general population. Many factors that predict Toxocara contact have been identified in human pediatric populations, but the results have been inconsistent [15],[29]. The results for gender have been contra- dictory. Specifically, the male gender has been observed to face increased risk [51],[57], to protect the subject [63],[64] and to show no association with Toxocara contact [52],[65],[66]. A young age [63], low socioeconomic status [16],[20],[60], low parental education [52], poor sanitation [53],[67], onychophagy [65],[66], geophagy [57],[59], an absence of hand washing before meals [54], eosinophilia [51],[58], and dog ownership [57],[58],[59],[68] are factors associated with Toxocara contact or infection. None of these known factors, except for geophagy and hand washing before meals, was significantly associated with Differences in seropositivity for IgG anti-Toxocara spp. antibod- ies according to income strata have been found in previous studies. These differences always indicate a greater impact on the lower income classes [16],[20],[60]. The same effect was observed, although to a lesser extent, for the appearance of clinical disease (only VT cases were observed), but the reason for this outcome is that the basis of the medical diagnosis was the serologic test [3],[7],[8]. Table 4. Frequency of parasites and commensals found in parasitological stool examinations from schoolchildren aged 1 to 12 years from urban areas of Fernando´polis, in the northwest of Sa˜o Paulo State, Brazil. Table 4. Frequency of parasites and commensals found in parasitological stool examinations from schoolchildren aged 1 to 12 years from urban areas of Fernando´polis, in the northwest of Sa˜o Paulo State, Brazil. Frequency{ % (CI95%){{ Anti-Toxocara spp. antibodies p-value{{{ Positive % (CI95%){{ Entamoeba histolytica/dı´spar 1 0.4 (0.0 – 2.2) 0 0.0 - Entamoeba coli 10 4.5 (2.3–7.9) 2 20.0 (3.5–51.9) 0.535 Giardia spp. Discussion Positive{ % cPR (95% CI) p-value` aPR (95% CI) p-value`` Socio-demographic characteristics Gender Female 17 14.2 0.85 (0.48–1.52) 0.584 1.27 (0.75–2.13) 0.357 Male 22 16.7 1.00 1.00 Overall 39 30.8 - - Age (years) $9 5 10.6 0.43 (0.15–1.21) 0.66 (0.40–1.07) 0.094 6 |- 9 8 11.6 0.46 (0.19–1.14) 0.040 0.82 (0.43–1.50) 0.555 4 |- 6 14 15.9 0.64 (0.29–1.38) 1.27 (0.87–1.86) 0.195 ,4 12 25.0 1.00 1.00 Overall 39 63.1 - - Income (head of household) $5 1 1.5 0.05 (0.01–0.35) 0.21 (0.00–10.68) 0.438 4 |- 5 1 2.7 0.08 (0.01–0.64) 1.11 (0.02–54.50) 0.957 3 |- 4 2 13.3 0.42 (0.10–1.81) #0.001J 1.07 (0.21–5.42) 0.931 2 |- 3 1 7.6 0.24 (0.03–1.81) 0.53 (0.07–4.21) 0.548 1 |- 2 18 25.7 0.80 (0.41–1.58) 1.00 (0.45–2.23) 0.954 ,1 16 32.0 1.00 1.00 Overall 39 82.9 - - Type of school Private 2 1.9 0.06 (0.1–0.22) #0.001 1.35 (0.04–52.71) 0.940 Public and philanthropic 37 24.8 1.00 1.00 Overall 39 26.8 - - SUS users only{{ Yes 34 22.2 4.40 (1.78–10.8) #0.001 0.82 (0.39–1.66) 0.560 No 5 5.1 1.00 1.00 Overall 39 27.2 - - Behaviors that increase soil ingestion Geophagy£ Yes 28 32.9 5.00 (2.49–10.05) #0.001 2.38 (1.36–4.18) 0.003 No 11 6.5 1.00 1.00 Overall 39 39.5 - - Onychophagy£ Yes 23 23.9 2.34 (1.23–4.42) 0.009 1.06 (0.63–1.78) 0.865 No 16 10.2 1.00 1.00 Overall 39 34.2 - - Hand washing habitm Yes 3 1.5 0.03 (0.01–0.08) #0.001 0.04 (0.01–0.11) #0.001 No 36 61.0 1.00 1.00 Overall 39 62.5 - - Clinical laboratory characteristics Relative eosinophilia Yes 16 30.1 2.61 (1.38–4.94) 0.003 1.51 (0.78–2.91) 0.204 No 23 11.5 1.00 1.00 Table 3. Crude and adjusted prevalence ratio with a 95% confidence interval (95% CI) based on independent variables for schoolchildren aged 1 to 12 years from urban areas of Fernando´polis, in the northwest of Sa˜o Paulo State, Brazil. May 2014 | Volume 8 | Issue 5 | e2830 PLOS Neglected Tropical Diseases | www.plosntds.org PLOS Neglected Tropical Diseases | www.plosntds.org 7 Toxocarariasis Risk Factors in Brazilian Children Table 3. Cont. Positive{ % cPR (95% CI) p-value` aPR (95% CI) p-value`` Overall 39 41.7 - - cPR - crude prevalence ratio (considering the design effect). aPR - adjusted prevalence ratio (considering the design effect). {ELISA anti-Toxocara spp. antibodies: positivity is the outcome. Discussion {{Refers to the families who have no health insurance at their disposal and are solely and exclusively dependent on the Brazilian Unified Health System. £Note these habits at least 3 times in the past month. mNote the habit of hand washing before meals (at least 3 times a day). `Pearson chi-squared test. ``Wald chi-squared test. JFisher exact test. doi:10.1371/journal.pntd.0002830.t003 Table 3. Cont. Other authors have stated that the duration of human IgG responses elicited by Toxocara larvae remains undetermined [61]. Viable larvae may persist in tissues and excrete/secrete antigens for several years, and no simple method is available to confirm parasite death after chemotherapy. Consequently, a single-sample IgG-ELISA titer cannot distinguish between past and current infection [62]. Based on this, the physician diagnosis was included in this evaluation to obtain more specific diagnoses of clinical disease. Thus, patients with a positive physician diagnosis received antiparasitic and/or anti-inflammatory therapy when needed, while those with positive serology without any apparent signs and symptoms were placed for clinical follow-up. of T. canis and T. cati [56], close relationships between pets and humans indoors [57],[58],[59], and the defecation habits of pets or infected stray animals in streets and public squares. These factors produce contamination of the environment, particularly the soil, and can create an environment suitable for human infection [55],[56]. However, certain factors do not represent the life cycle of the parasite or the natural history of the disease but rather the research design and the outcome of the diagnostic method. First, the sampling design should consider the principles of randomness, uniformity and stratification of individuals relative to the group composition to ensure that the exposure factors and outcome found for the sample will be similar to the population. Considerable variation among laboratory methods, technical modifications in the production of TES, and disagreement over the cutoff definition must also be considered [27]. These factors can generate results that do not necessarily reflect the prevalence in the general population. of T. canis and T. cati [56], close relationships between pets and humans indoors [57],[58],[59], and the defecation habits of pets or infected stray animals in streets and public squares. These factors produce contamination of the environment, particularly the soil, and can create an environment suitable for human infection [55],[56]. Toxocarariasis Risk Factors in Brazilian Children Toxocara exposure in the population investigated in the current study, with p-values ranging between 0.094 and 0.954 in the final Poisson model (Table 3). In terms of the impact of sensitivity and specificity, we do not expect variation beyond that expressed by the confidence interval for the seroprevalence, especially because false positive results may occur due to trichinosis and fascioliasis, which are unusual infections in this population. Strongyloidiasis and other parasite infections was observed but were not significantly associated with serological status. The literature also reports that false negative results are rare and only occur in certain localized early or very old infections (OLM) [77]. A previous study found that the ELISA technique, accompanied by absorption of the serum with Ascaris suum antigens, demonstrated a sensitivity of 80% and a specificity of 90% [78]. ( ) The significant associations between positivity of IgG anti- Toxocara spp. antibodies found in the univariate analysis, as well as the significant associations between this outcome and the magnitude of income, type of school, SUS users only, onychoph- agy, and relative eosinophilia, were lost after a multivariate analysis. The 2 variables that remained associated with this positivity variable represent the modifiable behavioral habits that underscore the proximity of humans to the parasite life cycle. The permanence of these in the multiple model, combined with the partial modification of geophagy and no modification of the habit of hand washing before meals (compare p-values and cPR vs. aPR in Table 3), suggests that income and the other sociodemographic attributes may be confounding factors in this relationship. The gender variable was retained in the final model, although it was not significantly associated in the univariate analysis because it implied a contradiction. For physician diagnosis, one laboratory parameter used by the physician was eosinophilia as determined from the CBC, which represented a non-specific test that could be altered for allergic conditions and parasitic infections. In this study, eosinophilia was used in conjunction with the presence of signs and symptoms and serology results to obtain a more specific clinical diagnosis. Additional studies that consider a longitudinal perspective, such as a prospective-cohort design or statistical models employing hierarchical or multilevel analyses, are needed to make new contributions to the discussion of the factors related to contact with Toxocara spp., as the vast majority of previous research has involved cross-sectional studies using classical models of statistical analysis. Acknowledgments As a simple example, we suggest that modifiable factors discussed here, such as the habit of washing hands, can be taught within schools. A study in Brazil with students from elementary school addressed health education in toxocariasis prevention; among the most interesting findings, the researchers suggested that the use of only 1 approach is insufficient to change risk behaviors in children, which reinforces the idea that the educational process should be continuous throughout the stages of child development [76]. The authors thank Professors Jeferson Leandro Paiva, Daiane Fernanda Pereira Mastrocola and Vaˆnia Sato from the Fernando´polis Educational Foundation for field organization and sample collection coordination; physician Marcio Cesar Reino Gaggini, infectious disease specialist, for the clinical evaluation of the children investigated in this survey; and statistician Joa˜o I´talo Dias Franc¸a for the substantive discussions about the appropriate methods and applications of data analysis used in the survey and in this paper. The strengths of this study are as follows: a complex probability sample with a multi-stage cluster design and stratified sampling of income, samples collected from different schools with the same methodology, and appropriate statistical analyses controlling for potential confounding factors and also consideration of the sampling design. The main limitations of this study is its cross- sectional design, which makes it impossible to establish causality and makes it difficult to relate the seropositivity found in the study to environmental contamination present in Fernando´polis. Toxocarariasis Risk Factors in Brazilian Children In practice, geophagy and a lack of hand washing before meals are behaviors that increase soil ingestion (directly or indirectly), facilitating human contact with the eggs of the parasites. We also suggest the hypothesis that hand washing before meals is more common in children whose socioeconomic status is high. For this reason, an association (due to a confounding effect) can be expected between poverty and the presence of IgG anti-Toxocara spp. antibodies. In summary, a confounding effect occurs if the association between exposure and outcome is distorted by the presence of another variable, the confounding factor [69],[70],[71]. Geophagy should be a phenomenon observed in both economic strata, in which hand washing demonstrates only a partial effect. 2. Hotez PJ, Wilkins PP (2009) Toxocariasis: America’s Most Common Neglected Infection of Poverty and a Helminthiasis of Global Importance? Plos Negl Trop Dis 3(3): 1–11. doi:10.1371/journal.pntd.0000400. Author Contributions Conceived and designed the experiments: AJFC GRE. Performed the experiments: AJFC JMPN ARdAL GRE. Analyzed the data: AJFC. Contributed reagents/materials/analysis tools: AJFC GRE. Wrote the paper: AJFC JMPN ARdAL GRE. Discussed the results: AJFC JMPN ARdAL GRE Approved the final, submitted version of the manuscript: AJFC JMPN ARdAL GRE. Conclusions The current study confirms that toxocariasis is a public health problem in urban areas of Fernando´polis, in northwestern Sa˜o Paulo State, southeast Brazil. The presence of modifiable behaviors that increase soil ingestion, such as the habits of geophagy and a lack of hand washing, contributes to the seroprevalence rates observed in the evaluated schoolchildren. In general, income and related sociodemographic variables have been strongly emphasized as causal factors in the human- Toxocara relationship based on interpretations of unadjusted [20],[60],[72] or adjusted models, considering few [65],[73] or no [16],[74],[75] attributes that are important and directly related to the parasite life cycle, such as soil contact and personal hygiene. 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Medronho RA, Bloch KV, Raggio R, Werneck GL (2009) Epidemiologia. 2nd ed. Sa˜o Paulo: Atheneu, 790p. 4. Fisher M (2003). Toxocara cati: an underestimated zoonotic 4. Fisher M (2003). Toxocara cati: an underestimated zoonotic agent. Trends Parasitol 19(4):167–70. doi:10.1016/S1471-4922(03)00027-8. Parasitol 19(4):167–70. doi:10.1016/S1471-4922(03)00027-8. , p 38. Deddens JA, Petersen MR (2008) Approaches for estimating prevalence ratios. Occup Environ Med 65:501–506. doi: 10.1136/oem.2007.034777. ( ) ( ) 5. Beaver P (1962). Toxocarosis (visceral larva migrans) in relation to tropical eosinophilia. Bull Soc Pathol Exot Filiales 55:555–76. 5. Beaver P (1962). Toxocarosis (visceral larva migrans) in eosinophilia. Bull Soc Pathol Exot Filiales 55:555–76. 39. Petersen MR, Deddens JA (2008) A comparison of two methods for estimating prevalence ratios. BMC Med Res Methodol 28;8:9. doi: 10.1186/1471-2288-8- 9. p 6. Miyazaki I (1991) An Illustrated Book Of Helminthic Zoonoses. Tokyo: International Medical Foundation of Japan, 503 p. International Medical Foundation of Japan, 503 p. 7. Magnaval JF, Glickman LT, Dorchies P, Morassin B (2001) Highlights of human toxocariasis. Korean J Parasitol 39:1–11. 7. Magnaval JF, Glickman LT, Dorchies P, Morassin B ( 40. Cummings P (2009). Methods for estimating adjusted risk ratios. Stata J 9(2) 175–196. toxocariasis. Korean J Parasitol 39:1–11. J 8. Despommier D (2003) Toxocariasis: clinical aspects, epidemiology, medical ecology, and molecular aspects. Clin Microbiol Rev 16(2):265–272. 41. Barros AJD, Hirakata V (2003). Alternatives for regression in cross-seccional studies: an empirical comparision of models that directly estimate the prevalence ratio. BMC Med Res Methodol 3:21. 9. Sharghi N, Schantz P, Hotez PJ (2000). Toxocariasis: an occult cause of childhood neuropsychological deficits and asthma? Semin Pediatr Infect Dis 11(4): 257–260. neuropsychological deficits and asthma? Semin Pediatr Infect Dis 11(4 42. Coutinho LMS, Scazufca M, Menezes PR (2008) Me´todos para estimar raza˜o de prevaleˆncia em estudos de corte transversal. Rev Saude Publica. 42(6): 992–998. doi: 10.1590/S0034-89102008000600003. 10. Xinou E, Lefkopoulos A, Gelagoti M, Drevelegas A, Diakou AI et al. (2003) CT and MR imaging findings in cerebral toxocaral disease. AJNR Am J Neuroradiol 24: 714–8. 43. Zocchetti C, Consonni D, Bertazzi P (1997) Relationship between prevalence rate ratios and odds ratios in cross-sectional studies. Int J Epidemiol 26: 220–3. 11. Toxocarariasis Risk Factors in Brazilian Children (1994) Toxocara seroprevalence in 5-year old elementary schoolchildren: relation with allergic asthma. Am J Epidemiol 140: 839–847. 18. Programa das Nac¸o˜es Unidas para o Desenvolvimento (PNUD). Atlas de Desenvolvimento Humano no Brasil. Available: http://www.pnud.org.br/atlas/ ranking/IDH_global_2011.aspx?indi ceAccordion = 1&li = li_Ranking2011. Accessed 25 January 2012. 51. Alonso J, Bojanich M, Chamorro M, Gorodner J (2000) Toxocara seroprev- alence in children from a subtropical city in Argentina. Rev Inst Med Trop Sao Paulo 42(4):235–7. doi: 10.1590/S0036-46652000000400010. 19. Diagno´stico do Municı´pio. Prefeitura Municipal de Fernando´polis. Diagno´stico do Municı´pio. Available: http://www.fernandopolis.sp.gov.br/portal/Principal. asp?ID = 103. Accessed 23 June 2007. 52. Sadjjadi SM, Khosravi M, Mehrabani D, Orya A (2000) Seroprevalence of Toxocara infection in school children in Shiraz, southern Iran. J Trop Pediatr 46(6):327–30. 20. Campos Ju´nior D, Elefant G, de Melo e Silva E, Gandolfi L, Jacob C et al. (2003) Frequency of seropositivity to Toxocara canis in children of different socioeconomic strata. Rev Soc Bras Med Trop 36(4): 509–13. doi: 10.1590/ S0037-86822003000400013. 53. Baboolal S, Rawlins SC (2002). Seroprevalence of toxocariasis in schoolchildren in Trinidad. Trans R Soc Trop Med Hyg 96: 139–143. 54. Fan C, Liao C, Kao T, Li M, Du W, Su K (2005) Sero-epidemiology of Toxocara canis infection among aboriginal schoolchildren in the mountainous areas of north-eastern Taiwan. Ann Trop Med Parasitol. 99(6):593–600. 21. Korn EL, Graubard BI (1991) Epidemiologic studies utilizing surveys: accounting for the sampling design. Am J Public Health 81 (9): 1666–1173. 22. Silva NN (2001) Amostragem Probabilı´stica: um curso introduto´rio. 2nd ed. Sa˜o Paulo: EDUSP, 120p. 55. Cassenote AJF (2010) Frequeˆncia de anticorpos anti-Toxocara spp em escolares do municı´pio de Fernando´polis-SP, Brasil e ana´lise da contaminac¸a˜o do solo por ovos do parasito. Sa˜ o Paulo.Dissertac¸a˜ o [Mestrado em Doenc¸as Infecciosas e Parasita´rias] – Faculdade de Medicina da Universidade de Sa˜o Paulo. 23. Williams WJ, Beutler E, Erslev AJ, Lichtman AM (1991) Hematology. 4th ed. New York: McGraw-Hill, 1882p. 24. Willis HH (1921) A simple levitation method for the detection of hookworm ova. Med J Aust 8: 375–6. 56. Damian M, Martins M, Sardinha J, Souza L, Chaves A et al. (2007) Frequency of the antibody anti-Toxocara canis in a community along the Uatuma˜ river, State of Amazonas. Rev Soc Bras Med Trop 40(6):661–4. doi: 10.1590/S0037- 86822007000600013. 25. Hoffman WA, Pons JA, Janer JL (1934). Sedimentation concentration method in Schistosomiasis mansoni. Puerto Rico J Publ Hlth 9: 283–298. 26. Toxocarariasis Risk Factors in Brazilian Children Arau´jo AJUS, Kanamura HY, Dias LCS, Gomes JF, Arau´jo SM (2003). Quantitative Coprotest: quantification of helminth eggs in fecal samples by commercial diagnostic kit. J Bras Patol Med Lab 39(2): 115–124. 57. Espinoza YA, Huapaya PH, Roldani WH, Jimenezi S, Arce Z et al. (2008) Clinical and serological evidence of Toxocara infection in school children from Morrope district, Lambayeque, Peru. Rev Inst Med Trop S Paulo 50(2): 101– 105. doi: 10.1590/S0036-46652008000200007. 27. Rubinsky-Elefant G, Shimizu S, Sanchez M, Jacob C, Ferreira A (2006) A serological follow-up of toxocariasis patients after chemotherapy based on the detection of IgG, IgA, and IgE antibodies by enzyme-linked immunosorbent assay. J Clin Lab Anal 20(4):164–72. 58. Figueiredo S, Taddei J, Menezes J, Novo N, Silva E et al. (2005) Estudo clı´nico- epidemiolo´gico da toxocarı´ase em populac¸a˜o infantil. J Pediatr 81(2):126–32. doi: 10.1590/S0021-75572005000300007. 28. Lowry OH, Rosebrough NJ, Farr AL, Randal RJ (1951). Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275. 59. Rolda´n WH, Espinoza YA, Huapaya PE, Huiza AF, Sevilla CR et al. (2009) Frequency of human toxocariasis in a rural population from Cajamarca, Peru determined by DOT-ELISA test. Rev Inst Med Trop Sao Paulo 51:67–71. doi: 10.1590/S0036-46652009000200002. 29. Rubinsky-Elefant G, da Silva-Nunes M, Malafronte RS, Muniz PT, Ferreira UM (2008) Human toxocariasis in rural Brazilian Amazonia: seroprevalence, risk factors, and spatial distribution. Am J Trop Med Hyg 79(1):93–8. 30. Efron B, Tibshirani RJ (1993) An Introduction to the Bootstrap. New York: Chapman & Hall/CRC, 436p. 60. Cilla G, Pe´rez-Trallero E, Gutie´rrez C, Part C, Goma´riz M (1996) Seroprevalence of Toxocara infection in middle-class and disadvantaged children in northern Spain (Gipuzkoa, Basque Country). Eur J Epidemiol 12(5):541–3. 31. Kish L (1965) Survey Sampling. New York: John Wiley & Sons, Inc, 646p. 32. Korn EL, Graubard BI (1991) Epidemiologic studies utilizing surveys: accounting for the sampling design. Am J Public Health 81(9):1166–1173. 61. Cypess RH, Karol MH, Zidian JL, Glickman LT, Gitlin D. (1977). Larva- specific antibodies in patients with visceral larva migrans. J Infect Dis 135: 633– 40. 33. Heo M, Kim Y, Xue X, Kim MY (2010) Sample size requirement to detect an intervention effect at the end of follow-up in a longitudinal cluster randomized trial. Statistics in Medicine 29 (3): 382–390. doi: 10.1002/sim.3806. 62. Rubinsky-Elefant G, Hirata CE, Yamamoto JH, Ferreira UM (2010) Human toxocariasis: diagnosis, worldwide seroprevalences and clinical expression of the systemic and ocular forms. References May 2014 | Volume 8 | Issue 5 | e2830 PLOS Neglected Tropical Diseases | www.plosntds.org 9 Toxocarariasis Risk Factors in Brazilian Children 63. Magnaval J, Baixench M (1993) Toxocariasis in the Midi Pyrene´es region. In Lewis JW, Maizels RM editors. Toxocara and Toxocariasis. London: British Society for Parasitology, pp.63–69. 71. Christenfeld NJ, Sloan RP, Carroll D, Greenland S (2004) Risk factors, confounding, and the illusion of statistical control. Psychosom Med 66(6):868– 75. 64. Theodoridis I, Frydas S, Papazahariadou M, Hatzistilianou M, Adamama et al. (2001) Toxocarosis as zoonosis. A review of literature and the prevalence of Toxocara canis antibodies in 511 serum samples. Int J Immunopathol Pharmacol 14: 17–23. 72. Demirci M, Kaya S, C¸ etin ES, Arıdog˘an BC, O¨ nal S et al. (2010) Seroepidemiological Investigation of Toxocariasis in the Isparta Region of Turkey. Iran J Parasitol 5(2): 52–59. 73. Dar ZA, Tanveer S, Yattoo GN, Sofi BA, Wani AS et al. (2008) Seroprevalence of Toxocariasis in Children in Kashmir, J&K State, India. Iranian J Parasitol 3 (4): 45–50. 65. Alderete JMS, Jacob CMA, Pastorino AC, Elefant GR, Castro APM et al (2003) Prevalence of Toxocara infection in schoolchildren from the Butanta˜ region, Sa˜o Paulo, Brazil. Mem Inst Oswaldo Cruz 98(5): 593–597. doi: 10.1590/S0074- 02762003000500002. 74. Jones J, Kruszon-Moran D, Won K, Wilson M, Schantz P (2008). Toxoplasma gondii and Toxocara spp. co-infection. Am J Trop Med Hyg 78(1):35–9. 66. Paranhos FR, Macedo MMB, Moreira LE, FEL Pereira (2011) Anti-Toxocara antibodies detected in children attending elementary school in Vitoria, State of Espı´rito Santo, Brazil: prevalence and associated factors. Rev Soc Bras Med Trop 44(4): 461–466. doi: 10.1590/S0037-86822011000400012. 75. Won KY, Kruszon-Moran D, Schantz PM, Jones JL (2008). National seroprevalence and risk factors for zoonotic Toxocara spp. infection. Am J Trop Med Hyg 79(4):552–7. 76. Santos MB (2003) Toxocarı´ase: avaliac¸a˜o do processo ensino-aprendizagem de recursos pedago´gicos aplicados a crianc¸as do ensino fundamental. Sa˜o Paulo. Dissertac¸a˜o [Mestrado em Medicina Veterina´ria e Sau´de Animal] – Faculdade de Medicina Veterina´ria e Zootecnia da Universidade de Sa˜o Paulo. 67. Santos GM, Almeida e Silva S, Passos Barbosa A, Campos DMB (2009). Investigac¸a˜o soroepidemiolo´gica sobre Larva Migrans Visceral por Toxocara canis em usua´rios de Servic¸os de Sau´de de Goiania, GO. Rev Pat Trop 38: 197–206. de Medicina Veterina´ria e Zootecnia da Universidade de Sa˜o Paulo. p 68. Fernando SD, Wickramasinghe VP, Kapilananda GM, Devasurendra RL, Amarasooriya JD et al. (2007) Epidemiological aspects and risk factors of toxocariasis in a pediatric population in Sri Lanka. Southeast Asian J Trop Med Public Health 38(6):983–90. 77. Toxocarariasis Risk Factors in Brazilian Children Ann Trop Med Parasitol 104:3–23. doi: 10.1179/ 136485910X12607012373957. 34. Park I, Lee H (2004) Design Effects for the weighted mean and total estimators under complex survey sampling. S Methodol 30:183–193. 35. Pereira JCR (2010) Bioestatı´stica em outras palavras. Sa˜o Paulo: Edusp, 424p. May 2014 | Volume 8 | Issue 5 | e2830 PLOS Neglected Tropical Diseases | www.plosntds.org PLOS Neglected Tropical Diseases | www.plosntds.org 10 PLOS Neglected Tropical Diseases | www.plosntds.org Toxocarariasis Risk Factors in Brazilian Children Pawlowski Z (2003) Toxocariasis in humans: clinical expression and treatment dilemma. J Helminthol. 75(4):299–305. 78. Camargo ED, Nakamura PM, Vaz AJ, Dasilva MV, Chieffi PP, Demelo EO (1992) Standardization of DOT-ELISA for the serological diagnosis of toxocariasis and comparison of the assay with ELISA. Rev Inst Med Trop Sao Paulo 34(1): 55–60. ( ) 69. Johnston SC (2001) Identifying Confounding by Indication through Blinded Prospective Review. Am J Epidemiol 154:276–84. 70. Howards PP, Schisterman EF, Poole C, Kaufman JS, Weinberg CR (2012) "Toward a clearer definition of confounding" revisited with directed acyclic graphs. Am J Epidemiol 15;176(6):506–11. May 2014 | Volume 8 | Issue 5 | e2830 PLOS Neglected Tropical Diseases | www.plosntds.org PLOS Neglected Tropical Diseases | www.plosntds.org 11
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Influence of the Ports of the Northern Sea Route on the Formation of Focal Zones for the Development of the Eastern Arctic
Arktika. Èkologiâ i èkonomika/Arktika: èkologiâ i èkonomika
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Экономика и управление народным хозяйством Арктической зоны Экономика и управление народным хозяйством Арктической зоны DOI: 10.25283/2223-4594-2021-1-6-18 УДК 338.27(571.60) DOI: 10.25283/2223-4594-2021-1-6-18 УДК 338.27(571.60) ВЛИЯНИЕ ПОРТОВ СЕВЕРНОГО МОРСКОГО ПУТИ НА ФОРМИРОВАНИЕ ОЧАГОВЫХ ЗОН ОСВОЕНИЯ ВОСТОЧНОЙ АРК­ТИКИ С. Н. Леонов, Е. А. Заостровских Институт экономических исследований Дальневосточного отделения РАН (Хабаровск, Российская Федерация) С. Н. Леонов, Е. А. Заостровских Статья поступила в редакцию 9 октября 2020 г. Экономика и управление народным хозяйством Рассмотрено влияние портов Северного морского пути на эффективность формирования очаговых зон освоения дальневосточной Арк­тики. Показано, что в дальневосточной Арк­тике транспорт выступает необходимым, но не достаточным условием возникновения и ускоренного развития локальных «полюсов роста». Ключевые слова: Арк­тика Дальнего Востока, СМП, транспортная инфраструктура, опорные зоны социально-эко- номического развития. 1 Указ Президента РФ «О национальных целях и стратегиче- ских задачах развития Российской Федерации на период до 2024 года» от 7 мая 2018 г. № 204. © Леонов С. Н., Заостровских Е. А., 2021 2 Распоряжение Правительства РФ «Об утверждении при- лагаемого плана развития инфраструктуры Северного морского пути на период до 2035 г.» от 21 декабря 2019 г. № 3120-р. — URL: https://www.garant.ru/products/ipo/prime/ doc/73261725/. 1 Указ Президента РФ «О национальных целях и стратегиче- ских задачах развития Российской Федерации на период до 2024 года» от 7 мая 2018 г. № 204. 2 Распоряжение Правительства РФ «Об утверждении при- лагаемого плана развития инфраструктуры Северного морского пути на период до 2035 г.» от 21 декабря 2019 г. № 3120-р. — URL: https://www.garant.ru/products/ipo/prime/ doc/73261725/. 3 Опорные зоны российской Арк­тики и их морские порты: 1. Кольская (Мурманск и Териберка), 2. Архангельская (Ар- хангельск), 3. Ненецкая (Нарьян-Мар, Индига), 4. Ворку- тинская (Варандей, Амдерма), 5. Ямало-Ненецкая (Сабетта, Харасавей, Новый Порт), 6. Таймыро-Туруханская (Диксон, Дудинка), 7. Северо-Якутская (Тикси), 8. Чукотская (Певек и Беринговский). Введение даже в летнее время арк­тических льдов у берегов Канады и Гренландии [5] (рис. 1). Восточная Арк­тика охватывает свыше трети пло- щади российского Дальнего Востока, включает половину крупнейшего в  мире административно- территориального образования Республики Саха (Якутия) и Чукотский автономный округ. Это самая северная, наименее заселенная и слабо освоенная в хозяйственном отношении часть Дальнего Восто- ка, характеризующаяся сложными демографически- ми и  миграционными процессами [1]. Фактически здесь нет ни заводов, ни крупных городов, а населе- ние самого крупного поселения (Анадыря) составля- ет лишь 15,6 тыс. человек. Вследствие этого возрастает значимость логисти- ки по маршруту Восточная Азия — Западная Европа через СМП. Объем перевозки грузов по трассе СМП должен возрасти с 31,5 млн т в 2019 г. до 80 млн т в 2024 г.1 и до 160 млн т к 2035 г.2 В результате растет важность развития транспортной инфра- структуры восточной Арк­тики, поскольку без этого существование СМП как современной целостной транспортной артерии невозможно. Второе обстоятельство связано с  вопросами на- циональной безопасности в  условиях санкционной войны. Одни авторы отмечают негативное влияние санкций [6], другие говорят об их позитивном воз- действии через концентрацию российских произ- водителей на завоевании региональных рынков [7; Кроме наличия природных ресурсов еще два об- стоятельства стимулируют интерес к освоению вос- точной Арк­тики. Первое связано с изменением климата. По оцен- кам ученых, уже в 2050 г. Северный морской путь (СМП) станет доступным для судов неледового класса, хотя протяженность и  толща льда будут по-прежнему затруднять навигацию [2—4]. В свою очередь, Северо-Западный проход, пролегающий через канадскую Арк­тику, останется ограниченным по проходимости морских судов из-за сохранения Арктика: экология и экономика, т. 11, № 1, 2021 6  Влияние портов Северного морского пути на формирование очаговых зон освоения восточной Арк­тики Рис. 1. Арк­тические порты мира. Картография выполнена В. Д. Хижняком на основании данных World Port Index 2019 (https://msi.nga.mil/Publications/WPI) Fig. 1. Arctic ports of the world. V. D. Khizhnyak based on the data from World Port Index 2019 made the map (https://msi.nga.mil/Publications/WPI) 20—60 1—20 менее 1 Ареалы наибольшего распространения сплошных льдов летом зимой Северный морской путь Северо-Западный проход Основные порты Размеры портов (млн т) действующие проектируемые расширяемые Рис. 1. Арк­тические порты мира. Картография выполнена В. Д. Хижняком на основании данных World Port Index 20 (https://msi.nga.mil/Publications/WPI) Fig. 1. Arctic ports of the world. V. D. 4 Под национальной грузовой базой понимаются грузы, про- изведенные в данной стране и перевезенные собственным морским флотом этой страны. 4 Под национальной грузовой базой понимаются грузы, про- изведенные в данной стране и перевезенные собственным морским флотом этой страны. 5 https://omorrss.ru/upload/files/Проект%20Транспортной%20 стратегии%20на%20период%20до%202035%20г..pdf. 5 https://omorrss.ru/upload/files/Проект%20Транспортной%20 стратегии%20на%20период%20до%202035%20г..pdf. Экономика и управление народным хозяйством * К арк­тическим относятся не только порты, расположенные в районе Северного полярного круга, но и находящи- еся в непосредственной близости и обеспечивающие поставки грузов в арк­тические регионы. ** Автономная территория Королевства Дания. Источник: рассчитано авторами на основе данных World Port Index 2019. * К арк­тическим относятся не только порты, расположенные в районе Северного полярного круга, но и находящи- еся в непосредственной близости и обеспечивающие поставки грузов в арк­тические регионы. ** Автономная территория Королевства Дания. Источник: рассчитано авторами на основе данных World Port Index 2019. навских странах развитие арк­тических портов ба- зируется на теории эндогенного экономического роста, в США и Канаде — на поиске инновационных возможностей экономического развития новоосва- иваемых территорий, в  России  — на триаде «раз- мещение производительных сил  — экономическое районирование  — территориально-производствен- ные комплексы» [9]. Сочетание названных факторов задает стратегические ориентиры развития как ре- гионов, так и портов. Названные обстоятельства в  значительной мере стимулируют развитие западной Арк­тики, но не дают ответа на вопрос, может ли активизация де- ятельности России в части развития СМП, развития и реконструкции арк­тических морских портов явить- ся стимулом развития локальных полюсов роста вокруг портов восточной Арк­тики. Попытаемся по- нять, насколько реалистичны эти ожидания и может ли транспорт выступить пропульсивной отраслью, ядром очаговых зон регионального развития Арк­ тики Дальнего Востока. Особенность развития портов российского арк­ тического бассейна состоит в  том, что 98% грузо­ оборота приходится на западный сектор и концен- трируется в  небольшом числе эффективно работа- ющих портов  — Мурманске (58%), Сабетте (17%), Варандее (7%). Порты восточной Арк­тики перераба- тывают лишь 2% арк­тического грузооборота стра- ны, что объясняется низким уровнем освоенности региона и  слабым внутренним спросом. Ситуацию не изменил даже существенный рост грузооборота портов восточной Арк­тики за последние три года (с 0,6 до 1,1 млн т), поскольку наращивание грузов в  западном сегменте Арк­тики происходило в  это время еще активнее. Состояние портов й Все порты восточной Арк­тики (Тикси, Певек, Эг- векинот, Провидения, Анадырь, Беринговский) от- носятся к  категории мелких (см. рис. 1) с  ограни- ченным числом механизированных причалов, они не способны принимать суда с большим дедвейтом [10]. Основной костяк портовой инфраструктуры, за- ложенный еще в советское время, морально устарел и нуждается в обновлении и модернизации. Бунке- ровка возможна только в Анадыре, в Эгвекиноте нет Методы и статистика Для обоснования результатов исследования авто- ры использовали идеи, высказанные в научных тру- дах отечественных и зарубежных ученых в области территориального кумулятивного роста, управления региональным развитием на основе теории форми- рования «полюсов роста». Методологической осно- вой исследования явились общенаучные методы, та- кие как формализация, группировка, анализ, синтез и сравнение. Информационной базой исследования послужили данные Росстата. Введение Khizhnyak based on the data from World Port Index 2019 made the map (https://msi.nga.mil/Publications/WPI) реализации проектов будут достигаться за счет взаимного стимулирующего влияния мероприятий по развитию арк­тической транспортной системы, ориентированной на СМП, и  промышленно-произ- водственных объектов. Соответственно развитие производственных объектов обеспечит загрузку транспорта национальной грузовой базой 4. В свою очередь, активное наращивание объектов транс- портной инфраструктуры должно способствовать снятию инфраструктурных ограничений и  привле- чению дополнительных транзитных грузов на трас- су СМП. Ожидается, что реализация этих планов обеспечит устойчивый экономический рост в АЗРФ и  территориальную связанность страны, что от- ражено в проекте новой «Транспортной стратегии Российской Федерации на период до 2035 года»5. 8]. Как следствие, в рамках региональной политики в  Арк­тике особое значение отводится формирова- нию механизмов социально-экономического раз- вития территорий в целях включения их потенциала в  хозяйственный оборот. В  Арк­тической зоне Рос- сийской Федерации (АЗРФ) на льготных условиях планируется создать восемь опорных зон в увязке с арк­тическими портами 3. Для развития транзитного потенциала и наращивания экспорта транспортных услуг предполагается создание двух арк­тических хабов (на входе и выходе трассы СМП — в Мурман- ске и Петропавловске-Камчатском). Формирование опорных зон развития АЗРФ пла- нируется осуществлять в увязке с развитием транс- порта, поскольку экономические эффекты при 7 Таблица 1. Российские и зарубежные морские арктические порты * по состоянию на начало 2020 г. Страна Крупные (20—60 млн т) Средние (1—20 млн т) Мелкие (менее 1 млн т) Число арк­тических портов во всей совокупности портов рассматриваемых стран Всего 1 11 70 82/1197 Россия 1 6 13 20/68 В том числе Восточная Арк­тика 0 0 6 6/68 Канада 0 2 2 4/286 США 0 0 3 3/666 Гренландия ** 0 1 16 17/17 Исландия 0 1 21 22/22 Норвегия 0 1 12 13/135 * К арк­тическим относятся не только порты, расположенные в районе Северного полярного круга, но и находящи- еся в непосредственной близости и обеспечивающие поставки грузов в арк­тические регионы. ** Автономная территория Королевства Дания. Источник: рассчитано авторами на основе данных World Port Index 2019. Таблица 1. Российские и зарубежные морские арктические порты * по состоянию на начало 2020 г. 7 Отметим, что в «Стратегии развития порта Петропавловск- Камчатский» заложен даже более оптимистичный вариант, рассматривающий начало в  2024  г. строительства здесь глубоководного контейнерного терминала мощностью 160 млн т (https://to-ros.info/?p=77223). 8 Например, в порту Беринговский рост экспорта угля в 2015 г. обеспечил прирост каботажных грузов на 30% (http://www. morvesti.ru/detail.php?ID=67799). 6 Утвержден план развития Северного морского пути до 2035 года.  — URL: https://rg.ru/2020/01/28/reg-szfo/utverzhden- plan-razvitiia-severnogo-morskogo-puti-do-2035-goda.html. Прогнозы развития грузовой базы портов восточной Арк­тики портов восточной Арк­тики Из 160  млн  т  6, прогнозируемых к  перевозке по СМП в  2035  г., объем грузов, перерабатываемых в  портах восточной Арк­тики, составит 32,7  млн  т (табл. 2). р дальневосточной Арк­тики Портовую инфраструктуру мировой Арк­тики со- ставляют 82 порта (табл. 1). Из них 85% относится к мелким портам и 14% — к средним. Крупным яв- ляется только один — российский Мурманск. Решение о развитии арк­тических портов в каждой стране зависит от их числа и  степени хозяйствен- ного освоения прилегающих регионов. В  Сканди- Арктика: экология и экономика, т. 11, № 1, 2021 8 Влияние портов Северного морского пути на формирование очаговых зон освоения восточной Арк­тики Таблица 2. Потенциальные объемы минерально-сырьевых грузов арк­тических портов Дальнего Востока (2035 г.) Опорная зона, порт, проект Груз Объем инвестиций, млрд руб. Сроки реализации Мощность, млн т Порты восточной Арк­тики 32,7 В том числе: Чукотская опорная зона, всего: 10,2 Певек: 0,2 Майское Руда Н. д. 2025—2028 * 0,1 Баимское Руда Н. д. 2025—2028 * 0,1 Беринговский (Угольный **) Уголь 23,0 2018—2022 10,0 Хаб Петропавловск-Камчатский, всего: 22,5 Ямал СПГ ** СПГ 70,0 2020—2023 21,7 Нефтетерминал ** Нефтепродукты 0,4 2020—2022 0,2 Терминал по переработке грузов ** Генеральные грузы 2,2 2020—2022 0,6 * Оценка авторов. ** Проект имеет преференции Свободного порта Владивосток или Территории опережающего развития. Источник: составлено по [11—17]. Таблица 2. Потенциальные объемы минерально-сырьевых грузов арк­тических портов Дальнего Востока (2035 г.) ц р ** Проект имеет преференции Свободного порта Владивосток или Территории опережающего развития. Источник: составлено по [11—17]. Для выполнения прогнозируемого объема работ потребуется завоз техники, оборудования, строи- тельных и  продовольственных товаров, что приве- дет к росту и каботажных грузов 8. Предполагается, что реализация перспективных проектов увеличит объем грузооборота портов Дальнего Востока, соз- даст дополнительные рабочие места и  будет спо- собствовать образованию дополнительных эконо- мических эффектов как в самих портах, так и за их пределами, способствуя формированию локальных узлов («точек роста»). пункта пропуска через государственную границу, в Певеке, Провидения, Эгвекиноте и Беринговском имеются проблемы с навигационным обслуживани- ем, существует дефицит электроэнергии. Отсутствие выхода на железнодорожную сеть страны постоян- но замедляет сроки реализации проектов. пункта пропуска через государственную границу, в Певеке, Провидения, Эгвекиноте и Беринговском имеются проблемы с навигационным обслуживани- ем, существует дефицит электроэнергии. Отсутствие выхода на железнодорожную сеть страны постоян- но замедляет сроки реализации проектов. Прогнозы развития грузовой базы Пропульсивный потенциал портовой инфраструктуры Пропульсивный потенциал портовой инфраструктуры Как видно из данных табл. 2, прирост грузооборо- та за счет освоения рудных и угольных месторожде- ний дальневосточной Арк­тики составит 10,2 млн т, 22,5 млн т связаны с перспективами развития порта Петропавловск-Камчатский, который территориаль- но не относится к АЗРФ, но является ключевым эле- ментом в развитии трассы СМП 7. Бесспорным является утверждение, что транс- портная инфраструктура определяет развитие ре- гиона и  его хозяйственного комплекса, выступая основой процесса освоения любого региона. Это верно и  для дальневосточной Арк­тики, где глав- ным отличием современного момента по сравнению с  ситуацией позднесоветского периода выступает опора на морскую логистику при реализации боль- шинства новых проектов ресурсного освоения [18]. Практически именно порты СМП являются реперны- ми точками транспортной системы севера Дальнего Востока, а  развитие арк­тических регионов в  силу 9 9 Экономика и управление народным хозяйством Арктической зоны Рис. 2. Арк­тические опорные зоны дальневосточной Арк­тики: Территория опережающего развития (ТОР) и Свободный порт Владивосток (СПВ). Карта выполнена В. Д. Хижняком на основании предоставленных данных Fig. 2. Arctic support zones of the Far Eastern Arctic: FGTs (faster growth territories) and FPV (Free port of Vladivostok). V. D. Khizhnyak based on the data provided made the map Рис. 2. Арк­тические опорные зоны дальневосточной Арк­тики: Территория опережающего развития (ТОР) и Свободный порт Владивосток (СПВ). Карта выполнена В. Д. Хижняком на основании предоставленных данных Fig. 2. Arctic support zones of the Far Eastern Arctic: FGTs (faster growth territories) and FPV (Free port of Vladivostok). V. D. Khizhnyak based on the data provided made the map огромности пространства и  слабой заселенности четко укладывается в  концепцию поляризации ре- гионального развития. ции роста промышленных центров, стимулирование индустриального развития региона [21, c. 123— 138]. Мировой опыт реализации идей теории «полю- сов роста» показал, что ключевое значение для их удачной реализации имел правильный выбор про- пульсивных отраслей или их сочетаний [20; 22]. Теоретическое обоснование неизбежности регио- нальной поляризации дал в середине XX в. Ф. Перру, отошедший в своих построениях от принципа гомо- генности (равномерности) территориального разви- тия [19]. Подобный подход позволил ему трактовать «полюса роста» как точки концентрации экономиче- ской и  инвестиционной активности, формирующи- еся вокруг «пропульсивных» отраслей и способные генерировать экономический рост в масштабах об- ширных территорий [20]. В  модели формирования «полюса роста» Ф. 9 Федеральный закон «О государственной поддержке пред- принимательской деятельности в Арк­тической зоне Россий- ской Федерации» от 13 июля 2020 г. № 193-ФЗ (http://www. consultant.ru/document/cons_doc_LAW_357078/); федераль- ный закон «О внесении изменений в часть вторую Налого- вого кодекса Российской Федерации в связи с принятием Федерального закона «О государственной поддержке пред- принимательской деятельности в Арк­тической зоне Россий- ской Федерации» от 13 июля 2020 г. № 195-ФЗ (https://www. garant.ru/products/ipo/prime/doc/74268808/). Пропульсивный потенциал портовой инфраструктуры Перру классифицировал отрасли производства по тенденциям развития на три груп- пы: деградирующие, с тенденцией снижения их доли в структуре экономики региона; с высоким темпом роста, но значительно не влияющие на развитие остальных отраслей территории; пропульсивные от- расли, для которых характерны как значительный рост производства, так и порождение цепной реак- у р В силу уникальности любого порта зачастую не- возможно с полной уверенностью утверждать, соче- тание каких факторов обязательно приведет к воз- никновению полюса роста портового региона [23]. В  современной научной литературе и  хозяйствен- ной практике пропульсивной признается динамично развивающаяся отрасль, являющаяся ведущей на данном цикле экономического развития и характе- ризующаяся высоким уровнем технологий и обшир- ным рынком, что обеспечивает индуцированный (по- ляризационный) эффект [8]. Другими словами, того, что порт и портовая инфраструктура выступают де- ловым и хозяйственным центром территории разме- щения, недостаточно для признания транспортной инфраструктуры пропульсивной отраслью. Арктика: экология и экономика, т. 11, № 1, 2021 10  Влияние портов Северного морского пути на формирование очаговых зон освоения восточной Арк­тики и Чукотская) расположены на Дальнем Востоке (см. рис. 2). Министерство развития Дальнего Востока и Арктики определило приоритеты каждой из этих опорных зон. Для Чукотки это Баимское месторож- дение и создание университета, для Якутии — раз- витие районов рек Анабара, Лены, Индигирки и Ко- лымы [29]. В Арк­тике транспортно-логистическая инфра- структура исторически всегда выступала центром экономической активности региона, а портам тради- ционно отводилась роль узлов поляризации хозяй- ственной жизни. Для формирования потенциально- го «полюса роста» крайне важно наличие пропуль- сивного потенциала у якорных, головных проектов, размещаемых на территории. Если рассматривать эту ситуацию в терминах теории Ф. Перру, наличие транспортно-логистической инфраструктуры на тер- ритории следует воспринимать как необходимое, но не достаточное условие возникновения реального полюса роста в регионе. Неразвитость транспортной и  энергетической инфраструктуры  — основная проблема обеих вос- точных опорных арк­тических зон, замедляющая их системное формирование и  устойчивое развитие [30]. Поэтому в качестве системообразующих в них выделяются проекты опережающей реконструкции и  развития транспортной и  энергетической инфра- структуры, играющие важную роль в хозяйственной интеграции двух соседствующих территорий. Транс- портная и энергетическая инфраструктура должна стягивать эти регионы в  устойчивое в  хозяйствен- ном плане пространственное образование — Даль- невосточную Арк­тику. Дальневосточная практика подтверждает данное утверждение. Так, вразрез с  традиционным под- ходом, согласно которому порты как важнейшие инфраструктурные активы служат катализатором экономического роста портовых регионов [24—26], в восточной Арк­тике наблюдается феномен сниже- ния вклада портов в создание валового региональ- ного продукта. Пропульсивный потенциал портовой инфраструктуры Сравнительный анализ динамики валового регионального продукта (ВРП) Чукотского автономного округа и  грузооборота портов Певек и Анадырь, являющихся основными портами Чукот- ки, не обнаруживает корреляции между приростом общих масштабов грузооборота и ВРП. Формирующаяся в  российской Арк­тике система льгот многообразна и в данное время еще не сло- жилась [31]. Льготы по развитию предприниматель- ства и  транспортной инфраструктуры предоставля- ются резидентам Свободного порта Владивосток в портах Певек и Петропавловск-Камчатский, а так- же резидентам территорий опережающего развития Беринговский и Камчатка (см. рис. 2). Сложившаяся ситуация подтверждает гипотезу, что в  реальности сам по себе пропульсивный эф- фект портового хозяйства невелик [27; 28]. В то же время в двух случаях может наблюдаться рост про- пульсивного эффекта транспортной инфраструкту- ры. Во-первых, если пропульсивный эффект приоб- ретает «наведенный» характер. Подобная ситуация характерна для портов, расположенных в  освоен- ных регионах, имеющих комфортные экономические и природные условия, когда на припортовой терри- тории создаются мощности по доработке продук- ции. Во-вторых, пропульсивный эффект транспорт- ной инфраструктуры может мультиплицироваться развитием транспортоемких добывающих отраслей. Так, у портов западной Арк­тики в условиях резкого наращивания грузооборота отмечается возникнове- ние кумулятивного эффекта на базе роста объемов транспортоемкой продукции и,  как следствие, — усиление пропульсивного потенциала. В дополнение к существующей системе преферен- ций в июле 2020 г. был принят пакет федеральных законов о  государственной поддержке предприни- мательской деятельности в  АЗРФ  9. Введено поня- тие резидента Арк­тической зоны, каковым может считаться любой зарегистрировавшийся в  Арк­тике предприниматель, готовый реализовать новый ин- вестиционный проект и вложить не менее 1 млн руб. Для резидентов Арк­тической зоны на 10 лет пред- лагается нулевая ставка налога на прибыль в феде- ральный бюджет при субсидиарной ответственности региона (если законом субъекта Федерации будет установлена пониженная ставка по налогу на при- быль в части зачисления в региональный бюджет). Они могут рассчитывать на 12-летний 50%-ный на- логовый вычет по налогу на добычу полезных иско- паемых на новых участках недр и ряд других льгот. При этом, хотя льготы вводятся для всей АЗРФ, следует ожидать, что их большинство окажется зна- чимым именно для резидентов западной части рос- сийской Арк­тики, стимулируя развитие ресурсодо- К сожалению, порты дальневосточной Арк­тики не обладают ни первым, ни вторым из условий по- вышения пропульсивного характера транспортной системы в регионе. Анализ современного состояния, а также перспектив развития минерально-сырьевой базы и  увеличения потенциального грузооборота в российской Арк­тике на 2035 г. (см. табл. 2) лишь подтверждает это утверждение. 10 Отметим, что технологически развитые порты мира спо- собствуют формированию различных видов логистической деятельности, доля которых оценивается на уровне 80% до- ходов портов [34]. Экономика и управление народным хозяйством к: рассчитано авторами. ние: знак «—» означает, что явление отсутствует (соответствующая величина равна нулю). бывающих отраслей и мультиплицируя пропульсив- ный эффект портового хозяйства данного региона. ясняется слаборазвитой инфраструктурой и  слож- ными условиями эксплуатации. Поэтому при оценке экономических эффектов от реализации портовых проектов на период до 2035  г. для дальневосточ- ных опорных зон и  арктического хаба в  расчетах предполагалось, что погрузо-разгрузочные работы в  перспективе составят основной объем доходов портов данного региона. Оценка перспективы развития портов восточной Арк­тики в этих условиях Особенность очагового развития восточной Арк­тики В целях повышения социально-экономической устойчивости российской Арк­тики Правительство России предлагает создать в регионе восемь опор- ных зон развития. Две из них (Северо-Якутская В целях повышения социально-экономической устойчивости российской Арк­тики Правительство России предлагает создать в регионе восемь опор- ных зон развития. Две из них (Северо-Якутская 11 Экономика и управление народным хозяйством Арктической зоны Таблица 3. Расчетные показатели экономических эффектов для опорных зон и арктического Показатель Всего 2019 2025 2030 2035 Объем перевалки грузов, млн т 2,9 35,1 35,3 35,3 В том числе: экспорт 0,4 32,6 32,7 32,7 каботаж 2,5 2,5 2,6 2,6 Экономические Объем дохода портов от перевалки грузов, млн руб. 8 254,2 49 576,8 49 877,0 49 924,8 В том числе от: экспорта, % 10,7 85,5 83,2 83,2 каботажа, % 89,3 14,5 16,8 16,8 Налог на прибыль, млн руб. 192,6 212,5 631,2 1 096,4 В том числе от экспорта — — 412,9 876,5 Численность занятых в портах, человек 264 619 620 630 В том числе связанных с экспортом 4 359 360 360 Источник: рассчитано авторами. Примечание: знак «—» означает, что явление отсутствует (соответствующая величина равна нулю). Оценка перспективы развития портов восточной Арк­тики в этих условиях Основные допущения: период прогноза составля- ет 15 лет (2020—2035 гг.), оценка стоимостных по- казателей выполнялась в ценах 2019 г. Группировка грузов осуществлялась по трем ка- тегориям: экспорт всего, экспорт с  учетом префе- ренциальных грузов (СПВ и ТОР), каботаж. Объем экспортных грузов определялся согласно табл. 2, объем каботажных грузов рассчитывался в  зави- симости от завоза грузов в  арк­тические регионы Дальнего Востока и дополнительного прироста ка- ботажа в  связи с  приростом объемов экспортных грузов. Транзитные грузы не учитывались из-за не- завершенности проектов арк­тических хабов СМП. Базой для оценки послужили статистические дан- ные о погрузочно-разгрузочной деятельности семи портов восточной Арк­тики по 11 наименованиям (уголь, нефть, СПГ, круглый лес, зерно, металлы, ма- шины, минерально-строительные материалы, руда, контейнеры и прочие тарно-штучные). Подробно оценка взаимосвязей между экономи- кой региона и портом описана в [32; 33], здесь же отметим следующее. Учитывалось, что в России в сравнении с основны- ми мировыми морскими державами логистические услуги формируют лишь 20% доходов портов 10, а ос- новная их доля (80%) приходится на погрузо-раз- грузочные работы. Такая структура доходов объ- При расчете налоговых льгот и преференций в от- ношении резидентов арк­тических портов учитыва- лось, что все экспортные грузы арк­тических морских портов Дальнего Востока реализуются в  рамках льготных режимов Свободного порта Владивосток, территорий опережающего развития и  налоговых льгот для резидентов Арк­тики. У большинства за- планированных экспортных проектов отсутству- ет общая оценка объема потребных инвестиций, Арктика: экология и экономика, т. Оценка перспективы развития портов восточной Арк­тики в этих условиях Основной прирост грузооборота обеспечит экспорт сырьевых грузов, объем которых увеличится с 0,4 до 10,2 млн т. Вместе с тем объем дохода портов от перевалки гру- зов по сравнению с 2019 г. вырастет в 7,7 раза до 24 955 млн руб. Экономические эффекты от реализа- ции преференциальных проектов будут наблюдаться на протяжении всего анализируемого периода, по- скольку проект был запущен в 2016 г. Объем дохода порта от перевалки экспорта бе- ринговского угля в 2019 г. составлял 882,3 млн руб., объем налога на прибыль проявится лишь в 2030 г. (286,8 млн руб.), так как проект реализуется в рамках С учетом описанных допущений выполнен расчет эффектов от реализации портовых проектов для опорных зон и арктического хаба на Дальнем Вос- токе до 2035 г. по пятилетиям. Полученные оценки представлены в  табл. 3 и  позволяют сделать ряд выводов. Чукотская опорная зона. Объем перевалки гру- зов в 2035 г. приблизится к отметке 11,3 млн т, уве- личившись по сравнению с 2019 г. в 8 раз. Основной прирост грузооборота обеспечит экспорт сырьевых грузов, объем которых увеличится с 0,4 до 10,2 млн т. Вместе с тем объем дохода портов от перевалки гру- зов по сравнению с 2019 г. вырастет в 7,7 раза до 24 955 млн руб. Экономические эффекты от реализа- ции преференциальных проектов будут наблюдаться на протяжении всего анализируемого периода, по- скольку проект был запущен в 2016 г. Чукотская опорная зона. Объем перевалки гру- зов в 2035 г. приблизится к отметке 11,3 млн т, уве- личившись по сравнению с 2019 г. в 8 раз. Основной прирост грузооборота обеспечит экспорт сырьевых грузов, объем которых увеличится с 0,4 до 10,2 млн т. Вместе с тем объем дохода портов от перевалки гру- зов по сравнению с 2019 г. вырастет в 7,7 раза до 24 955 млн руб. Экономические эффекты от реализа- ции преференциальных проектов будут наблюдаться на протяжении всего анализируемого периода, по- скольку проект был запущен в 2016 г. Особенности территориальной структуры прироста показателей арк­тических портов Дальнего Востока для опорных зон и хаба В целом грузооборот портов восточной Арк­тики будет развиваться более интенсивными темпа- ми, чем остальные показатели деятельности пор- тов. Если грузооборот за 2019—2035 гг. вырастет в  12  раз, то объем полученного дохода портов  — Особенности территориальной структуры прироста показателей арк­тических портов Дальнего Востока для опорных зон и хаба В целом грузооборот портов восточной Арк­тики будет развиваться более интенсивными темпа- ми, чем остальные показатели деятельности пор- тов. Если грузооборот за 2019—2035 гг. Оценка перспективы развития портов восточной Арк­тики в этих условиях 11, № 1, 2021 12  Влияние портов Северного морского пути на формирование очаговых зон освоения восточной Арк­тики хаба на Дальнем Востоке от реализации портовых проектов В том числе Северо-Якутская опорная зона Чукотская опорная зона Хаб Петропавловск-Камчатский 2019 2025 2030 2035 2019 2025 2030 2035 2019 2025 2030 2035 0,3 0,3 0,4 0,4 1,6 11,2 11,3 11,3 1,0 23,6 23,6 23,6 — — — — 0,4 10,1 10,2 10,2 — 22,5 22,5 22,5 0,3 0,3 0,4 0,4 1,2 1,1 1,1 1,1 1,0 1,1 1,1 1,1 эффекты 212,0 267,7 301,5 348,6 3 201,8 24 873,7 24 955,2 24 955,2 4 840,4 24 435,4 24 620,3 24 621,0 — — — — 27,6 88,7 88,7 88,7 — 79,4 78,8 78,8 100,0 100,0 100,0 100,0 72,4 11,3 11,3 11,3 100,0 20,6 21,2 21,2 6,5 8,2 9,2 10,7 60,3 73,2 360,0 647,2 125,8 131,1 262,0 438,5 — — — — — — 286,8 574,0 — — 126,1 302,5 20 20 20 20 124 231 232 242 120 368 368 368 — — — — 4 111 112 112 — 248 248 248 хаба на Дальнем Востоке от реализации портовых проектов в 6 раз, налоговые выплаты — в 5,7 раза, а числен- ность занятых — в 2,4 раза (см. табл. 3). а сроки их реализации практически приходятся на один и тот же период — 2025—2030 гг. Это может привести к  «инвестиционной усталости» инвесто- ров, а сроки завершения проектов могут оказаться растянутыми на несколько лет. Однако в процессе анализа данный факт нивелировался. Предполага- лось, что все проекты Чукотской опорной зоны, как и  хаба Петропавловск-Камчатский, будут заверше- ны к 2030 г. Для проектов СПВ и ТОР учитывалась нулевая ставка налога на прибыль в течение первых пяти лет и ставка в размере 12% в течение следую- щих пяти лет. Северо-Якутская опорная зона будет ориенти- рована на рост каботажных перевозок, вызванный повышенным спросом на технику, оборудование, строительные материалы, которые потребуется за- возить для разработки и освоения месторождений. В 2035 г. грузооборот зоны составит 0,4 млн т. Эко- номические эффекты, формируемые портом Тик- си, будут невелики: объем дохода порта составит 348,6 млн руб., налог на прибыль — 10,7 млн руб., численность занятых — 20 человек. численность занятых — 20 человек. Чукотская опорная зона. Объем перевалки гру- зов в 2035 г. приблизится к отметке 11,3 млн т, уве- личившись по сравнению с 2019 г. в 8 раз. 11 ТОР «Беринговский» и  Свободный порт на Чукотке пока- зали первые результаты. — URL: https://www.dvkapital.ru/ regionnow/chukotskij-avtonomnyj-okrug_03.10.2016_8827_ tor-beringovskij-i-svobodnyj-port-na-chukotke-pokazali- pervye-rezultaty.html. 12 В 2024 году на Камчатке начнут строить глубоководный контейнерный терминал. — URL: https://to-ros.info/?p=77223. Экономика и управление народным хозяйством 4. Традиционный взгляд на порты как на важней- шие инфраструктурные активы, которые являются деловым и  хозяйственным центром территориаль- ного развития, недостаточен для признания транс- портной инфраструктуры пропульсивной отраслью. Пропульсивный эффект портового хозяйства невы- сок и носит в основном либо наведенный характер, когда на припортовых территориях создаются мощ- ности по доработке продукции, что характерно для освоенных в  экономическом плане регионов, либо кумулятивный (накопительный) характер, когда транспортная инфраструктура стимулирует возник- новение транспортоемких добывающих отраслей, как это наблюдается, например, в западной части российской Арк­тики. 4. Традиционный взгляд на порты как на важней- шие инфраструктурные активы, которые являются деловым и  хозяйственным центром территориаль- ного развития, недостаточен для признания транс- портной инфраструктуры пропульсивной отраслью. Пропульсивный эффект портового хозяйства невы- сок и носит в основном либо наведенный характер, когда на припортовых территориях создаются мощ- ности по доработке продукции, что характерно для освоенных в  экономическом плане регионов, либо кумулятивный (накопительный) характер, когда транспортная инфраструктура стимулирует возник- новение транспортоемких добывающих отраслей, как это наблюдается, например, в западной части российской Арк­тики. Вместе с тем наиболее выгодным проектом хаба Петропавловск-Камчатский мог бы стать не рас- смотренный в  данных расчетах ввиду недостатка информации терминал по переработке транзитных контейнерных грузов, формируемых по направле- нию «Восточная Азия  — Западная Европа» через СМП, в объеме до 160 млн т 12. Очевидно, что хаб Петропавловск-Камчатский сможет достичь названных объемов обработки кон- тейнеров только в  случае обеспечения уровня ло- гистических услуг в  порту, сопряженного с  между- народными стандартами, что потребует уточнения и детальной проработки проекта в крайне сжатые сроки. При этом как минимум необходимо уточнить подход к исследованию проблем функционирования порта-хаба в условиях, когда логистические услуги должны будут создавать до 80% доходов порта. По- требуется разработка новых форм и методов оценки развития и функционирования морского транспорта в дальневосточной Арк­тике. 5. Для Северо-Якутской и Чукотской арк­тических опорных зон транспортная инфраструктура явля- ется важным условием развития, выступая в  каче- стве интегрирующего системообразующего про- екта, «стягивающего» эти территории в устойчивое в  хозяйственном плане пространственное образо- вание — Дальневосточную Арк­тику. При этом сама транспортная инфраструктура в восточной Арк­тике не является ядром экономической активности, так как не располагает достаточным потенциалом «про- пульсивности» для инициализации деятельности ло- кальных особых зон. Выводы 6. Наличие портов и транспортной инфраструкту- ры в восточной Арк­тике является важным условием функционирования СМП, но не может рассматри- ваться как достаточное условие развития региона. В  целом для дальневосточной Арк­тики транспорт- ная инфраструктура выступает необходимым, но не достаточным условием формирования и ускоренно- го развития очаговых зон региона. 1. Активизация деятельности государства в  вос- точной Арк­тике усиливает транспортно-сырьевую специализацию региона и пытается стимулировать его очаговое развитие. 2. Реализуемая государственная политика, приня- тие пакета нормативно-законодательных докумен- Оценка перспективы развития портов восточной Арк­тики в этих условиях вырастет в  12  раз, то объем полученного дохода портов  — 13 Экономика и управление народным хозяйством Арктической зоны Экономика и управление народным хозяйством Арктической зоны ТОР «Беринговский». В будущем велика вероятность того, что интенсивный рост перевалки угля обострит трудности, связанные с  загрязнением акватории, а также экологические и социальные проблемы. По- скольку порт Беринговский ориентирован на интере- сы австралийской угледобывающей компании «Tigers Realm Coal Ltd»11, из-за перетока доходов в зарубеж- ную экономическую систему и  усиления экологиче- ских проблем в регионе может получить развитие со- циально-экономический конфликт между бизнесом, региональной властью и населением. тов поддержки предпринимательской активности в  Арк­тике нацелены на рост объемов добычи ми- нерально-сырьевых ресурсов, что будет стимулиро- вать возникновение кумулятивного эффекта портов (увеличение грузооборота) и  скажется в  первую очередь на деятельности портов западной части Арк­тической зоны. 3. Основные эффекты дальневосточных арк­ тических портов в  перспективе будут связаны с переработкой экспортно ориентированных грузов и отсрочены на конец прогнозного периода. Среди дальневосточных портов, связанных с арк­тической акваторией, потенциал наибольшего влияния на ре- гион своего размещения имеет хаб Петропавловск- Камчатский при условии переработки транзитных контейнерных грузов, формируемых по направле- нию «Восточная Азия  — Западная Европа» через СМП, в объеме до 160 млн т. Хаб Петропавловск-Камчатский. Объем перевалки грузов в  диапазоне 15  предстоящих лет увеличится более чем в 20 раз и в 2035 г. со- ставит 23,6  млн  т. Экономические эффекты, про- дуцируемые камчатским хабом, выразятся в  ро- сте объема дохода порта в  5  раз (с 4840,4 до 24 621 млн руб.), налога на прибыль — в 3,4 раза (с 125,8 до 438,5 млн руб.), а численности занятых — в 3,4 раза (2019 г. — 120 человек, 2035 г. — 368). Экономический эффект от реализации преференци- альных проектов составит в 2030 г. 126,1 млн руб., в 2035 г. — 302,5 млн руб. Литература — C. 106—125. 5. Arctic Maps / The Arctic Inst. — URL: https://www. thearcticinstitute.org/arctic-maps/. 21. 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Преференциальные режимы соз- данных локальных точек роста и их влияние на эко- номику Дальнего Востока // Экон. и соц. перемены: факты, тенденции, прогноз. — 2020. — Т. 13, № 3. — С. 28—45. — DOI: 10.15838/esc.2020.3.69.3. 15. Павлов В. К., Селин В. С. Проблемы развития гру- зопотоков Северного морского пути и  методы их решения // Вестн. УГУЭС. Наука, образование, эко- номика. Сер. экономика. — 2015. — № 2 (12). 31. Fujita M., Mori T. The Role of Ports in the Making of Ma- jor Cities: Self-Agglomeration and Hub-Effect // J. of De- velopment Economics. — 1996. — № 49. — P. 93—120. 16. Перспективный план развития ТОР КАМЧАТ- КА. — URL: https://minfish.kamgov.ru/document/file/ download?id=55998. 16. Перспективный план развития ТОР КАМЧАТ- КА. — URL: https://minfish.kamgov.ru/document/file/ download?id=55998. 15 Информация об авторах ф р ц р Леонов Сергей Николаевич, доктор экономических наук, профессор, ведущий научный сотрудник, Инсти- тут экономических исследований Дальневосточного отделения РАН (680042, Россия, Хабаровск, Тихоокеан- ская ул., 153), e-mail: Leonov@ecrin.ru. Леонов Сергей Николаевич, доктор экономических наук, профессор, ведущий научный сотрудник, Инсти- тут экономических исследований Дальневосточного отделения РАН (680042, Россия, Хабаровск, Тихоокеан- ская ул., 153), e-mail: Leonov@ecrin.ru. у ) Заостровских Елена Анатольевна, кандидат экономических наук, научный сотрудник, Институт эконо- мических исследований Дальневосточного отделения РАН (680042, Россия, Хабаровск, Тихоокеанская ул., 153), e-mail: zaost@ecrin.ru. Заостровских Елена Анатольевна, кандидат экономических наук, научный сотрудник, Институт эконо- мических исследований Дальневосточного отделения РАН (680042, Россия, Хабаровск, Тихоокеанская ул., 153), e-mail: zaost@ecrin.ru. References (In Russian). 6. Dumnova N. A., Lazarenko A. L., Soldatova M. A. Prob- lemy i perspektivy razvitiya regional’noi ekonomiki v usloviyakh sanktsii. [Problems and Prospects of Regional Economy in Terms of Sanctions]. Fundam. issled., 2015, no. 2, pp. 2891—2894. (In Russian). 18. Pilyasov A. N., Putilova E. S. Novye proekty osvoe- niya rossiiskoi Arktiki: prostranstvo znachimo! [New projects for the development of Russian Arctic: space matters!]. Arktika i Sever, 2020, no. 38, pp. 21—43. (In Russian). 7. Balatskii E. Mobilizatsionnaya ekonomika v uslovi- yakh sanktsii. [Mobilization economy under sanctions]. 10.09.2020. Available at: http://geopolitics.by/analyt- ics/mobilizacionnaya-ekonomika-v-usloviyah-sankciy. (In Russian). 19. Perru F. Ekonomicheskoe prostranstvo: teoriya i prilozheniya. [Economic Space: Theory and Applica- tions]. Prostranstv. ekonomika, 2007, no. 2, pp. 77—93. (In Russian). 8. Rozanova L. I., Moroshkina M. V. Stimulirovanie raz- vitiya propul’sivnykh otraslei kak zadannyi impul’s strukturnykh sdvigov. [Stimulating the Development of Propulsive Industries as a Set Impulse for Structural Changes]. Vopr. bezopasnosti, 2015, no. 5, pp. 1—20. DOI: 10.7256/2409-7543.2015.5.16539. (In Russian). 8. Rozanova L. I., Moroshkina M. V. Stimulirovanie raz- vitiya propul’sivnykh otraslei kak zadannyi impul’s strukturnykh sdvigov. [Stimulating the Development of Propulsive Industries as a Set Impulse for Structural Changes]. Vopr. bezopasnosti, 2015, no. 5, pp. 1—20. DOI: 10.7256/2409-7543.2015.5.16539. (In Russian). 9. Pilyasov A. N., Zamyatina N. Yu. Development of the North 2.0: challenges of making a new theory. Arktika i Sever [Arctic and North], 2019, no. 34, pp. 57—76. DOI: 10.17238/issn2221-2698.2019.34.57. 20. Lasuen Kh. R. Urbanizatsiya i ekonomicheskoe razvitie: vremennoe vzaimodeistvie mezhdu geogra- ficheskimi i otraslevymi klasterami. [Urbanisation and Development  — the Territorial Interaction between Geografical and Sectoral Clusters]. Prostranstv. eko- nomika, 2009, no. 4, pp. 106—125. (In Russian). 21. Sapozhnikov A. D., Smol’nikov M. V. Burzhuazna- ya regional’naya teoriya i gosudarstvenno-mo- nopolisticheskoe regulirovanie razmeshcheniya proizvoditel’nykh sil: Kriticheskii analiz. [Bourgeois Re- gional Theory and State-Monopoly Regulation of the Placement of Productive Forces: A Critical Analysis]. Moscow, Mysl’, 1981, 251 p. (In Russian). 20. Lasuen Kh. R. Urbanizatsiya i ekonomicheskoe razvitie: vremennoe vzaimodeistvie mezhdu geogra- ficheskimi i otraslevymi klasterami. [Urbanisation and Development  — the Territorial Interaction between Geografical and Sectoral Clusters]. Prostranstv. eko- nomika, 2009, no. 4, pp. 106—125. (In Russian). 9. Pilyasov A. N., Zamyatina N. Yu. Development of the North 2.0: challenges of making a new theory. Arktika i Sever [Arctic and North], 2019, no. 34, pp. 57—76. DOI: 10.17238/issn2221-2698.2019.34.57. 21. Sapozhnikov A. D., Smol’nikov M. V. References avtonomnom okruge. [Analysis and prospects for the development of coal mining in the period up to 2035 in the Chukotka Autonomous Okrug]. Gor. prom-st’, 2016, no. 5, pp. 27—33. (In Russian). 1. Leonov S. N. Ispol’zovanie metodologii segmenta rynka mest dlya tselei geodemograficheskogo raion- irovaniya rossiiskoi Arktiki. [Using the methodology of the market segment of places for the purposes of geodemographic zoning of the Russian Arctic]. Dal’nii Vostok Rossii: ekonomicheskie, sotsial’nye, infrastruk- turnye faktory razvitiya. In-t ekon. issled. DVO RAN. Khabarovsk, 2018, pp. 103—112. (Uchenye zap.; vyp. 12). (In Russian). 1. Leonov S. N. Ispol’zovanie metodologii segmenta rynka mest dlya tselei geodemograficheskogo raion- irovaniya rossiiskoi Arktiki. [Using the methodology of the market segment of places for the purposes of geodemographic zoning of the Russian Arctic]. Dal’nii Vostok Rossii: ekonomicheskie, sotsial’nye, infrastruk- turnye faktory razvitiya. In-t ekon. issled. DVO RAN. Khabarovsk, 2018, pp. 103—112. (Uchenye zap.; vyp. 12). (In Russian). 13. Goloviznin A. A. Razvitie portov rossiiskoi Arktiki. [Development of ports in the Russian Arctic]. Available at: https://morproekt.ru/attachments/article/1116/ arctic_projects_2020_plus.pdf. (In Russian). 14. Dva ugol’nykh proekta na Taimyre — dve sud’by. [Two coal projects in Taimyr — two destinies]. Available at: https://portnews.ru/digest/print/21544/?backurl=/ digest/. (In Russian). 2. Aksenov Y., Popova E. E., Yool A., Nurser A. J. G., Wil- liams T. D., Bertino L., Bergh J. On The Future Navigabil- ity of Arctic Sea Routes: High-Resolution Projections of The Arctic Ocean and Sea Ice. Marine Policy, 2017, no. 75, pp. 300—317. 15. Pavlov V. K., Selin V. S. Problemy razvitiya gruzo- potokov Severnogo morskogo puti i metody ikh resh- eniya. [Problems of Development of Freght Traffics of the Northern Sea Route and Methods of Their Deci- sion]. Vestn. UGUES. Nauka, obrazovanie, ekonomika. Ser. ekonomika, 2015, no. 2 (12). (In Russian). 3. Smith L. C., Stephenson S. R. New Trans-Arctic Ship- ping Routes Navigable by Midcentury. Proc. Natl. Acad. Sci. USA, 2013, no. 13 (110), pp. 4871—4872. pp 4. Melia N., Haines K., Hawkins E. Sea Ice Decline and 21st Century Trans-Arctic Shipping Routes. Geophys. Res. Lett., 2016, no. 43 (18), pp. 9720—9728. 16. Perspektivnyi plan razvitiya TOR KAMChATKA. [Perspective development plan for ASEZ KAMCHATKA]. Available at: https://minfish.kamgov.ru/document/file/ download?id=55998. (In Russian). 5. Arctic Maps. The Arctic Inst. Available at: https:// www.thearcticinstitute.org/arctic-maps/. 17. Bunin I. Perspektivy porta, svyazannye s integratsiei v sistemu Sevmorputi. [Port prospects related to inte- gration into the Northern Sea Route system]. Morskie porty, 2019, no. 7. Available at: URL: http://www.mor- vesti.ru/analitika/1688/81547/). Abstract The authors consider propulsive potential of the ports of the eastern section of the Northern Sea Route (NSR) and indicate the influence of the NSR on increasing the efficiency of the key zones formation for the development of the Far Eastern Arctic. They base the work methodology on the theory of cumulative growth, in which the ideas of the “growth poles” formation occupy a decisive position. The authors use the grouping methods, logi- cal and comparative analysis. The Rosstat data serves as an information base of the study. It is shown that the state’s activity on the development of the NSR stimulates the development of the North Yakutsk and Chukotka support zones, enhancing the transport and raw materials specialization of the Eastern Arctic as a whole. At the same time, the authorities proceed from the assumption that the development of ports should reduce the costs of trade, increase employment, and have a multiplier effect both on the port system itself and on the sectors of the region interacting with it. The paper substantiates that in the Far Eastern Arctic, the traditional view of ports as the most important infrastructure assets, which are the business and economic center of territorial develop- ment, is insufficient for the recognition of transport infrastructure as a propulsion industry. The propulsive effect of the port economy is low and is mainly of an induced nature, when capacities for processing products are created in the port areas, or when the transport infrastructure stimulates the emergence of transport-intensive extractive industries, which is provided in the western part of the NSR. For the development of the North Yakutsk and Chukotka Arctic support zones, the transport infrastructure will be a backbone project that “connects” these regions in an economically sustainable spatial formation — the Far Eastern Arctic. Transport is a necessary but insufficient condition for the emergence and accelerated development of local “growth poles” in the Eastern Arctic Keywords: the Arctic of the Far East, the Northern Sea Route, ports, transport infrastructure, support zones for social and economic development. Арктика: экология и экономика, т. 11, № 1, 2021 16  Влияние портов Северного морского пути на формирование очаговых зон освоения восточной Арк­тики avtonomnom okruge. [Analysis and prospects for the development of coal mining in the period up to 2035 in the Chukotka Autonomous Okrug]. Gor. prom-st’, 2016, no. 5, pp. 27—33. (In Russian). Information about the authors Leonov Sergey Nikolaevich, Doctor of Economy, Professor, Leading Researcher, Economic Research Institute of Far Eastern Branch of the Russian Academy of Sciences (153, Tikhookeanskaya Street, Khabarovsk, Russia, 680042), e-mail: Leonov@ecrin.ru. Zaostrovskikh Elena Anatol’evna, PhD of Economy, Researcher, Economic Research Institute of Far Eastern Branch of the Russian Academy of Sciences (153, Tikhookeanskaya Street, Khabarovsk, Russia, 680042), e-mail: zaost@ecrin.ru. Bibliographic description of the articlel Bibliographic description of the articlel Bibliographic description of the article Leonov S. N., Zaostrovskikh E. A. Influence of the Ports of the Northern Sea Route on the Formation of Focal Zones for the Development of the Eastern Arctic. Arktika: ekologiya i ekonomika. [Arctic: Ecology and Economy], 2021, vol. 11, no. 1, pp. 6—18. DOI: 10.25283/2223-4594-2021-1-6-18. (In Russian). © Leonov S. N., Zaostrovskikh E. A., 2021 © Leonov S. N., Zaostrovskikh E. A., 2021 © Leonov S. N., Zaostrovskikh E. A., 2021 18 Арктика: экология и экономика, т. 11, № 1, 2021 g p p f Leonov S. N., Zaostrovskikh E. A. Influence of the Ports of the Northern Sea Route on the Formation of Focal Zones for the Development of the Eastern Arctic. Arktika: ekologiya i ekonomika. [Arctic: Ecology and Economy], 2021, vol. 11, no. 1, pp. 6—18. DOI: 10.25283/2223-4594-2021-1-6-18. (In Russian). Information about the authors Leonov Sergey Nikolaevich, Doctor of Economy, Professor, Leading Researcher, Economic Research Institute of Far Eastern Branch of the Russian Academy of Sciences (153, Tikhookeanskaya Street, Khabarovsk, Russia, 680042), e-mail: Leonov@ecrin.ru. Zaostrovskikh Elena Anatol’evna, PhD of Economy, Researcher, Economic Research Institute of Far Eastern Branch of the Russian Academy of Sciences (153, Tikhookeanskaya Street, Khabarovsk, Russia, 680042), e-mail: zaost@ecrin.ru. Арктика: экология и экономика, т. 11, № 1, 2021 References Burzhuazna- ya regional’naya teoriya i gosudarstvenno-mo- nopolisticheskoe regulirovanie razmeshcheniya proizvoditel’nykh sil: Kriticheskii analiz. [Bourgeois Re- gional Theory and State-Monopoly Regulation of the Placement of Productive Forces: A Critical Analysis]. Moscow, Mysl’, 1981, 251 p. (In Russian). 10. Leonov S. N., Zaostrovskikh E. A. Potentsial Vostoch- noi Arktiki kak katalizator razvitiya Dal’nego Vostoka Rossii. [The Eastern Arctic facilities accelerate the Russian Far East development]. Arktika: ekologi- ya i ekonomika, 2019, no. 4 (36), pp. 4—15. DOI: 10.25283/2223-4594-2019-4-4-15. (In Russian). 22. Neshchadin A., Trevu D.-S. O frantsuzskom opyte sozdaniya polyusov konkurentosposobnosti. [Adout the French Experience of Creation of Competitive- ness Poles]. Obshchestvo i ekonomika, 2012, no. 5, pp. 129—134. (In Russian). 11. Fishkin D. O., Grigor’ev M. N. Realizatsiya mineral’no- syr’evogo i logisticheskogo potentsiala Arkticheskoi zony Rossii — kak vypolnit’ “Maiskii” ukaz prezidenta Rossii. [Realization of Mineral Resource and Logistic Capacity of Russias Arctic Zone — How to Perform the Tasks Defined by the “May” Decree of the President of Russian]. Ros. Arktika, 2019, no. 7, pp. 41—48. (In Russian). 23. Giuliano G., Brien T. Responding to Increasing Port- Related Freight Volumes: Lessons from Los Angeles. Long Beach and Other US Ports and Hinterlands. Port Competition and Hinterland Connections. Round Table 143. [S. l.], OECD Publ., 2009, pp. 77—108. 23. Giuliano G., Brien T. Responding to Increasing Port- Related Freight Volumes: Lessons from Los Angeles. Long Beach and Other US Ports and Hinterlands. Port Competition and Hinterland Connections. Round Table 143. [S. l.], OECD Publ., 2009, pp. 77—108. 12. Plakitkina L. S. Analiz i perspektivy razvitiya dobychi uglya v period do 2035 goda v Chukotskom 12. Plakitkina L. S. Analiz i perspektivy razvitiya dobychi uglya v period do 2035 goda v Chukotskom 12. Plakitkina L. S. Analiz i perspektivy razvitiya dobychi uglya v period do 2035 goda v Chukotskom 17 Экономика и управление народным хозяйством Арктической зоны 24. Haezendonck E. Essays on Strategy Analysis for Seaports. Intern. J. of Maritime Economics, 2002, no. 4, pp. 185—187. 29. Minakir P. A., Leonov S. N. Problemy prognozirovani- ya razvitiya arkticheskikh regionov Dal’nego Vostoka. [Problems of forecasting the development of the Arc- tic regions of the Far East]. Arktika: ekologiya i eko- nomika, 2015, no. 1 (17), pp. 10—17. (In Russian). 25. Klink V. Towards the Borderless Main Port Rotter- dam: An Analysis of Functional, Spatial and Adminis- trative Dynamics in Port Systems. Tinbergen Institute Research Series, 1995, no. 104. pp 30. Leonov S. N. Экономика и управление народным хозяйством 33. Zaostrovskikh E. A. Ugol’nye porty Vanino i Vostoch- nyi, i ikh vliyanie na ekonomiku regiona. [The coal ports of Vanino and Vostochny and their influence on the re- gion’s economy]. Problemy razvitiya territorii, 2020, no. 1 (105), pp. 78—92. DOI: 10.15838/ptd.2020.1.105.6. (In Russian). 28. Minvostokrazvitiya oboznachilo prioritety raz- vitiya kazhdogo regiona Arktiki. [The Ministry for the Development of the Russian Far East out- lined the development priorities of each Arctic re- gion]. 20.05.2020. Available at: https://minvr.ru/ press-center/news/24381/?sphrase_id=1336418. In Russian). 28. Minvostokrazvitiya oboznachilo prioritety raz- vitiya kazhdogo regiona Arktiki. [The Ministry for the Development of the Russian Far East out- lined the development priorities of each Arctic re- gion]. 20.05.2020. Available at: https://minvr.ru/ press-center/news/24381/?sphrase_id=1336418. In Russian). 28. Minvostokrazvitiya oboznachilo prioritety raz- vitiya kazhdogo regiona Arktiki. [The Ministry for the Development of the Russian Far East out- lined the development priorities of each Arctic re- gion]. 20.05.2020. Available at: https://minvr.ru/ press-center/news/24381/?sphrase_id=1336418. In Russian). 34. Commercial Development of Regional Ports as Logistics Centers. New York, ESCAP; Korea Transport Inst.; United Nations, 2003, 109 p. References Preferential regimes of established local growth points and its impact on the economy of the Far East. Economic and Social Changes: Facts, Trends, Forecast, 2020, vol. 13, no. 3, pp. 28—45. DOI: 10.15838/esc.2020.3.69.3. pp 30. Leonov S. N. Preferential regimes of established local growth points and its impact on the economy of the Far East. Economic and Social Changes: Facts, Trends, Forecast, 2020, vol. 13, no. 3, pp. 28—45. DOI: 10.15838/esc.2020.3.69.3. 26. Vertakova Yu. V., Klevtsova M. G., Polozhentseva Yu. S. Formirovanie tochek klasternogo rosta ekonomi- cheskogo razvitiya territorii. [Formation of points of cluster growth of economic development of territo- ries]. Vestn. OrelGUET, 2015, no. 2 (32), pp. 56—60. (In Russian). 31. Fujita M., Mori T. The Role of Ports in the Making of Major Cities: Self-Agglomeration and Hub-Effect. J. of Development Economics, 1996, no. 49, pp. 93—120. 32. Zaostrovskikh E. A. Morskie porty Khabarovskogo kraya kak polyusa ekonomicheskogo rosta. [Seaports of Khabarovsk Krai as the Poles of Economic Growth]. Prostranstv. ekonomika, 2017, no. 4, pp. 170—183. DOI: 10.14530/se.2017.4.170-183. (In Russian). 31. Fujita M., Mori T. The Role of Ports in the Making of Major Cities: Self-Agglomeration and Hub-Effect. J. of Development Economics, 1996, no. 49, pp. 93—120. 32. Zaostrovskikh E. A. Morskie porty Khabarovskogo kraya kak polyusa ekonomicheskogo rosta. [Seaports of Khabarovsk Krai as the Poles of Economic Growth]. Prostranstv. ekonomika, 2017, no. 4, pp. 170—183. DOI: 10.14530/se.2017.4.170-183. (In Russian). 27. Kuznetsov S. V., Lachininskii S. S., Mikhailov A. S., Shendrik A. V. “Propul’sivnye otrasli” regionov Zapad- nogo porubezh’ya Rossii v usloviyakh geopoliticheskoi turbulentnosti. [“Propulsive Industries” of the Regions of Russia’s Western Borderland under of Geopolitical Turbulence]. Ekonomika regiona, 2019, vol. 15, no. 4, pp. 1253—1265. (In Russian). Information about the authors Leonov Sergey Nikolaevich, Doctor of Economy, Professor, Leading Researcher, Economic Research Institute of Far Eastern Branch of the Russian Academy of Sciences (153, Tikhookeanskaya Street, Khabarovsk, Russia, 680042), e-mail: Leonov@ecrin.ru. Zaostrovskikh Elena Anatol’evna PhD of Economy Researcher Economic Research Institute of Far Eastern
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HIGH-ORDER FINITE ELEMENTS FOR THE NEUTRON TRANSPORT EQUATION ON HONEYCOMB MESHES
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ERROR: type should be string, got "https://doi.org/10.1051/epjconf/202124703002 https://doi.org/10.1051/epjconf/202124703002 EPJ Web of Conferences 247, 03002 (2021)\nPHYSOR2020 ABSTRACT Presently, APOLLO3 R⃝/MINARET solves the transport equation using the multigroup Sn\nmethod with discontinuous finite elements on triangular meshes with Lagrange polyno-\nmial bases. The goal of this work is to solve the spatial problem on hexagonal geometries\nin the context of honeycomb lattice reactors, without further refining the computational\nmesh. The idea here is to construct high-order basis functions on the hexagonal element\nin order to improve the trade-off between computational cost and accuracy, in particu-\nlar for multiphysics simulations where, often, thermalhydraulic modelling requires only\nassembly-average cross-sections to be defined (e.g. severe accident of fast breeder reac-\ntors) i.e. the assemblies are assumed homogeneous. One approach to achieve this goal\nis through the use of generalised barycentric functions such as the Wachspress rational\nfunctions. This research endeavour deals with the application of Wachspress rational\nfunctions to the neutron transport equation for hexagonal geometries up to order 3. With\nthis method, it is possible to decrease the number of spatial unknowns required for the\nsame accuracy, and thus the computational burden for complex geometries, such as hon-\neycomb lattices is reduced. KEYWORDS: Discontinuous Galerkin, Wachspress, Discrete ordinates, Polygons HIGH-ORDER FINITE ELEMENTS FOR THE NEUTRON\nTRANSPORT EQUATION ON HONEYCOMB MESHES\nAnsar Calloo1, Romain Le Tellier2 and David Labeurthre1\n1DEN - Service d’´etudes des r´eacteurs et de math´ematiques appliqu´ees (SERMA)\nCEA, Universit´e Paris-Saclay, F-91191 Gif-sur-Yvette, France 2DEN - DTN/STMA/LMAG\nCEA Cadarache, F-13108 Saint-Paul-lez-Durance, France ansar.calloo@cea.fr, romain.le-tellier@cea.fr, david.labeurthre@cea.fr © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0\n (http://creativecommons.org/licenses/by/4.0/). KEYWORDS: Discontinuous Galerkin, Wachspress, Discrete ordinates, Polygons DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0\nses/by/4.0/). 1. INTRODUCTION The neutron transport equation [1] is solved increasingly in the framework of multiphysics cou-\npling schemes. It has been observed that high-fidelity calculations for such cases are still computa-\ntionally expensive for the neutronics part, especially in the case of parametric or accidental studies. One possible way of dealing with this situation is through the use of modern computing techniques\nto benefit from new computer architectures. A second means before optimising the code itself is\nby working on the discretisation scheme (in the present work, the spatial one). This work lies in the framework whereby the neutron transport equation is solved with discon-\ntinuous Galerkin finite elements, but on meshes that are not traditional triangles or quadrilater-\nals. Presently, APOLLO3 R⃝/MINARET [2,3] solves the transport equation using the multigroup Sn EPJ Web of Conferences 247, 03002 (2021)\nPHYSOR2020 https://doi.org/10.1051/epjconf/202124703002 method with discontinuous finite elements on triangular meshes with Lagrange polynomial bases. The goal of this work is to solve the spatial problem on hexagonal geometries in the context of\nhoneycomb lattice reactors, without further refining the computational mesh. Indeed, despite the\nlack of regularity of the angular flux, it has been shown in past works [4,5](especially on Adap-\ntive Mesh Refinement techniques) that increasing the discretization order is an effective means to\nsignificantly improve the accuracy of the solution. method with discontinuous finite elements on triangular meshes with Lagrange polynomial bases. The goal of this work is to solve the spatial problem on hexagonal geometries in the context of\nhoneycomb lattice reactors, without further refining the computational mesh. Indeed, despite the\nlack of regularity of the angular flux, it has been shown in past works [4,5](especially on Adap-\ntive Mesh Refinement techniques) that increasing the discretization order is an effective means to\nsignificantly improve the accuracy of the solution. The idea here is then to construct high-order basis functions on the hexagonal element in order to\nimprove the trade-off between computational cost and accuracy. One approach to achieve this goal\nis through the use of generalised barycentric functions such as the Wachspress rational functions. They have been employed in the past in neutron transport for quadrilaterals and arbitrary polygo-\nnals [6,7]. This research endeavour deals with the application of Wachspress rational functions [8]\nto the neutron transport equation for hexagonal geometries up to order 3. 1. INTRODUCTION With this method, it is\npossible to decrease the spatial unknowns required for the same accuracy, and thus the computa-\ntional burden for complex geometries, such as honeycomb lattices is reduced [9]. ∗It contains the external sources and the scattering source from other directions. 2.1. Discontinuous Galerkin form of the neutron transport equation Z\nκ\n((Ωn·∇ψn)vh + Σg\ntψnvh)dV +\nZ\n∂κ−\n(n · Ωn)ψ+\nn v+\nh dS Z\nκ\n((Ωn·∇ψn)vh + Σg\ntψnvh)dV +\nZ\n∂κ−\n(n · Ωn)ψ+\nn v+\nh dS\n=\nZ\nqnvhdV −\nZ\n∂\n(n · Ωn)ψ−\nn v+\nh dS\n(2) =\nZ\nκ\nqnvhdV −\nZ\n∂κ−\n(n · Ωn)ψ−\nn v+\nh dS\n(2) (2) Usually for meshes with triangular or quadrilateral elements, P is the space of polynomials of\ndegree k. However, in the general case of convex polygons, such as hexagons, it cannot be the case\nif P were the space of C0(κ) functions and such that at order k, the approximation space Pk ⊂P. Usually for meshes with triangular or quadrilateral elements, P is the space of polynomials of\ndegree k. However, in the general case of convex polygons, such as hexagons, it cannot be the case\nif P were the space of C0(κ) functions and such that at order k, the approximation space Pk ⊂P. 2.1. Discontinuous Galerkin form of the neutron transport equation Considering an open spatial domain D of R2 with boundary ∂D, meshed in a set Mh of Nκ\nhexagonal elements κ, the discrete-ordinates equation for the monoenergetic neutron transport\nequation in a given direction Ωn is expressed as: Ωn · ∇ψn(r) + Σt(r)ψn(r) = qn(r)∀r ∈D\n(1) (1) with Σt being the macroscopic total cross section and qn(r) being the neutron source in direction\nΩn ∗. Given n(r) the unit outward normal to ∂D at r ∈∂D, ∂D−is the inflow boundary defined\nas with Σt being the macroscopic total cross section and qn(r) being the neutron source in direction\nΩn ∗. Given n(r) the unit outward normal to ∂D at r ∈∂D, ∂D−is the inflow boundary defined\nas as ∂D−= {r ∈∂D | Ωn · n(r) < 0} The boundary conditions for r ∈∂D−are of Dirichlet type for ψn(r). Taking P(κ) as the space of\nreal-valued functions on κ, the set Vh is defined as The boundary conditions for r ∈∂D−are of Dirichlet type for ψn(r). Taking P(κ) as the space of\nreal-valued functions on κ, the set Vh is defined as Vh = {v ∈L2(D)|∀κ ∈Mh, v|κ = P(κ)} The unknown angular flux ψn(r) is thus expanded over the functions vh ∈Vh. These functions\nform a basis of the approximation space and are non-zero over κ and discontinuous across κ|κ′\ninterfaces. Defining ψ+\nn and ψ−\nn as the internal and external traces respectively, Eq. (2), the weak\nformulation for the transport equation, is obtained by multiplying Eq. (1) by vh, integrating by parts\nover element κ, applying the divergence theorem to the streaming term, and using the upwinding The unknown angular flux ψn(r) is thus expanded over the functions vh ∈Vh. These functions\nform a basis of the approximation space and are non-zero over κ and discontinuous across κ|κ′\ninterfaces. Defining ψ+\nn and ψ−\nn as the internal and external traces respectively, Eq. (2), the weak\nformulation for the transport equation, is obtained by multiplying Eq. (1) by vh, integrating by parts\nover element κ, applying the divergence theorem to the streaming term, and using the upwinding 2 2 EPJ Web of Conferences 247, 03002 (2021)\nPHYSOR2020 https://doi.org/10.1051/epjconf/202124703002 approximation for the numerical flux at the interfaces. More details can be found in [10]. 2.2. Wachspress rational functions on the regular hexagon Thus, the equation of the straight line\nd′\ni through ai−1,i and ai,i+1 is given as l′\ni(x) = 0. Using these geometrical constructions, and the\npreviously-defined ones, it is now possible to build the second-order Wachspress shape functions: Furthermore, the midpoint of ai and ai+1 is defined as ai,i+1. Thus, the equation of the straight line\nd′\ni through ai−1,i and ai,i+1 is given as l′\ni(x) = 0. Using these geometrical constructions, and the\npreviously-defined ones, it is now possible to build the second-order Wachspress shape functions: (\nwi\n= ci\nli+2li+3li+4li+5l′\ni\nq\nwi,i+1\n= ci,i+1\nli+2li+3li+4li+5li\nq\n(4) (\nwi\n= ci\nli+2li+3li+4li+5l′\ni\nq\nwi,i+1\n= ci,i+1\nli+2li+3li+4li+5li\nq\n(4)\nwhere ci and ci i+1 are chosen such that wi(ai) = 1 and wi i+1(ai i+1) = 1 (4) where ci and ci,i+1 are chosen such that wi(ai) = 1 and wi,i+1(ai,i+1) = 1. where ci and ci,i+1 are chosen such that wi(ai) = 1 and wi,i+1(ai,i+1) = 1. Besides, the nodes ai+1,i+1,i (respectively ai,i,i+1) are defined as 1\n3(2ai+1+ai) (respectively 1\n3(2ai+\nai+1)). The equation of the straight lines through ai+1,i+1,i and ai+3,i+3,i+4 is l′\ni+1,i+3(x) = 0. Finally, γ(x) = 0 is the equation of the circle Γ through the 12 points ai+1,i+1,i and ai,i,i+1. With\nthis last set of geometrical elements, the third-order Wachspress basis functions are defined: Besides, the nodes ai+1,i+1,i (respectively ai,i,i+1) are defined as 1\n3(2ai+1+ai) (respectively 1\n3(2ai+\nai+1)). The equation of the straight lines through ai+1,i+1,i and ai+3,i+3,i+4 is l′\ni+1,i+3(x) = 0. Finally, γ(x) = 0 is the equation of the circle Γ through the 12 points ai+1,i+1,i and ai,i,i+1. With\nthis last set of geometrical elements, the third-order Wachspress basis functions are defined: \n\n\n\n\n\n\nwi\n= ci\nli+2li+3li+4li+5γ\nq\nwi,i,i+1\n= ci,i,i+1\nli+2li+3li+4li+5lil′\ni+1,i+3\nq\nwi+1,i+1,i\n= ci+1,i+1,i\nli+2li+3li+4li+5lil′\ni,i+4\nq\n(5) (5) where ci, ci,i,i+1 and ci+1,i+1,i are chosen such that wi(ai) = 1, wi,i,i+1(ai,i,,i+1) = 1 and\nwi+1,i+1,i(ai+1,i+1,,i) = 1 respectively. Fig. 2 shows the typical shape functions for order 3 for\ni = 2. where ci, ci,i,i+1 and ci+1,i+1,i are chosen such that wi(ai) = 1, wi,i,i+1(ai,i,,i+1) = 1 and\nwi+1,i+1,i(ai+1,i+1,,i) = 1 respectively. Fig. 2 shows the typical shape functions for order 3 for\ni = 2. J. L. 3. NUMERICAL RESULTS In this section, we provide numerical results for a 2D one-group transport benchmark which has\nbeen set up in [12]. 2.2. Wachspress rational functions on the regular hexagon Gout proved that these rational functions form a basis for spanning space P, and such that at\norder k, Pk(κ) ⊂P and Pk+1(κ) ̸⊂P [8,11]. Besides, given that these functions are anchored at\nnodes on the sides only, they can be assimilated to serendipity functions [7]. 2.2. Wachspress rational functions on the regular hexagon J. L. Gout has defined Wachspress rational basis functions on the regular hexagon up to order 3 in\n[11]. For this particular case, the following geometrical elements are given as shown in Fig. 1 and\nfollowing the same notations as in [11]. x\ny\nO\na4\na5\na6\na1\na2\na3\nb4\nb5\nb6\nb1\nb2\nb3\na34\na665\na556\na332\na223\nFigure 1: Geometrical elements to define Wachspress shape functions on a regular hexagon. Figure 1: Geometrical elements to define Wachspress shape functions on a regular hexagon. Let us consider a regular “flat-top” hexagon with centre (0,0), a pitch (distance between two parallel\nsides) of p and thus, a sidelength of\np\n√\n3. The vertices ai of the hexagon are numbered as shown in Let us consider a regular “flat-top” hexagon with centre (0,0), a pitch (distance between two parallel\nsides) of p and thus, a sidelength of\np\n√\n3. The vertices ai of the hexagon are numbered as shown in 3 EPJ Web of Conferences 247, 03002 (2021)\nPHYSOR2020 https://doi.org/10.1051/epjconf/202124703002 Fig. 1, such that ai and ai+1 are consecutive. For any i in Z/6Z, li(x) = 0 is the equation of the\nstraight line di through ai−1 and ai. The intersection of straight lines di−1 and di+1 is the point bi,\nand q(x) = 0 is the equation of the circle Q through all the points bi (this is also called the adjoint\ncircle of the regular hexagon). These geometrical elements are sufficient to define the first-order\nWachspress shape functions wi which are anchored at the vertices of the hexagon: Fig. 1, such that ai and ai+1 are consecutive. For any i in Z/6Z, li(x) = 0 is the equation of the\nstraight line di through ai−1 and ai. The intersection of straight lines di−1 and di+1 is the point bi,\nand q(x) = 0 is the equation of the circle Q through all the points bi (this is also called the adjoint\ncircle of the regular hexagon). These geometrical elements are sufficient to define the first-order\nWachspress shape functions wi which are anchored at the vertices of the hexagon: wi = ci\nli+2li+3li+4li+5\nq\n(3) (3) where ci is a constant chosen such that wi(ai) = 1. where ci is a constant chosen such that wi(ai) = 1. Furthermore, the midpoint of ai and ai+1 is defined as ai,i+1. Table 1: Benchmark cross sections Table 1: Benchmark cross sections Table 1: Benchmark cross sections\nMixture\nΣt (cm−1)\nΣs0 (cm−1)\nΣs1 (cm−1)\nνΣf (cm−1)\n1\n0.025\n0.013\n0.0\n0.0155\n2\n0.025\n0.024\n0.006\n0.0\n3\n0.075\n0.0\n0.0\n0.0 ‡Throughout this analysis, the computational cost is not measured as time or arithmetic inten-\nsity. The WSF solver has not been implemented optimally with this goal. 3.2. Results analysis The one-group benchmark is analysed in two different settings: within the APOLLO3 R⃝/MINARET\nframework and using the Wachspress shape functions finite elements. Both cases use discrete\nordinates with product angular hexagonal quadratures shortened as HQ(n,m) in the results tables - n\nand m are the polar and azimuthal orders respectively, leading to 6nm directions in 2D geometries. APOLLO3 R⃝/MINARET[3] solves the neutron transport equations with a Discontinuous Galerkin\nformulation with the usual Lagrange shape functions on unstructured triangular meshes. Thus,\neach hexagonal cell within the meshed geometry is at least split in 6 equilateral triangles. In\nTable 2, splits of s (1, 2 and 3) are given and means that the side of a triangular mesh in the meshed\ngeometry should not exceed sidelength\ns\n, thereby leading to an unstructured grid of triangles. The\nsolutions are computed for spatial orders of 1 and 2. The reference case is the best-estimate case\ncomputed with HQ(4,5) angular quadrature, order 2, and a split of 3 - number of spatial unknowns\nNunk amounting to 38100. The best-estimate case with the Wachspress shape functions solver\n(WSF) is HQ(4,5) angular quadrature and spatial basis order of 3, equivalent to Nunk = 2286. For each case, the effective multiplication factor keff is given and compared to the best-estimate\nresult. Furthermore, the absorption rates Rabs are also analysed †. The maximum and average\nerrors are defined as in [12]. The results are summarised in Table 2 for APOLLO3 R⃝/MINARET\nand in Table 3 for WSF. The keff for the reference APOLLO3 R⃝/MINARET and WSF solutions\ndiffers by less than 1 pcm whereas the ϵmax and ϵ are both less than 0.1%. From Table 2, it can be deduced that APOLLO3 R⃝/MINARET satisfactorily predicts the keff for\nlow-order quadratures and coarser spatial discretisation, although the errors on the absorption rates\nare higher. It can also be observed that as the computational mesh is refined, the errors on the\nabsorption rates decreases, yet at the expense of more unknowns in the system, and thus a higher\ncomputational cost ‡. For a given computational mesh, increasing the order of the method is more\nefficient to decrease the error. Thus, these numerical results hint that increasing the spatial basis\norder would be a better option for solving the transport problem efficiently. †The absorption cross section is computed as Σt −Σs0.\n‡ 3.1. Benchmark description The benchmark consists of three mixtures with a radial distribution as given in Fig. 3. The cross\nsections for the mixtures are as given in Table 1. As stated by A. H´ebert, this benchmark is not\nrepresentative of a real-life problem but has been designed to magnify transport and anisotropy\neffects (as much as 2500 pcm!) within the domain and on the vacuum boundary condition. In this\nhoneycomb lattice, the length of the side of a hexagonal cell is 19 cm. 4 https://doi.org/10.1051/epjconf/202124703002 EPJ Web of Conferences 247, 03002 (2021)\nPHYSOR2020 (a) w2\n(b) w223\n(c) w332\nFigure 2: Example of Wachspress basis functions of order 3. (b) w223 (a) w2 (a) w2\n(b) w\n(c) w332 (c) w332 Figure 2: Example of Wachspress basis functions of order 3. Figure 3: 2D hexagonal benchmark problem proposed by [12]. Mixture 0 is here only to\ncomplete the hexagonal geometry description, and is equivalent to void. Figure 3: 2D hexagonal benchmark problem proposed by [12]. Mixture 0 is here only to\ncomplete the hexagonal geometry description, and is equivalent to void. 5 https://doi.org/10.1051/epjconf/202124703002 EPJ Web of Conferences 247, 03002 (2021)\nPHYSOR2020 3.2. Results analysis Increasing the order is cost effective in terms of Nunk to the\nquality of the result. For this problem, an order of 2 for a given angular quadrature is satisfactory\nwith respect to the solution obtained. Considering a purely computational aspect of using Wachs-\npress basis functions on unrefined hexagons, a coarser mesh is sufficient, and thus Nunk depends\nonly the order of the method, which increases linearly. Indeed, the matricial problem solved locally\ngrows as N 2\ndofs and increasing the order for better accuracy remains competitive so long the com-\nputation time remains reasonably low compared to refining meshes. Hence, the ideal framework\nwould be to increase the order locally to avoid increasing the global Nunk of the problem. All these results are consistent with TRIVAC and SNATCH results published in [12], and the new\ncapabilities of DRAGON for hexagonal geometries [13]. 3.2. Results analysis From Table 3, solutions with first-order Wachspress rational functions are satisfactory considering From Table 3, solutions with first-order Wachspress rational functions are satisfactory considering 6 6 https://doi.org/10.1051/epjconf/202124703002 EPJ Web of Conferences 247, 03002 (2021)\nPHYSOR2020 Table 2: Solutions from APOLLO3 R⃝/MINARET\nHQ(n,m)\norder\nsplit\nNunk = Nκ × Ndofs\nkeff\n∆keff (pcm)\nϵmax (%)\nϵ (%)\nHQ(2,3)\n1\n1\n762 × 3\n1.002160\n-16.9\n7.16\n0.60\nHQ(2,3)\n1\n2\n2540 × 3\n1.002235\n-9.4\n0.71\n0.11\nHQ(2,3)\n1\n3\n6350 × 3\n1.002251\n-7.8\n1.57\n0.13\nHQ(2,3)\n2\n1\n762 × 6\n1.002262\n-6.6\n2.55\n0.17\nHQ(2,3)\n2\n2\n2540 × 6\n1.002255\n-7.3\n2.12\n0.15\nHQ(2,3)\n2\n3\n6350 × 6\n1.002257\n-7.2\n1.83\n0.14\nHQ(4,5)\n1\n1\n762 × 3\n1.002224\n-10.4\n7.23\n0.53\nHQ(4,5)\n1\n2\n2540 × 3\n1.002304\n-2.4\n2.09\n0.13\nHQ(4,5)\n1\n3\n6350 × 3\n1.002324\n-0.5\n0.47\n0.03\nHQ(4,5)\n2\n1\n762 × 6\n1.002330\n0.1\n0.12\n0.02\nHQ(4,5)\n2\n2\n2540 × 6\n1.002330\n0.1\n0.13\n0.01\nHQ(4,5)\n2\n3\n6350 × 6\n1.002328\n–\n–\n– Table 2: Solutions from APOLLO3 R⃝/MINARET Table 3: Solutions with Wachspress shape functions\nHQ(n,m)\norder\nNunk = Nκ × Ndofs\nkeff\n∆keff (pcm)\nϵmax (%)\nϵ (%)\nHQ(2,3)\n1\n127 × 6\n1.001664\n-65.8\n3.94\n1.89\nHQ(2,3)\n2\n127 × 12\n1.002161\n-16.2\n0.32\n0.07\nHQ(2,3)\n3\n127 × 18\n1.002152\n-17.0\n0.20\n0.08\nHQ(4,5)\n1\n127 × 6\n1.001829\n-49.3\n3.50\n1.78\nHQ(4,5)\n2\n127 × 12\n1.002324\n0.2\n0.29\n0.10\nHQ(4,5)\n3\n127 × 18\n1.002323\n–\n–\n– Table 3: Solutions with Wachspress shape functions the Nunk involved in the problem. Increasing the order is cost effective in terms of Nunk to the\nquality of the result. For this problem, an order of 2 for a given angular quadrature is satisfactory\nwith respect to the solution obtained. Considering a purely computational aspect of using Wachs-\npress basis functions on unrefined hexagons, a coarser mesh is sufficient, and thus Nunk depends\nonly the order of the method, which increases linearly. Indeed, the matricial problem solved locally\ngrows as N 2\ndofs and increasing the order for better accuracy remains competitive so long the com-\nputation time remains reasonably low compared to refining meshes. Hence, the ideal framework\nwould be to increase the order locally to avoid increasing the global Nunk of the problem. the Nunk involved in the problem. 4. CONCLUSIONS In this work, a high-order discontinuous Galerkin formulation using Wachspress rational basis\nfunctions has been applied to the Sn neutron transport equation on 2D honeycomb lattices. In 7 7 EPJ Web of Conferences 247, 03002 (2021)\nPHYSOR2020 https://doi.org/10.1051/epjconf/202124703002 the context of precise solutions to the transport equation on coarse meshes as it is the case for\nmultiphysics calculations on hexagonal reactor geometries (e.g. fast-neutron reactor application),\nit is sufficient to use high-order finite element solutions on unrefined honeycomb meshes. This\nwork contributes to that effort by demonstrating that Wachspress rational basis functions of order\n2 or 3 on honeycomb lattices are well adapted. Further work on the use of these functions is to\nextend them in a polygonal framework and set up p-refinement strategies to reduce the number of\nunknowns. the context of precise solutions to the transport equation on coarse meshes as it is the case for\nmultiphysics calculations on hexagonal reactor geometries (e.g. fast-neutron reactor application),\nit is sufficient to use high-order finite element solutions on unrefined honeycomb meshes. This\nwork contributes to that effort by demonstrating that Wachspress rational basis functions of order\n2 or 3 on honeycomb lattices are well adapted. Further work on the use of these functions is to\nextend them in a polygonal framework and set up p-refinement strategies to reduce the number of\nunknowns. REFERENCES ] A. HEBERT. Applied Reactor Physics. Presses Internationales Polytechnique (2009). [2] D. SCHNEIDER, F. DOLCI, F. GABRIEL, and J.-M. P. et. al. “APOLLO3 R⃝: CEA/DEN\ndeterministic multipurpose platform for core physics analysis.” In Proc. of Int. Mtg. on the\nPhysics of Fuel Cycles and Advanced Nuclear Systems PHYSOR 2016. Sun Valley, USA\n(2016). [3] J. Y. MOLLER and J. J. LAUTARD. “MINARET, a deterministic neutron transport solver for\nnuclear core calculations.” In Proc. of Int. Conf. on Mathematics and Computational Methods\nApplied to Nuclear Science and Engineering. Rio de Janeiro, Brazil (2011). [4] D. FOURNIER and R. LE TELLIER and C. SUTEAU. “Analysis of an a posteriori Error\nEstimator for the Transport Equation with SN and Discontinuous Galerkin Discretizations.”\nAnnals of Nuclear Energy, volume 38, pp. 221–231 (2011). [5] Y. WANG and J. C. RAGUSA. “On the Convergence of DGFEM Applied to the Discrete\nOrdinates Transport Equation for Structured and Unstructured Triangular Meshes.” Nuclear\nScience and Engineering, volume 163, pp. 56–72 (2009). [6] G. G. DAVIDSON and T. S. PALMER. “Finite Element Transport Using Wachspress Ratio-\nnal Basis Functions on Quadrilaterals in Diffusive Regions.” Nuclear Science and Engineer-\ning, volume 159, pp. 242–255 (2008). [7] M. W. HACKEMACK and J. C. RAGUSA. “Quadratic serendipity discontinuous finite ele-\nment discretization for SN transport on arbitrary polygonal grids.” Journal of Computational\nPhysics, volume 374, pp. 188 – 212 (2018). [8] E. L. WACHSPRESS. A Rational Finite Element Basis. Academic Press (1975). [9] C. TALISCHI, G. H. PAULINO, and C. H. LE. “Honeycomb Wachspress finite elements\nfor structural topology optimization.” Structural and Multidisciplinary Optimization, vol-\nume 37(6), pp. 569–583 (2009). [10] L. GASTALDO, R. LE TELLIER, C. SUTEAU, D. FOURNIER, J. M. RUGGIERI. “High-\norder discrete ordinate transport in non-conforming 2D Cartesian meshes.” In Proceedings\nof International Conference on Mathematics, Computational Methods & Reactor Physics. M&C 2009, Saratoga Springs, New York (2009). [11] J. L. GOUT. “Rational Wachspress-type Finite Elements on Regular Hexagons.” IMA Jour-\nnal of Numerical Analysis, volume 5, pp. 59–77 (1985). [12] A. HEBERT. “A Raviart-Thomas-Schneider implementation of the Simplified Pn method in\n3D hexagonal geometry.” In Proc. of Int. Mtg. on Advances in Reactor Physics to Power the\nNuclear Renaissance PHYSOR 2010. ANS, Pittsburgh, USA (2010). [13] A. A. CALLOO and A. HEBERT. “Comparing the discontinuous Galerkin and high-order\ndiamond differencing methods for the discrete ordinates transport equation in hexagonal ge-\nometry.” In Proc. of this meeting (2020). REFERENCES 8 8"
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Evaluation of cebranopadol, a dually acting nociceptin/orphanin FQ and opioid receptor agonist in mouse models of acute, tonic, and chemotherapy-induced neuropathic pain
Inflammopharmacology
2,017
cc-by
10,807
Abstract Background Cebranopadol (a.k.a. GRT-6005) is a dually acting nociceptin/orphanin FQ and opioid receptor agonist that has been recently developed in Phase 2 clinical trials for painful diabetic neuropathy or cancer pain. It also showed analgesic properties in various rat models of pain and had a better safety profile as compared to equi-anal- gesic doses of morphine. Since antinociceptive properties of cebranopadol have been studied mainly in rat models, in the present study, we assessed analgesic activity of sub- cutaneous cebranopadol (10 mg/kg) in various mouse pain models. Conclusion Cebranopadol may have significant potential for the treatment of various pain types, including inflam- matory and chemotherapy-induced neuropathic pain. Keywords Cebranopadol  Pain models  Neurogenic inflammation  Chemotherapy-induced peripheral neuropathy  Oxaliplatin  Simvastatin Keywords Cebranopadol  Pain models  Keywords Cebranopadol  Pain models  Neurogenic inflammation  Neurogenic inflammation  Chemotherapy-induced peripheral neuropathy  Methods We used models of acute, tonic, and chronic pain induced by thermal and chemical stimuli, with a particular emphasis on pharmacoresistant chronic neuropathic pain evoked by oxaliplatin in which cebranopadol was used alone or in combination with simvastatin. Evaluation of cebranopadol, a dually acting nociceptin/orphanin FQ and opioid receptor agonist in mouse models of acute, tonic, and chemotherapy-induced neuropathic pain Kinga Sałat1 • Anna Furgała1 • Robert Sałat2 Received: 25 July 2017 / Accepted: 6 October 2017 / Published online: 25 October 2017  The Author(s) 2017. This article is an open access publication that of morphine. Used in combination and administered simultaneously, 4 or 6 h after simvastatin, cebranopadol did not potentiate antiallodynic activity of this cholesterol- lowering drug. Cebranopadol did not induce any motor deficits in the rotarod test. that of morphine. Used in combination and administered simultaneously, 4 or 6 h after simvastatin, cebranopadol did not potentiate antiallodynic activity of this cholesterol- lowering drug. Cebranopadol did not induce any motor deficits in the rotarod test. 2 Faculty of Production Engineering, Warsaw University of Life Sciences, 164 Nowoursynowska St, 02-787 Warsaw, Poland Inflammopharmacol (2018) 26:361–374 https://doi.org/10.1007/s10787-017-0405-5 Inflammopharmacol (2018) 26:361–374 https://doi.org/10.1007/s10787-017-0405-5 Inflammopharmacology ORIGINAL ARTICLE Evaluation of cebranopadol, a dually acting nociceptin/orphanin FQ and opioid receptor agonist in mouse models of acute, tonic, and chemotherapy-induced neuropathic pain ORIGINAL ARTICLE 1 Chair of Pharmacodynamics, Department of Pharmacodynamics, Jagiellonian University Medical College, 9 Medyczna St, 30-688 Krakow, Poland Introduction evidence from animal and human studies for the involve- ment of peripheral opioid receptors in analgesia, especially in the presence of inflammation (Sehgal et al. 2011), or neuropathy (Plein and Rittner 2017). The nociceptin/orphanin FQ opioid peptide receptor (NOP receptor) is the most recently discovered member of the opioid receptor family. Together with its endogenous ligand–nociceptin, also known as orphanin FQ (N/OFQ), it forms the fourth member of the opioid receptor family which is abundantly expressed in various body tissues. A large body of evidence shows that the activation of N/OFQ-NOP system regulates functions of the central nervous system being implicated in feeding, body weight homeostasis, stress, stress-related psychiatric disorders— depression, anxiety, drug, and alcohol dependence (Witkin et al. 2014). Data from preclinical studies are also in line with these findings showing that N/OFQ plays an important role in comorbid neuropathic pain and post-traumatic stress disorder (Zhang et al. 2015), acute and chronic restraint stress responses (Delaney et al. 2012), depression (Vitale et al. 2017), and other stress-related conditions (Leggett et al. 2006; Witkin et al. 2014). The data presented above come from rat studies and there is limited knowledge about antinociceptive properties of cebranopadol in mice. Moreover, these previous studies investigated the influence of cebranopadol on tactile allo- dynia but not thermal (i.e., heat or cold) allodynia and hyperalgesia. Hence, in the present study, we utilized mouse models of acute, tonic, and chronic pain induced by thermal or chemical (inflammatory) stimuli, with a partic- ular emphasis on pharmacoresistant chronic neuropathic pain evoked by oxaliplatin. Oxaliplatin is a third-genera- tion platinum-based anti-tumor drug used to treat advanced colorectal cancer. Compared to other platinum-based drugs, it has lower incidence of hematological adverse effects and gastrointestinal toxicity, but in approximately 95% of patients, oxaliplatin causes severe neuropathic pain episodes and increased sensitivity to cold (Manji 2013) which often lead to dose reduction or even treatment dis- continuation. These neuropathic pain episodes can be effectively attenuated by l opioid peptide (MOP) and NOP receptor agonists (Micheli et al. 2015). Apart from this, the N/OFQ-NOP pathway is also involved in the modulation of inflammatory and immune responses of the body by influencing migration of leuco- cytes, cytokine secretion, and lymphocyte proliferation. Introduction The International Association for the Study of Pain (IASP) defines pain as an unpleasant sensory and emotional experience associated with actual or potential tissue dam- age, or described in terms of such damage. The sensation of pain involves multiple signaling pathways, numerous neurotransmitters, and other mediators that are involved in the inhibitory or facilitatory control of pain intensity. These mechanisms affect the perception of stimuli as non-painful or painful, respectively, but their positive or negative modulation of pain signaling is strongly dependent on the receptor type involved and its location in the target tissue (Argoff 2011). Key results As shown in the hot plate test, the analgesic activity of cebranopadol developed more slowly as com- pared to morphine (90–120 min vs. 60 min). Cebranopadol displayed a significant antinociceptive activity in acute pain models, i.e., the hot plate, writhing, and capsaicin tests. It attenuated nocifensive responses in both phases of the formalin test and reduced cold allodynia in oxaliplatin- induced neuropathic pain model. Its efficacy was similar to In living organisms, endogenous opioids (endorphins, enkephalins, and dynorphins) are key molecules in the descending pain suppression pathways. Recently, it has been discovered that opioid receptors are widely dis- tributed not only in the central but also peripheral nervous system and in the non-neuronal tissues. There is also 1 Chair of Pharmacodynamics, Department of Pharmacodynamics, Jagiellonian University Medical College, 9 Medyczna St, 30-688 Krakow, Poland 12 3 3 362 K. Sałat et al. inflammatory pain (CFA-induced arthritis model), bone cancer pain model (Raffa et al. 2017), and neuropathic pain: chronic constriction injury (CCI) and diabetic neu- ropathic pain models (Raffa et al. 2017) after intraplantar, intracerebroventricular, intrathecal, intravenous (Tzschen- tke et al. 2017), subcutaneous, or oral route (Linz et al. 2014). Compared to selective MOP receptor agonists, ce- branopadol was more potent in models of chronic neuropathic than acute nociceptive pain and its duration of action was long (Linz et al. 2014). Noteworthy, safety pharmacology studies with cebranopadol demonstrated that the development of analgesic tolerance in cebranopadol- treated animals subjected to CCI procedure was delayed as compared to equi-analgesic doses of morphine (Sałat et al. 2015a), and at analgesic doses, cebranopadol did not cause respiratory depression in a rat whole-body plethysmogra- phy model, or motor coordination deficits in the rat rotarod test (Linz et al. 2014, 2017; Lambert et al. 2015; Gu¨nther et al. 2017). Introduction Recent findings showing the involvement of N/OFQ in inflammatory responses (Gavioli and Roma˜o 2011) and the evidence for a role of NOP receptors and N/OFQ in the modulation of neurogenic inflammation, migraine (Tajti et al. 2015), and airway tone (Singh et al. 2016) led to the hypothesis that N/OFQ-NOP system might be an important drug target for analgesic drugs. This is in part supported by the previous findings showing that the blockade of NOP receptors can attenuate inflammation (Gavioli et al. 2015). On the other hand, this issue is not completely clear and unequivocally explored as there is also evidence for anal- gesic efficacy of NOP agonists in neuropathic and inflammatory pain, both in animal models (Sukhtankar et al. 2013) and clinical trials (Sałat et al. 2015a). In the present study, we investigated the effect of cebranopadol on cold nociceptive threshold of oxaliplatin- treated mice. We used two protocols of its administration: the first one which utilized this drug alone, and the second one in which cebranopadol was used in combination with simvastatin. Available data show potential effectiveness of simvastatin in several animal models of pain (Shi et al. 2011; Miranda et al. 2011; Chen et al. 2013; Mansouri et al. 2017), including inflammatory (Chen et al. 2013) and neuropathic pain models (Shi et al. 2011). First, in the previous studies (Bhalla et al. 2015), simvastatin effec- tively reversed vincristine-induced neuropathic pain by anti-inflammatory effects and it was able to attenuate vincristine-induced increase in myeloperoxidase activity. Cebranopadol (a.k.a. GRT-6005; trans-60-fluoro-40,90- dihydro-N,N-dimethyl-4-phenyl-spiro[cyclohexane- 1,10(30H)-pyrano[3,4-b]indol]-4-amine) is a dually acting nociceptin/orphanin FQ and opioid receptor agonist (Ki (nM)/EC50 (nM)/relative efficacy (%): human NOP receptor 0.9/13.0/89; human mu-opioid peptide (MOP) receptor 0.7/1.2/104; human kappa-opioid peptide (KOP) receptor 2.6/17/67; human delta-opioid peptide (DOP) receptor 18/110/105) (Linz et al. 2014) that has been recently developed in Phase 2 clinical trials for painful diabetic neuropathy or cancer pain (reviewed in Sałat et al. 2015a). It showed analgesic properties in various rat models of acute thermal pain, i.e., tail-flick model, chronic 123 Evaluation of cebranopadol, a dually acting nociceptin/orphanin FQ and opioid receptor… 363 respective Polish and EU regulations (Directive No. 86/609/EEC). respective Polish and EU regulations (Directive No. 86/609/EEC). Second, anti-inflammatory and anti-oxidant effects of this drug also resulted in reduction of cisplatin-induced nephrotoxicity and hepatotoxicity in rats (Is¸eri et al. 2007) and simvastatin protected Sertoli cells against cisplatin cytotoxicity (Wang et al. 2015). Chemicals Cebranopadol and morphine hydrochloride were purchased from MedChem Express (NJ, USA) and Polfa Kutno (Poland), respectively. These drugs at a fixed dose of 10 mg/kg were administered subcutaneously before behavioral tests. This dose was selected based on our previous pilot study which revealed that full antinocicep- tive efficacy of morphine used as a reference drug was observed at doses 6–10 mg/kg. For in vivo tests, cebra- nopadol was prepared in a mixture of 100% DMSO (Polskie Odczynniki Chemiczne, Poland) and 0.9% saline (1:1), then being diluted in saline to achieve a proper concentration and was injected 120 min before testing (for details, see Sect. 3.1.1). Morphine hydrochloride was dis- solved in 0.9% saline solution and it was administered 60 min before the tests. Control animals received vehicle. Acetic acid, ethanol, 0.9% natrium chloride solution, 5% glucose solution, and 37% formaldehyde solution were provided by Polskie Odczynniki Chemiczne (Poland). Capsaicin, simvastatin, and oxaliplatin were purchased from Sigma-Aldrich (Germany). For the in vivo experi- ments, capsaicin was dissolved in ethanol (100%) at 5% of the final desired volume, and then, 0.9% saline was added. This mixture was vortexed for 10 min (Sałat et al. 2014). Simvastatin was suspended in 0.9% saline solution. The dose of simvastatin used in the present research (100 mg/ kg, p.o.) was chosen on the basis of the previous studies published by other authors (Mansouri et al. 2015). Introduction Third, it has been also shown that simvastatin was able to attenuate neuropathic pain induced by CCI in rats and it significantly decreased the ratio of membrane/cytosolic RhoA by reducing RhoA/ LIMK/cofilin pathway activity (Qiu et al. 2016). Further- more, it exerted antihyperalgesic and antiallodynic effects through the inhibition of spinal RhoA activation and its daily intrathecal administration before nerve injury pre- vented the development of neuropathy in nerve-ligated mice (Ohsawa et al. 2016). Interestingly, the RhoA-de- pendent pathway is implicated in the regulation of Transient receptor potential melastatin subtype 8 (TRPM8), a cold-sensing cation channel (Sun et al. 2014) which is also required for cold-related symptoms of oxaliplatin-induced peripheral neurotoxicity (Knowlton et al. 2011; Kono et al. 2012). Taken together, these data clearly suggest that statins are effective in neuropathic pain conditions and they might modulate pain sensitivity of cold-exposed subjects. This justifies the rationale to undertake this part of research which aimed to assess if combined use of simvastatin and cebranopadol could attenuate cold hypersensitivity of oxaliplatin-treated mice. Animals and housing conditions Experiments were carried out at the Department of Phar- macodynamics, Faculty of Pharmacy, Jagiellonian University Medical College in Krakow. The investigators involved in behavioral assays were blinded to the experi- mental groups to avoid potential bias in data recording. Adult male Albino Swiss (CD-1) mice weighing 18–22 g were purchased from the Animal Breeding Farm of the Jagiellonian University Faculty of Pharmacy. Before behavioral tests, the animals were kept in groups of 10 mice in standard plastic cages and housed under controlled conditions (room temperature of 22 ± 2 C, light/dark (12:12) cycle, lights on at 8.00 AM, humidity 50–60% and free access to food and water). Experimental groups con- sisted of 8–10 animals/dose. For the tests, the animals were selected randomly. After the assay, the mice were imme- diately euthanized by cervical dislocation. All experiments were performed between 9 AM and 3 PM. The procedures for in vivo tests were approved by the Local Ethics Com- mittee of the Jagiellonian University in Krakow (Approval No. 4/2016; 22.03.2016) and the treatment of animals was in full accordance with ethical standards laid down in Acute pain models (thermal pain, inflammatory, and chemogenic pain models) Thermal pain—hot plate test Antinociceptive properties of cebranopadol and morphine in the hot plate test were assessed as described previously (Eddy and Leimbach 1953) with some minor modification (Sałat et al. 2015b). Briefly, 1 day before the proper pharmacological experi- ment, the animals were tested to establish baseline pain sensitivity threshold (baseline latency) for each animal. For further pain tests, only mice with baseline latencies B 20 s were used. On the test day, the animals were subcuta- neously treated either with the test drugs, or vehicle 60 min before placing the animal on a hot/cold plate apparatus (Bioseb, France). This apparatus can generate heat or cold and is supplied with a temperature controller that maintains surface temperature to a set point. Herein, the temperature was set at 55–56 C. Latency time to pain reaction, i.e., the 12 3 364 K. Sałat et al. oxaliplatin (10 mg/kg prepared in 5% glucose solution). Control mice received 5% glucose solution as vehicle. Pain threshold of experimental animals was assessed using the cold plate test 3 h and 7 days after oxaliplatin adminis- tration to establish its effect on acute-phase and late-phase cold allodynia, respectively. time until the animal licked its hind paws or jumped was recorded by means of a stop-watch (Q&Q HS-46, Japan, precision: 1/100 s). In this assay, a cut-off time (60 s) was enforced to avoid paw tissue damage, and mice not responding within 60 s were removed from the apparatus and assigned a score of 60 s. Inflammatory acute pain—writhing test In this test, mice were placed individually into glass beakers and were allowed to habituate for the next 30 min. Then, each mouse was weighed, injected with the test compound or vehicle, and then placed back into the glass beaker for 60 min. To induce inflammatory pain, 0.9% acetic acid solution pre- pared in saline was injected by the intraperitoneal route. Mice were placed in the beakers once again and were observed continuously for the next 30 min. Stereotypical writhes (lengthwise constrictions of the torso with a con- comitant concave arching of the back) were counted over this period in drug-treated and control mice (Sałat et al. 2013a). Influence on cold allodynia (cold plate test)—single-drug administration protocol The cold plate test was per- formed using the hot/cold plate apparatus set at 2 C. In this assay (Fig. Acute pain models (thermal pain, inflammatory, and chemogenic pain models) 1a), the animals were tested first to obtain baseline latencies to pain reaction (i.e., lifting, biting, shaking of hind paws, jumping, and movement deficits) before oxaliplatin injection (referred to as latencies ‘before oxaliplatin’). Then, oxaliplatin was injected and latencies to pain reaction were measured again (referred to as ‘pre- drug latencies’). Subsequently, test drugs were adminis- tered and post-drug latencies were measured again. In this assay, a cut-off time of 60 s was established to avoid potential paw tissue damage and animals not responding within 60 s were removed from the apparatus and assigned a score of 60 s. Neurogenic pain—capsaicin test After the adaptation period (15 min), 1.6 lg of capsaicin dissolved in 20 ll of a mixture containing 0.9% saline and ethanol (5% of the final volume) was injected intraplantarly in the ventral surface of the right-hind paw of each mouse. The animals were observed individually for 5 min following capsaicin injection. In all experimental groups, the amount of time spent on licking, biting, or lifting the injected paw was recorded with a chronometer and was considered as an indicator of nociception (Sałat et al. 2014). Influence on cold allodynia (cold plate test)—combined drug administration protocol (simvastatin and cebra- nopadol) In the cold plate test, we additionally investigated the antiallodynic activity of cebranopadol used in combination with simvastatin. The protocol of the administration of these both drugs (alone or in combina- tion) is shown in Fig. 1. It served to establish whether cebranopadol could enhance antiallodynic properties of simvastatin in the oxaliplatin model of neuropathic pain. Influence on motor coordination—rotarod test In rodents, the injection of diluted formalin solution evokes a biphasic nocifensive behavioral response (licking or bit- ing the injected paw) of experimental animals. The first (acute) nociceptive phase of the test lasts for 5 min, whilst the second (late) one occurs between 15 and 30 min after formalin injection. In this assay, 20 ll of 5% formalin solution was injected into the dorsal surface of the right- hind paw of each mouse. Then, the mice were placed separately in glass beakers and were observed for the next 30 min. Total time spent on licking or biting the formalin- treated paw was measured during the first 5 min of the test, and then between 15 and 30 min of the test in drug-treated and vehicle-treated mice (Sałat et al. 2015b). Before the test, the animals were trained for 3 consecutive days on the rotarod apparatus (Rotarod apparatus, May Commat RR0711, Turkey; rod diameter: 2 cm) that was rotating at a fixed speed of 18 rotations per minute (rpm). In each session, the mice were placed on the rotating rod for 3 min with an unlimited number of trials. The proper experiment was performed 24 h after the last training session. After the administration of the test drugs or vehi- cle, the mice were tested on the rod that revolved at 6, 18, or 24 rpm. Motor impairments in mice were defined as the inability to remain on the rotarod apparatus for 1 min. The results are expressed as the mean time spent on the rotarod (Sałat et al. 2015b). Data analysis 1 Protocol of the administration of cebranopadol alone (a), simvastatin alone (b), or both drugs in combination (c–e) in oxaliplatin-induced neuropathic pain model. The scheme shows specific time- points at which the data measured as latencies to pain reaction in the cold plate test were collected. CPT cold plate test, OXA oxaliplatin, CEB cebranopadol, SIM simvastatin Evaluation of cebranopadol, a dually acting nociceptin/orphanin FQ and opioid receptor… 365 Evaluation of cebranopadol, a dually acting nociceptin/orphanin FQ and opioid receptor… 365 A CPT Post-Drug latency 2h 7d CPT Pre-Drug latency CPT Post-Drug latency 2h 3h CPT Pre-Drug latency CEB CEB OXA CPT Latency before OXA A B C D E 1-6d SIM + CEB CPT Post-Drug latency 2h 7d CPT Pre-Drug latency CPT Post-Drug latency 2h 3h CPT Pre-Drug latency CPT Latency before OXA CPT Post-Drug latency 2h 7d CPT Pre-Drug latency CPT Post-Drug latency 2h 3h CPT Pre-Drug latency SIM SIM OXA CPT Latency before OXA CPT Post-Drug latency 2h 1d CPT Pre-Drug latency CPT Post-Drug latency SIM CEB 2h CPT Post-Drug latency SIM 4h CPT Pre-Drug latency 7d CPT Post-Drug latency CEB 2h CPT Post-Drug latency SIM 6h CPT Pre-Drug latency 7d CPT Post-Drug latency B C D 1-6d SIM + CEB CPT Post-Drug latency 2h 7d CPT Pre-Drug latency CPT Post-Drug latency 2h 3h CPT Pre-Drug latency SIM SIM OXA CPT Latency before OXA CPT Post-Drug latency 2h 1d CPT Pre-Drug latency CPT Post-Drug latency SIM B CPT Post-Drug latency 2h 7d CPT Pre-Drug latency CPT Post-Drug latency 2h 3h CPT Pre-Drug latency SIM SIM OXA CPT Latency before OXA B B D E CEB 2h CPT Post-Drug latency SIM 6h CPT Pre-Drug latency 7d CPT Post-Drug latency that the antinociceptive activity of cebranopadol was signifi- cant 90 min and 120 min after its administration (p \ 0.01 and p \ 0.001, respectively; Fig. 2). Taking this into con- sideration, further pain tests were performed 90 and 120 min after cebranopadol injection. that the antinociceptive activity of cebranopadol was signifi- cant 90 min and 120 min after its administration (p \ 0.01 and p \ 0.001, respectively; Fig. 2). Taking this into con- sideration, further pain tests were performed 90 and 120 min after cebranopadol injection. the mean ± SEM. Data analysis Induction phase To develop chemotherapy-induced peripheral neuropathy (CIPN) and neuropathic pain mice were injected intraperitoneally with a single dose of Data analysis was carried out using GraphPad Prism soft- ware (v.5.0, CA, USA). Numerical results are expressed as 123 123 A B C D E 1-6d SIM + CEB CPT Post-Drug latency 2h 7d CPT Pre-Drug latency CPT Post-Drug latency 2h 3h CPT Pre-Drug latency CEB CEB OXA CPT Latency before OXA CPT Post-Drug latency 2h 7d CPT Pre-Drug latency CPT Post-Drug latency 2h 3h CPT Pre-Drug latency SIM SIM OXA CPT Latency before OXA CPT Post-Drug latency 2h 1d CPT Pre-Drug latency CPT Post-Drug latency SIM CEB 2h CPT Post-Drug latency SIM 4h CPT Pre-Drug latency 7d CPT Post-Drug latency CEB 2h CPT Post-Drug latency SIM 6h CPT Pre-Drug latency 7d CPT Post-Drug latency ig. 1 Protocol of the dministration of cebranopadol one (a), simvastatin alone (b), both drugs in combination –e) in oxaliplatin-induced europathic pain model. The heme shows specific time- oints at which the data easured as latencies to pain action in the cold plate test ere collected. CPT cold plate st, OXA oxaliplatin, CEB ebranopadol, SIM simvastatin valuation of cebranopadol, a dually acting nociceptin/orphanin FQ and opioid receptor… 365 the mean ± SEM. Statistical analysis was carried out using one-way analysis of variance (ANOVA), followed by Tukey’s, or Dunnett’s post hoc comparisons, or repeated- measures analysis of variance, followed by Bonferroni’s multiple comparison. P \ 0.05 was considered significant. that the antinociceptive activity of cebranopadol was signifi- cant 90 min and 120 min after its administration (p \ 0.01 and p \ 0.001, respectively; Fig. 2). Taking this into con- sideration, further pain tests were performed 90 and 120 min after cebranopadol injection. Previously obtained results for subcutaneous morphine in A B C D E 1-6d SIM + CEB CPT Post-Drug latency 2h 7d CPT Pre-Drug latency CPT Post-Drug latency 2h 3h CPT Pre-Drug latency CEB CEB OXA CPT Latency before OXA CPT Post-Drug latency 2h 7d CPT Pre-Drug latency CPT Post-Drug latency 2h 3h CPT Pre-Drug latency SIM SIM OXA CPT Latency before OXA CPT Post-Drug latency 2h 1d CPT Pre-Drug latency CPT Post-Drug latency SIM CEB 2h CPT Post-Drug latency SIM 4h CPT Pre-Drug latency 7d CPT Post-Drug latency CEB 2h CPT Post-Drug latency SIM 6h CPT Pre-Drug latency 7d CPT Post-Drug latency Fig. Data analysis Statistical analysis was carried out using one-way analysis of variance (ANOVA), followed by Tukey’s, or Dunnett’s post hoc comparisons, or repeated- measures analysis of variance, followed by Bonferroni’s multiple comparison. P \ 0.05 was considered significant. Previously obtained results for subcutaneous morphine in the hot plate test performed under the same experimental conditions (Sałat et al. 2009) demonstrated its significant antinociceptive properties. The dose of 6 mg/kg was highly effective and this activity was noted earlier than that of cebranopadol (between 30 and 60 min after administration). Acute pain models Thermal acute pain—hot plate test Neurogenic pain—capsaicin test In the intraplantar capsaicin test (Fig. 3b), the pretreatment with cebranopadol and morphine completely abolished the licking response of capsaicin-treated mice (F[2,21] = 50.18, p \ 0.0001). This confirmed strong antinociceptive properties of both agents in this pain model. Inflammatory acute pain—writhing test To establish at which time-points subcutaneous cebranopadol exerts antinociceptive activity, the mouse hot plate test was used. In this assay, an overall effect of treatment was observed (F[4,28] = 6.676, p \ 0.001). The post hoc analysis revealed As shown in Fig. 3a, cebranopadol and morphine were equally effective in reducing acetic acid-induced writhing behavior (F[2,20] = 52.86, p \ 0.0001). 12 3 3 K. Sałat et al. 366 Fig. 2 Antinociceptive activity of subcutaneous cebranopadol in the mouse hot plate test measured at various time-points. Results are shown as latency time to pain reaction (±SEM) in response to thermal stimulation (temperature of 55–56 C). Statistical analysis: repeated- measures analysis of variance, followed by Bonferroni’s multiple comparison. Significance vs. pre-drug latency: **p \ 0.01, ***p \ 0.001 attenuate neurogenic pain (F[2,21] = 14.92, p \ 0.0001) with morphine being slightly more effective (Fig. 4a). The antinociceptive activity of both tested agents was also shown in the second (inflammatory) phase which demon- strated that cebranopadol and morphine significantly (at p \ 0.01 and p \ 0.001, respectively) reduced pain responses in formalin-treated animals (F[2,21] = 11.81, p \ 0.001; Fig. 4b). Influence on cold allodynia (combined treatment protocol: cebranopadol and simvastatin) Preliminary experiments (data not shown) performed using the cold plate test in mice not treated with oxaliplatin (i.e., non-neuropathic mice) revealed overall effects of single- dose and repeated-dose simvastatin treatments (single-dose simvastatin: F[5,32] = 8.366, p \ 0.0001; repeated-dose simvastatin: F[7,46] = 4.209, p \ 0.01). Post hoc analyses Influence on cold allodynia (single-drug administration protocol) Fig. 2 Antinociceptive activity of subcutaneous cebranopadol in the mouse hot plate test measured at various time-points. Results are shown as latency time to pain reaction (±SEM) in response to thermal stimulation (temperature of 55–56 C). Statistical analysis: repeated- measures analysis of variance, followed by Bonferroni’s multiple comparison. Significance vs. pre-drug latency: **p \ 0.01, ***p \ 0.001 In this experiment, an overall effect of treatment was observed (F[16,127] = 15.58, p \ 0.0001). As shown in Fig. 5, during the early phase cold allodynia, both cebra- nopadol and morphine demonstrated strong and statistically significant (p \ 0.01) antiallodynic properties. Their antiallodynic activity was also confirmed during the late phase of cold allodynia—7 days after oxaliplatin injection (significant at p \ 0.05 and p \ 0.01 for cebra- nopadol and morphine, respectively). Neurogenic pain—capsaicin test Inflammatory tonic pain model—formalin test The results obtained in the first phase of the intraplantar formalin test confirmed that both drugs were able to response (±SEM) (b). Statistical analysis: one-way analysis of variance, followed by Dunnett’s post hoc test. Significance vs. vehicle-treated mice: ***p \ 0.001 Fig. 3 Antinociceptive activity of cebranopadol (CEB) and morphine (MOR) in the acetic acid-induced writhing test (a) and in capsaicin- induced neurogenic pain model (b). Results are shown as the mean number of writhes (±SEM) (a), or duration of the licking/biting response (±SEM) (b). Statistical analysis: one-way analysis of variance, followed by Dunnett’s post hoc test. Significance vs. vehicle-treated mice: ***p \ 0.001 response (±SEM) (b). Statistical analysis: one-way analysis of variance, followed by Dunnett’s post hoc test. Significance vs. vehicle-treated mice: ***p \ 0.001 Fig. 3 Antinociceptive activity of cebranopadol (CEB) and morphine (MOR) in the acetic acid-induced writhing test (a) and in capsaicin- induced neurogenic pain model (b). Results are shown as the mean number of writhes (±SEM) (a), or duration of the licking/biting 123 367 Evaluation of cebranopadol, a dually acting nociceptin/orphanin FQ and opioid receptor… Fig. 4 Antinociceptive activity of cebranopadol (CEB) and morphine (MOR) in the tonic pain model (i.e., the intraplantar formalin test). Influence on the first, neurogenic phase responses (a) and on the second, inflammatory phase responses (b). Statistical analysis: one- way analysis of variance, followed by Dunnett’s post hoc test. Significance vs. vehicle-treated mice: **p \ 0.01, ***p \ 0.001 second, inflammatory phase responses (b). Statistical analysis: one- way analysis of variance, followed by Dunnett’s post hoc test. Significance vs. vehicle-treated mice: **p \ 0.01, ***p \ 0.001 second, inflammatory phase responses (b). Statistical analysis: one- way analysis of variance, followed by Dunnett’s post hoc test. Significance vs. vehicle-treated mice: **p \ 0.01, ***p \ 0.001 Fig. 4 Antinociceptive activity of cebranopadol (CEB) and morphine (MOR) in the tonic pain model (i.e., the intraplantar formalin test). Influence on the first, neurogenic phase responses (a) and on the second, inflammatory phase responses (b). Statistical analysis: one- way analysis of variance, followed by Dunnett’s post hoc test. Significance vs. vehicle-treated mice: **p \ 0.01, ***p \ 0.001 Influence on motor coordination (rotarod test) In general, the use of cebranopadol as ‘add-on’ therapy with simvastatin in neuropathic, oxaliplatin-treated mice showed no benefits. However, we noted some differences among groups treated with ‘add-on’ cebranopadol used simultaneously (F[9,70] = 6.657, p \ 0.0001; Fig. 6B: sim ? ceb), 4 h (F[9,65] = 11.37, p \ 0.0001; Fig. 6C: In the rotarod test, neither cebranopadol nor morphine influenced animals’ motor coordination at 6, 18, or 24 rpm which means that the drugs tested did not cause motor deficits in mice. Fig. 6 Influence of oxaliplatin (10 mg/kg, i.p.; OXA), simvastatin (100 mg/kg, oral route; SIM) alone, or in combination with subcu- taneous cebranopadol (10 mg/kg, s.c.; CEB) on cold sensitivity threshold and cold allodynia measured in the cold plate test in oxaliplatin-induced neuropathic pain model. Effect of oxaliplatin on the development of cold allodynia (a). Effect of 7-day administration of oral simvastatin alone (OXA ? SIM), or in combination with single-dose cebranopadol added on day 7: simultaneously (b OXA ? SIM ? CEB), 4 h (c OXA ? SIM ? CEB (4 h)), or 6 h (d OXA ? SIM ? CEB (6 h)) after simvastatin. Results are shown as latency time to pain reaction (±SEM) in response to thermal (cold) stimulation (2 C). In this test, the data were collected at two time-points: on day 1 to measure the latencies to pain reaction during the acute-phase cold allodynia that developed after oxaliplatin injection, and on day 7 to measure the latencies to pain reaction during the late-phase cold allodynia. Day 1: grey bars indicate baseline latencies of oxaliplatin-treated mice measured before drug treatment (i.e., before vehicle or simvastatin administration; defined as ‘pre-drug’) and black bars depict latencies after drug administra- tion (i.e., after vehicle or simvastatin administration; referred to as ‘post-drug’ values). Day 7: grey bars indicate baseline latencies of oxaliplatin-treated mice measured before drug treatment (i.e., before vehicle, simvastatin or cebranopadol administration; defined as ‘pre- drug’) and black bars depict latencies after drug administration (i.e., after vehicle, simvastatin only, or simvastatin and cebranopadol administration; referred to as ‘post-drug’ values). A detailed protocol used is also shown in Fig. 1. Statistical analysis: one-way analysis of variance, followed by Tukey’s post hoc test. Significance vs. latency to pain reaction before oxaliplatin injection: ## p \ 0.01, ### p \ 0.001. Fig. 368 sim ? ceb (4 h)) or 6 h (F[9,65] = 9.875, p \ 0.0001; Fig. 6d: sim ? ceb (6 h)) after simvastatin. Post hoc analyses showed no significance of these results but interestingly, we observed a trend towards the prolongation of latency time to pain reaction in mice treated with combined simvastatin and cebranopadol, when cebra- nopadol was administered 4 h (Fig. 6c), or 6 h after simvastatin (Fig. 6d), but not if these two drugs were given simultaneously (Fig. 6b). to pain reaction after a single dose of this drug. Notewor- thy, comparing pre-drug latencies in the cold plate test (grey bars—Figs. 6b–d: sim ? oxa) during the early (on day 1) and the late (on day 7) phases of cold allodynia to the latency of untreated controls (a white bar—Figs. 6b–d: sim ? oxa), it can be concluded that the repeated (7-day) administration of simvastatin partially reversed the decrease of pain threshold caused by oxaliplatin and par- tially restored it to values of mice not treated with oxaliplatin. Fig. 5 Antiallodynic activity of cebranopadol and morphine in oxaliplatin-induced neuropathic pain model. Cold allodynia was measured using the cold plate test in the early phase (3 h after oxaliplatin administration) and in the late phase (7 days after oxaliplatin administration). Results are shown as latency time to pain reaction (±SEM) in response to thermal stimulation (temperature of 2 C). Statistical analysis: one-way analysis of variance, followed by Tukey’s post hoc test. Significance vs. latencies of vehicle-treated mice (i.e., mice not treated with oxaliplatin): ###p \ 0.001, and vs. pre-drug latencies at the respective time-point: *p \ 0.05, **p \ 0.01, ***p \ 0.001 Fig. 5 Antiallodynic activity of cebranopadol and morphine in oxaliplatin-induced neuropathic pain model. Cold allodynia was measured using the cold plate test in the early phase (3 h after oxaliplatin administration) and in the late phase (7 days after oxaliplatin administration). Results are shown as latency time to pain reaction (±SEM) in response to thermal stimulation (temperature of 2 C). Statistical analysis: one-way analysis of variance, followed by Tukey’s post hoc test. Significance vs. latencies of vehicle-treated mice (i.e., mice not treated with oxaliplatin): ###p \ 0.001, and vs. pre-drug latencies at the respective time-point: *p \ 0.05, **p \ 0.01, ***p \ 0.001 p \ 0.0001). Significantly decreased latency time to pain reaction (p \ 0.001 vs. values before oxaliplatin adminis- tration) was observed 3 h after oxaliplatin injection. This indicated for the development of cold allodynia in oxali- platin-treated, neuropathic mice (Fig. 6a). Cold allodynia was maintained even for 7 days (significant at p \ 0.05 vs. values before oxaliplatin administration). revealed that there were significant (p \ 0.001) differences between the latencies of naı¨ve mice and those of mice treated with a single dose of simvastatin. The latencies of the latter group were significantly reduced as compared to naı¨ve mice. Similar effects were observed in mice repeat- edly treated with simvastatin (p \ 0.01 vs. naı¨ve mice). No significant differences were noted when the latencies of mice treated with single-dose and repeated-dose simvas- tatin were compared (F[5,46] = 0.3205, p [ 0.05). On day 1 in neuropathic (oxaliplatin-treated) mice, simvastatin administration (black bars—Figs. 6b–d: sim ? oxa) did not influence cold allodynia significantly, although we observed a slight prolongation of latency time The administration of oxaliplatin significantly reduced cold sensitivity threshold in mice (F[8,31] = 11.48, 123 12 3 K. Sałat et al. Influence on motor coordination (rotarod test) 6 Influence of oxaliplatin (10 mg/kg, i.p.; OXA), simvastatin (100 mg/kg, oral route; SIM) alone, or in combination with subcu- taneous cebranopadol (10 mg/kg, s.c.; CEB) on cold sensitivity threshold and cold allodynia measured in the cold plate test in oxaliplatin-induced neuropathic pain model. Effect of oxaliplatin on the development of cold allodynia (a). Effect of 7-day administration of oral simvastatin alone (OXA ? SIM), or in combination with single-dose cebranopadol added on day 7: simultaneously (b OXA ? SIM ? CEB), 4 h (c OXA ? SIM ? CEB (4 h)), or 6 h (d OXA ? SIM ? CEB (6 h)) after simvastatin. Results are shown as latency time to pain reaction (±SEM) in response to thermal (cold) stimulation (2 C). In this test, the data were collected at two time-points: on day 1 to measure the latencies to pain reaction during the acute-phase cold allodynia that developed after oxaliplatin injection, and on day 7 to measure the latencies to pain reaction during the late-phase cold allodynia. Day 1: grey bars indicate baseline latencies of oxaliplatin-treated mice measured before drug treatment (i.e., before vehicle or simvastatin administration; defined as ‘pre-drug’) and black bars depict latencies after drug administra- tion (i.e., after vehicle or simvastatin administration; referred to as ‘post-drug’ values). Day 7: grey bars indicate baseline latencies of oxaliplatin-treated mice measured before drug treatment (i.e., before vehicle, simvastatin or cebranopadol administration; defined as ‘pre- drug’) and black bars depict latencies after drug administration (i.e., after vehicle, simvastatin only, or simvastatin and cebranopadol administration; referred to as ‘post-drug’ values). A detailed protocol used is also shown in Fig. 1. Statistical analysis: one-way analysis of variance, followed by Tukey’s post hoc test. Significance vs. latency to pain reaction before oxaliplatin injection: ## p \ 0.01, ### p \ 0.001. 12 3 Discussion In this study, we implemented mouse models of acute, tonic, and chronic pain to assess antinociceptive properties of cebranopadol, a dually acting nociceptin/orphanin FQ and opioid receptor agonist. Antinociceptive efficacy of cebranopadol was compared to that of morphine used at an equal dose. A summary of results obtained for both drugs in various mouse pain tests is presented in Table 1. In line with these findings, in our present study, both cebranopadol and morphine attenuated chemogenic, inflammatory acute pain responses induced by acetic acid. Since the writhing test is regarded a rodent model of pain mediated by peripheral mechanisms related to inflamma- tion (Vogel and Vogel 1997), it seems plausible that peripheral NOP and MOP receptors might play a role in the observed activity of both drugs. The involvement of NOP receptors in the pathophysiology of inflammation, arterial hypertension, and cardiac or brain circulatory ischemia has been reported previously (reviewed in Schro¨der et al. 2014). In the rat model of carrageenan-induced inflam- mation, i.t. N/OFQ inhibited thermal hyperalgesia (Yamamoto et al. 1997b; Hao et al. 1998), and NOP receptors and their endogenous ligand—N/OFQ modulated neurogenic inflammation and other functions, such as air- way tone and caliber (Singh et al. 2016). In the hot plate test, both cebranopadol and morphine demonstrated strong and statistically significant antinoci- ceptive properties. Of note, the activity of cebranopadol was delayed as compared to that previously shown for morphine (90–120 min vs. 30–60 min for cebranopadol and morphine, respectively) (Gades et al. 2000; Sałat et al. 2012). The hot plate assay is a rodent model of acute pain. The paws of mice are very sensitive to heat at temperatures that are not harmful to the skin. The characteristic responses such as jumping, licking of the paws are of central origin and it is thought that drugs with antinoci- ceptive properties in the hot plate test act primarily in the spinal medulla and/or higher central nervous system levels (Vogel and Vogel 1997). In this assay, peripherally acting analgesics are generally not active (Vogel and Vogel 1997). Thus, the results obtained in the present study confirmed the role of central opioidergic system in medi- ating analgesia caused by cebranopadol (and morphine). The influence of cebranopadol and morphine on peripherally expressed opioid receptors might also explain their activity observed in the capsaicin test. This pain test reflects acute inflammatory pain responses related to neu- rogenic inflammation. 123 23 Evaluation of cebranopadol, a dually acting nociceptin/orphanin FQ and opioid receptor… 369 (King et al. 1997). Moreover, spinal N/OFQ potentiated analgesia caused by systemic morphine (Tian et al. 1997), while a selective non-peptide NOP receptor agonist, SCH- 221510, showed anti-inflammatory and analgesic proper- ties in a mouse model of TNBS-induced inflammatory bowel disease after systemic administration (Sobczak et al. 2013, 2014). Discussion Capsaicin is an exogenous activator of the TRPV1 channels present in sensory neurons, mainly in C-fibers and, to a lesser extent, Ad. It shows a biphasic effect, i.e., it stimulates TRPV1 located in sensory neurons, resulting in a rapid phase of neurogenic pain with a burning sensation, local vascular and extravascular responses, after which persistent desensitization with concomitant long lasting analgesia appears (reviewed in Sałat et al. 2013b; Marwaha et al. 2016). Previously, it was shown that NOP receptor activation abolished capsaicin-induced contraction of guinea pig airways (Shah et al. 1998; Corboz et al. 2000), reduced capsaicin-induced bronchoconstriction, and increased airway hyper-responsiveness in ovalbumin- Available literature data indicate that functional NOP and MOP receptors are expressed not only at spinal and supraspinal sites of the ascending and descending pain pathways but also in the periphery. NOP receptors and their ligand—N/OFQ have been found in many peripheral organs (e.g., airways and cardiovascular system) and in the immune system in rodents and humans (Schro¨der et al. 2014). In a rat model of inflammatory pain (i.e., trini- trobenzene sulfonic acid (TNBS)-induced colonic hyperalgesia), N/OFQ demonstrated antihypersensitive effects after peripheral administration and it was antinociceptive in the capsaicin test in mice (Sakurada et al. 2005). N/OFQ exerted analgesic properties in the tail- flick test in rats (Xu et al. 1996; Tian et al. 1997) and mice Table 1 Comparison of antinociceptive properties of cebranopadol and morphine in mouse models of pain Pain test Efficacy: cebranopadol (CEB) vs. morphine (MOR) Figure Hot plate test (acute thermal pain) CEB = MORa Figure 2 Writhing test (acute inflammatory pain) CEB = MOR Figure 3a Capsaicin test (acute neurogenic pain) CEB = MOR Figure 3b Formalin test (tonic—neurogenic and inflammatory pain) CEB \ MOR Figure 4a,b Cold plate test (oxaliplatin-induced neuropathic pain) CEB C MOR Figure 5 aData from Gades et al. 2000; Sałat et al. 2012 Table 1 Comparison of antinociceptive properties of cebranopadol and morphine in mouse models of pain 370 K. Sałat et al. sensitized mice (D’Agostino et al. 2010). Of note, in the peripheral nervous system, N/OFQ inhibited neurotrans- mitter release (Giuliani et al. 2000) and inhibited substance P-mediated nociception in mice (Inoue et al. 1999). Con- sidering an important role of substance P in neurogenic inflammation caused by capsaicin, the antinociceptive activity of cebranopadol in the capsaicin test can be explained in terms of its influence on neurogenic inflammation. responses in mice. Discussion In addition, some non- peptide NOP receptor agonists (SR14150 and SR16835) displayed NOP receptor-dependent antiallodynic activity in SNL model in mice (Khroyan et al. 2011), whereas in the mouse CCI model, GRT-TA2210 and Ro65-6570 demon- strated strong antiallodynic effects after spinal, supraspinal, and systemic administration (Linz et al. 2013). Strong anti-inflammatory activity of cebranopadol, as well as the previously reported role of MOP, NOP recep- tors and N/OFQ in the attenuation of inflammation led us to investigate, if cebranopadol might potentiate/modulate the antiallodynic activity of other drugs with anti-inflammatory properties in chronic pain conditions. For this purpose, we chose the oxaliplatin neuropathic pain model and we used simvastatin as a potential novel treatment option for this pharmacoresistant pain type. Among numerous pathome- chanism inflammation has been discovered as one of the key factors underlying neurotoxicity of oxaliplatin (Mas- sicot et al. 2013; Waseem and Parvez 2016). Anti- inflammatory and analgesic properties of simvastatin in neuropathic pain conditions have been widely described in the literature (Miranda et al. 2011; Jaiswal and Sontakke 2012). Moreover, it has been shown that statins have pro- tective effect on ultrastructural alterations induced by cold stress in rats (Bombig et al. 2003). They are also able to attenuate mitochondrial injury induced by cold exposure, inhibit the elevation of blood pressure in cold-treated mice via the downregulation of Bcl-2 pathway (Liang et al. 2017), and they are effective in the attenuation of Ray- naud’s phenomenon (Baumha¨kel and Bo¨hm 2010). This activity of simvastatin is complex and it involves various pleiotropic effects, such as modulation of endothelial functions and Rho-kinase inhibition, which, in turn, affects TRPM8 activity (Sun et al. 2014). Taken together, these g In animals treated with oxaliplatin, the pain threshold for cold nociception is significantly lower as compared to non-treated mice. This was confirmed in our study in the cold plate test and indicated for the development of cold allodynia in oxaliplatin-treated mice. Recently, it has been shown that tactile allodynia and cold allodynia in rodents treated with oxaliplatin are mediated by TRPA1 stimula- tion (Nassini et al. 2011; Zhao et al. 2012; Sałat et al. 2013b; Marwaha et al. 2016) and a single dose of oxali- platin induces acute cold hypersensitivity associated with an enhanced responsiveness of TRPA1 channels (Zhao et al. 2012). Discussion To the best of our knowledge, this drug has not been evaluated in these pathological conditions previously and our present study confirmed its potential utility for patients suffering from oxaliplatin-induced neu- ropathic pain. This is a potentially interesting finding as classical opioid drugs have limited efficacy in neuropathic pain conditions and they are regarded the third-line (mor- phine) or the second-line (tramadol) treatment for neuropathic pain (Finnerup et al. 2015), which is in part due to a diversity of mechanisms underlying the develop- ment of neuropathic pain (Torrance et al. 2013). In the formalin test—a tonic pain model, cebranopadol was slightly less active than morphine. This was observed both in the acute (neurogenic) phase, and in the late (in- flammatory) phase of this test. Intraplantar formalin induces chemogenic pain that results from neurogenic inflammation, sensory C-fibers activation, as well as sen- sitization within the spinal cord dorsal horn and the brain (Hunskaar and Hole 1987; Tjølsen et al. 1992; Yashpal and Coderre 1998), but there is also evidence that formalin is a potent stimulator of TRPA1 channels (McNamara et al. 2007; Nassini et al. 2015; Sałat and Filipek 2015). Our present study shows that NOP/MOP receptor activation might also mediate analgesia in the formalin test. It was previously demonstrated that in the formalin test, N/OFQ was antinociceptive after intrathecal administration, whereas it exerted pronociceptive effects and antagonized opioid analgesia when administered intracerebroventricu- larly (Erb et al. 1997; Yamamoto et al. 1997a; Zhu et al. 1997; Hao and Ogawa 1998; Wang et al. 1999). This bidirectional and site-specific modulation of nociception was also confirmed in the mouse formalin test in which UFP-101, a peptide antagonist selective for NOP receptors, exerted antinociceptive and pronociceptive effects after intracerebroventricular and intrathecal administration, respectively (Rizzi et al. 2006). Antihyperalgesic activity in the formalin test was also observed after systemic administration of selective, non-peptide NOP receptor agonist GRT-TA2210 (Linz et al. 2013). The expression of NOP receptors is up-regulated in chronic (neuropathic and inflammatory) pain conditions (Briscini et al. 2002; Chen and Sommer 2006; Schro¨der et al. 2014) and N/OFQ showed antihypersensitive effects in various rodent models of neuropathic pain, including rat CCI model (Yamamoto et al. 1997b; Corradini et al. 2001; Courteix et al. 2004), spinal nerve ligation (SNL) model (Ju et al. 2013), as well as the mouse diabetic neuropathic pain model (Kamei et al. 1999). References Argoff C (2011) Mechanisms of pain transmission and pharmacologic management. Curr Med Res Opin 27(10):2019–2031. doi:10. 1185/03007995.2011.614934 Baumha¨kel M, Bo¨hm M (2010) Recent achievements in the management of Raynaud’s phenomenon. Vasc Health Risk Manag 6:207–214 Bhalla S, Singh N, Jaggi AS (2015) Dose-related neuropathic and anti-neuropathic effects of simvastatin in vincristine-induced neuropathic pain in rats. 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Chen Y, Sommer C (2006) Nociceptin and its receptor in rat dorsal root ganglion neurons in neuropathic and inflammatory pain models: implications on pain processing. J Peripher Nerv Syst 11:232–240. doi:10.1111/j.1529-8027.2006.0093.x Chen XY, Li K, Light AR, Fu KY (2013) Simvastatin attenuates formalin-induced nociceptive behaviors by inhibiting microglial RhoA and p38 MAPK activation. J Pain 14:1310–1319. doi:10. 1016/j.jpain.2013.05.011 To conclude, in this study using mouse models of acute, tonic, and chronic pain, we demonstrated antinociceptive and antiallodynic properties of cebranopadol—a novel, first-in- class agonist at nociceptin/orphanin FQ and opioid receptors. Of note, the antiallodynic activity of cebranopadol in neuro- pathic pain related to CIPN was shown for the first time, indicating a potential novel treatment option for this type of chronic pain. Apart from this, the results from our present study suggest that NOP receptors might be an important drug target for analgesics used not only in neuropathic pain con- ditions but also in inflammatory pain. This is of particular relevance in terms of pharmacoresistance of these types of chronic pain to currently available analgesic drugs. Corboz MR, Rivelli MA, Egan RW et al (2000) Nociceptin inhibits capsaicin-induced bronchoconstriction in isolated guinea pig lung. Eur J Pharmacol 402:171–179. Compliance with ethical standards data clearly suggest that statins might modulate body functioning of cold-exposed subjects. Conflict of interest None declared. Conflict of interest None declared. In the cold plate test in oxaliplatin-treated mice com- pared to non-treated controls, a significant reduction of latency time to pain reaction was observed both 3 h and 7 days after its administration. This indicated the devel- opment of cold allodynia in oxaliplatin-treated mice. Cold allodynia in neuropathic mice was not influenced by a single dose of simvastatin administered alone (see results for the early phase), but in contrast to this, a 7-day treat- ment with this drug partially reversed allodynia induced by cold and the difference between latencies of non-neuro- pathic mice and oxaliplatin ? repeated simvastatin-treated mice was no longer significant. Thus, it might indicate that the repeated administration of simvastatin elevated pain threshold of animals with CIPN. The addition of cebra- nopadol to simvastatin did not demonstrate additional benefits. Of note, a simultaneous treatment with both drugs reduced latency time to pain reaction. Numerous studies have shown that the hot/cold plate test latencies might decrease with a repeated testing and this decrease may involve learning abilities, differences in animals’ weight, habituation time, and other unknown factors (Czopek et al. 2016). Of note, cebranopadol added to oxaliplatin ? re- peated simvastatin-treated mice 4 h or 6 h after the last dose of simvastatin (day 7) did not reduce the latency time to pain reaction and a tendency towards the prolongation of this parameter was noted. This is an interesting finding which requires further studies, but it may potentially sug- gest some pharmacodynamic interaction at the common site of action. Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the present study was conducted. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Acknowledgements This study was financially supported by the National Science Centre grant UMO-2015/17/B/NZ7/02937. 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CHALLENGES OF THE FIRST REVOLUTIONARY JACQUERIE IN FRANCE AND RESPONSES OF THE NATIONAL ASSEMBLY
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УДК 944.04.1 УДК 944.04.1 Блуменау С.Ф., доктор исторических наук, профессор, Брянский государствен- ный университет имени академика И.Г. Петровского (Россия) Вестник Брянского государственного университета. 2020 (4) Вестник Брянского государственного университета. 2020 (4) ВЫЗОВЫ ПЕРВОЙ РЕВОЛЮЦИОННОЙ ЖАКЕРИИ ВО ФРАНЦИИ И ОТВЕТЫ НАЦИОНАЛЬНОГО СОБРАНИЯ Взятие Бастилии 14 июля 1789г. вызвало множество восстаний и выступлений по всей Франции, наиболее масштабным из которых явилась первая крестьянская «жакерия» революционной поры. Война с деревенской Францией, жители которой составляли две трети населения всей страны, была бесперспективной для революционных властей. Сама «жакерия» и сопутствовав- шая ей неуплата сеньориальных цензов и оброков, церковной десятины и государственных нало- гов угрожали экономическим коллапсом. Столкнувшись с проблемой такой сложности, Ассам- блея смогла найти выход. Она решилась на реформы, отменившие часть сеньориальных прав и церковную десятину. Одновременно революционеры упразднили сословный строй и обеспе- чили гражданское равенство. В длительной исторической перспективе это стало впечатляющим началом формирования во Франции либеральной демократии. Ключевые слова: Учредительное собрание, крестьянское восстание, сеньориальный режим, церковная десятина, сословный строй. DOI: 10.22281/2413-9912-2020-04-04-20-30 Взятие Бастилии дало толчок для восстаний и выступлений по всей Франции. В городах и бургах развер- нулась «муниципальная революция», приведшая к установлению на местах новой власти. Массовой базой движе- ния являлся городской мелкий люд. Его выступления мотивировались двумя началами: революционными настроениями и голодом. То были бес- корыстные и политизированные сан- кюлоты- революционеры, боровшиеся против реакции, угрожавшие умерен- ным, поддерживавшие реформатор- ское большинство Собрания. ликвидации прерогатив сеньора, выте- кавших из сословного неравноправия и личной зависимости держателей. Остальные тяготы селяне соглашались выкупить, следуя правовым идеям вре- мени, выводившим их из первоначаль- ной уступки земли господами. Избира- тельная кампания и первые шаги Ас- самблеи породили у крестьян «вели- кую надежду». Но за несколько меся- цев дело не сдвинулось с мертвой точки. Между тем, события середины июля свидетельствовали об «обруше- нии всех сил общественного порядка» (16, р.9). Образовавшийся вакуум вла- сти не предполагал, казалось, суровых карательных мер за преступления про- тив личности и собственности. Это подтолкнуло крестьян к масштабному восстанию, получившему в литературе название «жакерии», напоминавшее о грозных событиях XIV в. В ходе вос- стания, продолжившегося с 20-х чисел июля до конца первой декады августа, выступавшие уверяли самих себя и В отличие от них крестьян забо- тило не торжество революции, а насущные материальные интересы (1, с.95). К концу Старого порядка фран- цузская деревня осознала несправед- ливость сеньориального режима, а в наказах Генеральным штатам изло- жила конкретные претензии. В «жало- бах» ставился вопрос о безвозмездной 20 Исторические науки и археология в основном территорию восточной Франции. Причин тому было немало. В тамошних провинциях сохранялись пе- режитки крепостничества. Во Франш- Конте и Бургундии встречалось право «мертвой руки» (мэнморта), ограничи- вавшее возможности передачи кре- стьянского участка и имуществ по наследству (2, с.142; 4,с.56). В Эльзасе продолжали требовать сеньориальную барщину. ВЫЗОВЫ ПЕРВОЙ РЕВОЛЮЦИОННОЙ ЖАКЕРИИ ВО ФРАНЦИИ И ОТВЕТЫ НАЦИОНАЛЬНОГО СОБРАНИЯ В отдельных областях повин- ности забирали до четверти урожая держателей. «Сеньориальная реакция» приводила к более строгому взиманию натуральных оброков и денежных пла- тежей, к захватам общинных угодий, к покушениям на коллективные права пользования. других в расположенности к их устремлениям Собрания и короля. Во- жаки фабриковали ложные декреты и воззвания правящих кругов, якобы одобрявших «экспедиции» деревень против сеньорий. Наряду с «великой надеждой» в душах земледельцев поселился и «Ве- ликий страх», вызванный к жизни тре- вожной обстановкой и подпитываемый исторической памятью о разбойниках прошлого. Тысячи нищих, бродивших по дорогам и промышлявших воров- ством, готовых собрать даже незрелое зерно, пугали их. За спинами голодных людей усматривали заговор привилеги- рованных, а начавшаяся эмиграция по- рождала слухи о «походе» против Франции брата короля графа В большинстве провинций во- стока действовало правило: «нет сень- ора без законного титула» и, чтобы из- бавиться от повинностей, крестьяне пытались уничтожить документ, куда были занесены их обязательства. Но для этого приходилось применять насилие или хотя бы угрозы, что вело к жесткому варианту антисеньориаль- ной борьбы с разгромом и сожжением замков. На крайнем же севере, в обла- стях Эно и Камбрези работала иная формула: «нет земли без сеньора» и, пусть там тоже существовал «суровый феодализм» с личными повинностями и барщиной, от господина требовали лишь формального отречения от своих прав. Отсутствие здесь паник «Вели- кого страха» также понижало градус напряженности. В результатедо край- ностей доходило редко. д' Артуа во главе армии преступ- ников, нанятых в разных странах (11, р.157, 187). Паники «Великого страха» усиливали агрессию крестьян в отно- шении своих обидчиков и их иму- ществ. «Жакерия» распадалась на мно- жество разрозненных, но синхронных восстаний, происходивших по схо- жему сценарию. Главной целью вос- ставших было уничтожение описей повинностей - терье, что лишало сень- оров доказательства правомерности их требований. Одновременно при «посе- щениях» домов и замков, церковных учреждений крестьяне настаивали на возврате общинных земель, уменьше- нии или даже упразднении десятин, возмещении трат за проигранные су- дебные процессы. Нередко участники «акций» предавались безобразиям и воровству: ломали мебель и утварь, стремительно опустошали винные по- греба хозяев, присваивали приглянув- шиеся вещи (1, с.114). Другое дело - очаг «жакерии» на западе Франции - в Нормандском бо- каже (области) и Нижнем Мэне. Не- уступчивость усиливалась в регионе «Великим страхом». Нападения на Летняя «жакерия»1789 г. охватила 21 Вестник Брянского государственного университета. 2020 (4) ственному образу и сделался бы похо- жим на своего антипода - деспотизм. замки сочетались с поношением гос- под. Но убийства сеньоров были ред- костью по стране. ВЫЗОВЫ ПЕРВОЙ РЕВОЛЮЦИОННОЙ ЖАКЕРИИ ВО ФРАНЦИИ И ОТВЕТЫ НАЦИОНАЛЬНОГО СОБРАНИЯ Побоям подверга- лись агенты сеньории: управляющие, откупщики повинностей. Насилие против личности не было определяю- щим в действиях восставших; урон се- ньоров носил материальный и мораль- ный характер. замки сочетались с поношением гос- под. Но убийства сеньоров были ред- костью по стране. Побоям подверга- лись агенты сеньории: управляющие, откупщики повинностей. Насилие против личности не было определяю- щим в действиях восставших; урон се- ньоров носил материальный и мораль- ный характер. Главное, что отвращало от жест- кого курса в отношении восставших, - преобладание крестьянства в населе- нии Франции, конфронтация с ним была бесперспективной для революци- онных властей. К тому же деревенские массы являлись естественным союзни- ком последних в борьбе за гражданское равенство. Восстание селян против привилегированных облегчало ликви- дацию сословных границ во француз- ском обществе после их упразднения в Ассамблее. С другой стороны, антисе- ньориальные устремления крестьян встречали понимание, а, порой, и одоб- рение формировавшегося в недрах Конституанты левого большинства. Однако выступления сельской Фран- ции, обстановка анархии и экономиче- ского кризиса в стране привычно скло- няли к политике усмирения. Именно в таком духе началась 3 августа дискус- сия о крестьянских волнениях. От Ко- митета докладов выступил его член Са- ломон. На основе полученных из раз- ных концов Франции писем он нарисо- вал безрадостную картину. Представ- ляется, заявлял докладчик, что «соб- ственность, какой бы она ни была по природе, стала жертвой самого пре- ступного разбоя… Замки - сожжены, монастыри - разрушены, фермы - поки- нуты на расхищение». Сеньориальные платежи и налоги не вносятся, «законы лишены силы, магистраты - власти, правосудие - только призрак, который бесполезно искать в судах» (7, р.336). Вывод выступавшего: не может быть принято никакое объяснение отказа от своих обязательств перед сеньором и государством. Но судебных санкций в Главное, что отвращало от жест- кого курса в отношении восставших, - преобладание крестьянства в населе- нии Франции, конфронтация с ним была бесперспективной для революци- онных властей. К тому же деревенские массы являлись естественным союзни- ком последних в борьбе за гражданское равенство. Восстание селян против привилегированных облегчало ликви- дацию сословных границ во француз- ском обществе после их упразднения в Ассамблее. С другой стороны, антисе- ньориальные устремления крестьян встречали понимание, а, порой, и одоб- рение формировавшегося в недрах Конституанты левого большинства. Однако выступления сельской Фран- ции, обстановка анархии и экономиче- ского кризиса в стране привычно скло- няли к политике усмирения. Именно в таком духе началась 3 августа дискус- сия о крестьянских волнениях. От Ко- митета докладов выступил его член Са- ломон. ВЫЗОВЫ ПЕРВОЙ РЕВОЛЮЦИОННОЙ ЖАКЕРИИ ВО ФРАНЦИИ И ОТВЕТЫ НАЦИОНАЛЬНОГО СОБРАНИЯ На основе полученных из раз- ных концов Франции писем он нарисо- вал безрадостную картину. Представ- ляется, заявлял докладчик, что «соб- ственность, какой бы она ни была по природе, стала жертвой самого пре- ступного разбоя… Замки - сожжены, монастыри - разрушены, фермы - поки- нуты на расхищение». Сеньориальные платежи и налоги не вносятся, «законы лишены силы, магистраты - власти, правосудие - только призрак, который бесполезно искать в судах» (7, р.336). Вывод выступавшего: не может быть принято никакое объяснение отказа от своих обязательств перед сеньором и государством. Но судебных санкций в отношении неплательщиков, а тем бо- лее силовых мер к восставшим он не Реакция Собрания на случивше- еся не была быстрой. Депутаты пыта- лись учесть разные факторы. Новости о восстании приходили с опозданием. Полемика началась лишь 3 августа, что объяснялось и совсем недавним (28 июля) созданием Комитета докла- дов, которому поручили предвари- тельно разобраться с проблемой. Весомые доводы побуждали Ас- самблею высказаться за подавление «жакерии», ведь она была масштаб- ным покушением на право собствен- ности и безопасность личности. Вос- ставшие отказывались также платить налоги, усугубляя без того тяжелейшее финансовое положение страны. Но и силовой вариант требовал определен- ной законодательной базы. Еще 20 июля «умеренный» Лалли-Толлендаль сформулировал текст воззвания, гро- зившего бунтовщикам задержанием и передачей в руки правосудия (7, р.252;15,р.39), однако Собрание огра- ничилось приглашением «всех фран- цузов к миру, к поддержанию порядка и общественного спокойствия» (7,р.267). Репрессии требовали поли- цейской силы, но формирование наци- ональной гвардии не везде заверши- лось, а ее возможности не были апро- бированы. Провозгласив курс на по- давление «жакерии», французский парламент сильно повредил бы соб- 22 Исторические науки и археология предлагал. права несправедливыми, необозначен- ный депутат предположил, что одно только упоминание о них может уси- лить крестьянские волнения. Речь вызвала разные суждения. Представитель третьего сословия Му- жен де Рокфор призвал принять проект Комитета. Некий депутат от дворян по- считал, что к декларации «с приглаше- нием народа к миру», надо добавить еще одну, дабы запустить в действие старые законы. Решительней выска- зался неназванный консерватор. Ссы- лаясь на драматизм экономической си- туации, он потребовал прекратить «войну бедных против богатых» (sic!) и наказывать неплательщиков налогов на основе соответствующих ордонан- сов. А «всех тех, кто покушается на свободу и собственность индивида», должен преследовать королевский прокурор, опирающийся на местные судебные органы (7, р.336-337).Этот суровый план не был принят, а предло- жение Комитета докладов передали в Редакционный комитет для выработки окончательного варианта. На деле обе позиции мало отлича- лись друг от друга. Вестник Брянского государственного университета. 2020 (4) Гневные филиппики в адрес фео- дализма вкупе с осторожными кон- кретными соображениями успокоили и ободрили депутатов, стимулировали отречения от разных прав. Двое из них настояли на упразднении сеньориаль- ной юстиции. Епископ Шартра де Лю- берзак увидел в исключительном праве охоты «бич для разоренных де- ревень», потребовал его отмены и сам подал пример. Кто-то призвал отка- заться от мэнморта во Франш- Конте, Бургундии и других местах, где он еще существовал. Архиепископ Экса Буа- желен говорил о неприемлемости фео- дальных элементов в договорах сень- ора с крестьянином. Но бедность за- ставляла последнего соглашаться с по- добными пунктами. Следовало зара- нее аннулировать условия в докумен- тах, способные привести к возрожде- нию феодализма (7, р.346). соответствовали пожеланиям сеньоров и должны были в 30 раз превышать сумму ежегодных доходов от повинно- стей. Лично инициаторы всего этого мало что теряли. Ноай именовался в шутку Жаном Безземельным, то есть взять у него было нечего. А д'Эгийон, напротив, один из богатейших дворян Собрания (17, р.36) составил себе со- стояние, в основном, за счет владений в Сан- Доминго. Оба депутата, как и Тарже, высту- пили на вечернем заседании 4 августа. Можно ли считать их речи неким оза- рением, передавшим особую энерге- тику остальным законодателям? По- хоже, что события, оказались, отчасти, спланированными. Накануне вечером Бретонский клуб, предварявший у себя тематику грядущих заседаний Ассам- блеи, занялся обсуждением вопроса. Алгоритм будущих действий облег- чался тем, что бретонский лидер Ле Шапелье стал с 3 августа председате- лем Учредительного собрания. Воз- можно, в Клубе окончательно созрела- мысль о выступлении депутата от тре- тьего сословия из Нижней Бретани, скромного торговца полотном и вином Легана де Керангаля. Несколько депутатов- священни- ков обратились к Собранию с прось- бой разрешить им пожертвовать сво- ими случайными правами. В ответ представитель дворянства предложил дать им компенсацию в виде увеличе- ния «скромной доли», то есть жалова- нья кюре. Епископ Нанси Ла Фар хотел распространить практику выкупа по- винностей у светских сеньоров на цер- ковную десятину. То был ловкий ма- невр с целью спасти богатства церкви. В свете новых юридических представ- лений положение сеньориальной и церковной собственности не являлось одинаковым. Первая ассимилирова- лась с индивидуальной частной соб- ственностью, а основные повинности и платежи цензитариев приравнива- лись к обычной арендной плате. Вто- рая была корпоративной, а корпорации рассматривались революционерами Отталкиваясь от Декларации прав человека и гражданина, над проектами которой уже работали законодатели, оратор противопоставлял ей сеньори- альный режим, заставлявший человека впрягаться в повозку, словно рабочий скот. ВЫЗОВЫ ПЕРВОЙ РЕВОЛЮЦИОННОЙ ЖАКЕРИИ ВО ФРАНЦИИ И ОТВЕТЫ НАЦИОНАЛЬНОГО СОБРАНИЯ Декларация Коми- тетов вызвала бы дальнейшее раздра- жение и, возможно, распространение «жакерии» на другие регионы Фран- ции. Платформа несогласных, пусть и сочувствовавшая селянам, не предла- гала им реального облегчения. Ситуа- ция, казалось, зашла в тупик. Новый парламент впервые встретился с соци- альной проблемой такого масштаба. Простые решения здесь не подходили, а большинство законодателей - люди городские - не были искушены в аграр- ной сфере. Многим дворянам эти во- просы давались легче, а зарево горя- щих замков заставляло мысль работать быстрей и продуктивней. Реформиро- вание сеньориальных отношений не могло исходить от ретроградов и оно стало инициативой либеральной знати. От его имени подготовленный текст зачитал выдающийся юрист Г.- Ж.- Б. Тарже. В документе подчеркива- лось, что платежи сеньорам должны вноситься, как прежде, пока Ассамблея не внесет изменений. То же касалось и налогов, которые могут лишь в буду- щем стать другими – менее обремени- тельными. Но в проекте ничего не гово- рилось, ни о применении силы против крестьянских выступлений, ни о судеб- ном преследовании восставших, непла- тельщиков и должников (7, р.343). Многие отнеслись к предложениям Ко- митетов настороженно и даже враж- дебно. Одни считали письма с мест ма- лодоказательным источником, другие сетовали на несоблюдение регламента, третьи оспаривали содержание плани- руемого постановления и саму его необходимость. Назвав феодальные Два ее представителя - молодые виконт де Ноай и герцог д' Эгийон разо- брались в причинах «жакерии» и отыс- кали путь восстановления обществен- ного спокойствия. Виконт предложил план из 4 пунктов. Первые два – преду- сматривали равенство граждан в фис- кальной сфере и уплату налогов про- порционально доходам, третий пункт касался выкупа сеньориальных прав, а последний - отмену тех из них, что вы- текали из личной зависимости кре- стьян. Политичная речь д Эгийона пре- увеличивала жертвы дворянства: «Они уже отказались от своих привилегий и налоговых изъятий; ясно, что… нельзя от них требовать безоговорочного от- каза от… феодальных прав» (3.Т.2, с.11). Герцог поставил вопрос о необхо- димой компенсации. Размеры выкупа 23 Вестник Брянского государственного университета. 2020 (4) Вестник Брянского государственного университета. 2020 (4) Солидаризуясь с восставшими, он просил парламентариев устроить «ис- купительный костер» из феодальных грамот. Но смелые призывы сочетались у него с умеренными планами выкупа повинностей и баналитетов по выгод- ной для сеньоров цене, схожей с пред- ложенной Эгийоном (7, р.345). 24 Исторические науки и археология ных наказах (6, с.45-48). Партикуля- ризму провинций противилась центра- лизаторская тенденция, подпитанная идеей Нации, распространенной в предвыборной публицистике 1789г. (5, с.62-63;18). Превращение Генераль- ных штатов в единое Национальное собрание усилило эту линию. негативно как учреждения феодаль- ного прошлого, обреченные на исчез- новение вместе со своей собственно- стью. Церковные владения имели осо- бое предназначение; доходы с них сле- довало во многом направлять на под- держку неимущих и малоимущих. Это тоже учел Ла Фар, заметивший, что из предполагаемого выкупа «могла быть извлечена польза и для бедных» (3.Т.2, с.12). негативно как учреждения феодаль- ного прошлого, обреченные на исчез- новение вместе со своей собственно- стью. Церковные владения имели осо- бое предназначение; доходы с них сле- довало во многом направлять на под- держку неимущих и малоимущих. Это тоже учел Ла Фар, заметивший, что из предполагаемого выкупа «могла быть извлечена польза и для бедных» (3.Т.2, с.12). В обстановке подъема законода- тели решились на отказ от местных привилегий ради укрепления Фран- ции, а заодно и собственного имиджа. Отречение от особых прав первыми провозгласили депутаты от Дофине, а их почин быстро подхватили бретонцы во главе с Ле Шапелье. Тот объявил, что наставший после периода ожида- ний и надежд «день счастья и безопас- ности, позволил растворить древние права и мечты Бретани в еще более прочных и священных правах, кото- рые… Ассамблея обеспечивает в дан- ный момент всей французской импе- рии».Эстафета «жертв» перешла к Провансу, затем к Бургундии и Ланге- доку (7, р.347). К концу обсуждения, перешедшего за полночь, в зале насту- пила эйфория. Парламентарии обни- мались и плакали от счастья. Яркое и плодотворное заседание, сплотившее, пусть и на короткое время, едва ли не весь депутатский корпус, получило название «ночи чудес». «Жакерия» сфокусировала внима- ние депутатов на сеньориальных по- винностях и десятине. Но изначально третье сословие и революционные круги стремились покончить с сослов- ными привилегиями (9,р.131). Сломав перегородки между духовенством, дво- рянством и разночинцами в самой Ас- самблее, следовало логикой вещей пе- ренести новый порядок на все француз- ское общество. Утверждение граждан- ского равенства было тем более необхо- димо, что улучшило бы самочувствие крестьянской массы и содействовало бы прекращению волнений и анархии в стране. Вестник Брянского государственного университета. 2020 (4) В ходе заседания виконт Бо- гарне потребовал равенства наказаний «для граждан всех классов и их доступа ко всем церковным, гражданским и во- енным должностям» (7, р.346). Расставание с привилегиями про- должили различные территориальные единицы страны. Определенными пре- имуществами, во многом фискаль- ными, пользовались недавно, по исто- рическим меркам, присоединенные к Франции провинции. То были области со штатами, сохранившие особый ста- тус. В момент предреволюционного кризиса, пользуясь ослаблением госу- дарственной власти, они даже попыта- лись увеличить свою самостоятель- ность, что нашло отражение в отдель- На следующий день наступило не- которое «отрезвление», а отдельные за- конодатели даже заговорили об «ограб- лении». Другие оказались более прозор- ливыми. Крайне правый маркиз де Фе- рьер объяснял в письме своим избирате- лям, что в момент мятежа «было бы бес- полезно и даже опасно противиться все- общей воле нации» (3.т.2, с.171-172). Предложения «ночи чудес» систе- матизировали и зачитали на заседании 5 августа. В статьях с 3-ей по 6-ую 25 Вестник Брянского государственного университета. 2020 (4) Вестник Брянского государственного университета. 2020 (4) соответствующей статьи декрета и обо- злили оппонентов обструкционистской позицией по поводу решений «ночи чу- дес». Уже 6 августа один депутат – кюре стал грозить законодателям коро- левской санкцией, другой - квалифици- ровал «жертвы», принесенные от имени сословий, недействительными, третий - призвал советоваться с избира- телями (7, р.353-354). Выведенный из себя, представитель формировавшейся в Собрании «крайне левой» (13, р.134- 135) Бюзо попенял духовенству за не- способность даже изобразить готов- ность к уступкам и заявил, что «церков- ные имущества принадлежат нации» (3.т.1, с.376). уничтожались исключительные права сеньоров на содержание голубятен и кроличьих садков, охоту и рыбную ловлю, а также упразднялась сеньори- альная юстиция. Особое значение имели две первые статьи. В соответ- ствии с одной - безвозмездно отменя- лись мэнморты, барщины и другие, связанные с пережитками феодализма права. По другой - предусматривалась возможность выкупа денежных цензов и натуральных оброков. Но перечень выкупаемого открывали баналитеты, то есть монополии сеньоров на владе- ние мельницами, печами, виноград- ными прессами, бойнями, кузницами. Однако эти привилегии подчеркивали неравноправное положение крестьян, а значит, по юридической логике того времени, должны были уничтожаться без компенсации, что не планирова- лось. Одна статья касалась десятин: все они обращались в денежную форму и могли выкупаться. ( , ) Второй раз эта формула возникла в связи с обсуждением вопроса о 30-ти миллионном займе, предложенном Неккером (8, р.341). 8 августа в Собра- нии выступили маркиз Лакост и шева- лье А.Ламет. Первый сетовал на огромный долг и обремененность народа налогами. Вестник Брянского государственного университета. 2020 (4) Выход из положения намечался за счет десятины и церков- ных земель. «Пусть все имущества, называемые церковными, какого бы характера они ни были, принадлежат нации». А, начиная с 1790г., отменя- лись десятины. Ламет поддержал мне- ние коллеги: у индивидов есть священ- ные права, независимые от общества, а корпорации существуют только благо- даря ему. Оно может упразднить эти институты, а их средства направить на общее благо. Депутат потребовал ис- пользовать церковные имущества в ка- честве залога для кредиторов государ- ства (7, р.370-371). Декрет начинался с преамбулы из трех пунктов, формулировавших за- дачи перемен. В первом говорилось, что в свободном государстве собствен- ность свободна так же, как и люди. Она избавлялась от феодальных черт, а се- ньориальный режим был поколеблен. Во втором пункте подчеркивалось ра- венство прав и обязанностей францу- зов. В третьем - прокламировался от- каз различных территорий от прежних преимуществ, что являлось важным шагом в деле единения Нации (7,р.352). По решению депутатов дальше допускалась правка только формы проекта, но не его содержания. Наиболее острая полемика развер- нулась из- за церковной десятины. Здесь духовные лица допустили «фаль- старт»: выступили еще до обсуждения Только 10 августа законодатели добрались до поправок в статью о де- сятинах. Против отмены десятины первоначально выступили почти все ораторы от первого сословия, причем Наиболее острая полемика развер- нулась из- за церковной десятины. Здесь духовные лица допустили «фаль- старт»: выступили еще до обсуждения 26 Исторические науки и археология Действительно, для крупных зе- мельных собственников это станови- лось значимым приращением доходов. Другие землевладельцы - миллионы крестьян - были обременены и сеньо- риальными тяготами, и государствен- ными налогами, и церковной десяти- ной. При этом в развитых областях се- вера, в Парижском районе сеньориаль- ные повинности отнимали у крестьян немного. Так, в Нормандии, в областях Брэ и Ко они составляли соответ- ственно 3% чистого продукта (10,р.357) и менее 2% урожая (брутто) (12,р.52), тогда как десятина обходи- лась селянам в 7-8% их доходов. Это значило немало, и отмена десятины могла удержать многие деревни от присоединения к восставшим провин- циям. прежние различия между низшим ду- ховенством и прелатами стерлись. Кюре Ле Франсуа и епископ Перпинь- яна утверждали, что отмена лишит церковь возможности помочь бедным - «классу несчастных». Епископ Лангра Ла Люзерн назвал десятину священ- ной собственностью, которой не мо- жет обладать Нация, ибо имеет не ма- териальную, а моральную сущность. Между тем оппоненты клира вносили в декрет принципиальные правки. Вестник Брянского государственного университета. 2020 (4) Де- путат де Вийе обратил внимание на разницу между церковными и свет- скими десятинами, которые были соб- ственностью, передававшейся в се- мьях из поколения в поколение. Он просил упразднить первые, а вторые - инфеодизированные, «считающиеся поземельными рентами, сделать выку- паемыми» (7,р.382-383,385). На следующий день характер пре- ний изменился, он диктовался свет- скими лицами. Гупиль де Префельн представил дело так, будто депутаты от клира, как и их оппоненты, желали от- мены десятины, но посредством вы- купа, оказавшегося, увы, дороже ее са- мой. Суровый Рикар все же обвинил де- ятелей духовенства в лицемерии: они с горячностью осуждали сеньориальные права, а от себя ограничились предло- жением выкупа десятины, обремени- тельным для Нации, но способным принести им рост благосостояния. В ходе речи оратор получил документы от священнослужителей уже с безвоз- мездным отказом от данного источника их доходов. Появилась и декларация, подписанная прелатами и кюре о пере- даче десятины в руки Нации. Затем от имени церкви выступил архиепископ Парижский, поддержанный кардина- лом Ларошфуко. Атмосфера в зале, ка- залось, напоминала ту, что была в Ас- самблее в «ночь чудес» Но заседание Тогда же о десятине с противопо- ложных позиций высказались два по- литических тяжеловеса: граф Мирабо и аббат Сийес. Первый справедливо от- метил, что собственник тот, кто распо- ряжается не только доходами, но и ка- питалом. Получатель десятины тако- вым не является, а сама она выступает как оплата за поддержание морали и развитие образования. Надо и дальше оплачивать данный полезный труд, но делать это «менее расточительным и более справедливым способом». Сийес, напротив, возмутился упраздне- нием десятины без возмещения. Ведь она останется в руках того, кто должен ее платить - должника. Аббат не только продемонстрировал абсурдность ситу- ации, но и обвинил крупных землевла- дельцев, включая часть депутатов, в по- лучении выгоды: «Вы рассчитываете увеличить с ее (десятины - С.Б.) помо- щью ваше личное состояние» (3.Т.1, с.380-381). 27 Вестник Брянского государственного университета. 2020 (4) рией, прибегая, порой, к помощи регу- лярных войск, они наносили бунтарям серьезный военный урон, а захвачен- ных подвергали суровым наказаниям: смертной казни через повешение, от- правке на галеры и в тюрьму (1,с.122;14,р.105). по поводу десятины носило не искрен- ний, а постановочный характер. Пред- ставители духовенства осознали тщет- ность сопротивления левому большин- ству Собрания и опасались оконча- тельно уронить себя в глазах обще- ственности. Поправленный текст декрета при- няли в тот же день - 11 августа. Депу- татскому корпусу удалось справиться с непосредственной и срочной задачей: не дать хаосу восторжествовать. С со ер ур 1. Адо А. В. Крестьяне и Великая французская революция. М., 1987. 2. Блуменау С. Ф. Правовые отношения вокруг сеньории во Франции Ста- рого порядка / / Право: история, теория, практика. Выпуск 3. Брянск, 1999. 3. Документы истории Великой французской революции. Т. 1-2. М., 1990- 1992. 4. Саньяк Ф. Гражданское законодательство Французской революции: 1789- 1804. М., 1928. 5. Тырсенко А. В. Эмманюэль Жозеф Сийес и французская либеральная мысль его времени. М., 2005. 6. Шампьон Э. Франция накануне революции по наказам 1789г. СПб., 1906. CHALLENGES OF THE FIRST REVOLUTIONARY JACQUERIE I FRANCE AND RESPONSES OF THE NATIONAL ASSEMBLY Storming of the Bastille on July 14, 1789 caused many uprisings and revolts throughout France, the largest of which was the first peasant "Jacquerie" of the revolutionary period. The war with the rural France whose inhabitants made up two-thirds of the population of the whole country, was hopeless for the revolutionary authorities. The "Jacquerie" itself and the accompanying non-payment of sei- gneurial censuses and taxes, Church tithes and state taxes could lead to the economic collapse. Facing a problem of such complexity, the Assembly was able to find a way out. They ventured to undertake reforms that abolished some of the seigneurial rights and Church tithes. At the same time, the revo- lutionaries abolished the estate system and ensured civil equality. In a long historical perspective, this was an impressive start to the formation of a liberal democracy in France. Keywords: Constituent Assembly, peasant revolt, seigneurial regime, Church tithes, estate system. Вестник Брянского государственного университета. 2020 (4) de Necker ou la faillité de la vertu. P.,1987. 8. Diesbach Ch. de Necker ou la faillité de la vertu. P.,1987. 9. Furet F. Nuit du 4 août // Dictionnaire critique de la Révolution française. P.,1988. 9. Furet F. Nuit du 4 août // Dictionnaire critique de la Révolution française P.,1988. 10. Goujard Ph. L’abolition de la féodalité dans le district de Neufchâtel (Seine- Inférieure) // Contributions à l’ histoire paysanne de la révolution française.P., 1977. 10. Goujard Ph. L’abolition de la féodalité dans le district de Neufchâtel (Seine- Inférieure) // Contributions à l’ histoire paysanne de la révolution française.P., 1977. 11. Lefebvre G. La Grande Peur de 1789. P., 1932. 12. Lemarchand G.La Fin du féodalisme dans le pays de Caux. P., 1989. 12. Lemarchand G.La Fin du féodalisme dans le pays de Caux. P., 1989. 13. Leuwers H. Robespierre. P., 2014. 13. Leuwers H. Robespierre. P., 2014. 14. Méthivier H. La fin de l’Ancien Régime. P., 1986. 15. Robespierre M. Oeuvres. T.6. P., 1950. 16. Tackett T. La Grande Peur et le complot aristocratique sous la Révolution française // Annales historiques de la Révolution française, 2004, N.1( 335). 16. Tackett T. La Grande Peur et le complot aristocratique sous la Révolution française // Annales historiques de la Révolution française, 2004, N.1( 335). q ( ) 17. Tackett T. Par la volonté du peuple: Comment les deputės de 1789 sont devenusrévolutionnaires. P., 1997. 17. Tackett T. Par la volonté du peuple: Comment les deputės de 1789 sont devenusrévolutionnaires. P., 1997. 18. Thouret J.- G. Avis de bons normands à leurs frѐres tous les bons françois de toutes les provinces et de tous les Ordres. P., 1789. 18. Thouret J.- G. Avis de bons normands à leurs frѐres tous les bons françois de toutes les provinces et de tous les Ordres. P., 1789. CHALLENGES OF THE FIRST REVOLUTIONARY JACQUERIE IN FRANCE AND RESPONSES OF THE NATIONAL ASSEMBLY Вестник Брянского государственного университета. 2020 (4) Упразднение мэнморта и личных по- винностей помешали подпитке «жаке- рии» в самом ее эпицентре - в восточ- ной Франции. Отмена церковной деся- тины переломила настроения крестьян и не позволила волнениям переки- нуться на другие регионы страны. Не- смотря на размах движения, оно распа- далось на множество отрядов, пресле- довавших узкую цель - «разобраться» с собственным сеньором: сжечь описи повинностей или заставить господина отречься от своих прав. После этого вы- ступление нередко «выдыхалось». Но «рейды» больших соединений восстав- ших приносили немалый вред. Против них жестко сработали муниципалитеты и руководимые ими национальные гвардейцы. В тесном контакте со ста- рыми королевскими учреждениями - превотальной юстицией и жандарме- Каково же значение декрета 11 ав- густа в длительной перспективе? Исто- рики иногда иронизируют над предло- жением, открывавшим текст знамени- того закона: «Национальное собрание полностью уничтожает феодальный порядок» (7,р.397), напоминая, что се- ньория тогда уцелела. Но под нее осу- ществили подкоп, а указанная выше формула стимулировала перемены, приведшие за 4 года к обрушению се- ньориального режима. Вторым резуль- татом декрета стала стремительно по- следовавшая за безвозмездной отменой секуляризация имуществ первого со- словия, разрушившая материальное бо- гатство и социальное могущество церкви. Третьим сдвигом сделалось упразднение сословной системы и установление гражданского равенства. Отсюда - торжество в будущем либе- рально- демократического строя во Франции, оказавшегося также приме- ром для многих государств. И, наконец, закон способствовал ускоренному сплочению французской нации. 1. Адо А. В. Крестьяне и Великая французская революция. М., . В. Крестьяне и Великая французская революция. М., 1987. 1. Адо А. В. Крестьяне и Великая французская революция. М., 1987. 2. Блуменау С. Ф. Правовые отношения вокруг сеньории во Франции Ста- рого порядка / / Право: история, теория, практика. Выпуск 3. Брянск, 1999. 3 Д В й ф й Т 1 2 М 1990 р р р р , р , р у р , 3. Документы истории Великой французской революции. Т. 1-2. М., 1990- 1992. 4. Саньяк Ф. Гражданское законодательство Французской революции: 1789- 1804. М., 1928. 5. Тырсенко А. В. Эмманюэль Жозеф Сийес и французская либеральная мысль его времени. М., 2005. р 6. Шампьон Э. Франция накануне революции по наказам 1789г. СПб., 1906. 28 Исторические науки и археология 7. Archives parlementaires dé 1787 à 1860. Premiѐre série. T. VIII. P.,1875. 7. Archives parlementaires dé 1787 à 1860. Premiѐre série. T. VIII. P.,1875. 7. Archives parlementaires dé 1787 à 1860. Premiѐre série. T. VIII. P.,1875 7. Archives parlementaires dé 1787 à 1860. Premiѐre série. T. VIII. P.,1875. 8. Diesbach Ch. References 1. Ado A. V. Krest'yane i Velikaya francuzskaya revolyuciya. M., 1987. 1. Ado A. V. Krest'yane i Velikaya francuzskaya revolyuciya. M., 1987. 2. Blumenau S. F. Pravovye otnosheniya vokrug sen'orii vo Francii Starogo p yadka / / Pravo: istoriya, teoriya, praktika. Vypusk 3. Bryansk, 1999. 2. Blumenau S. F. Pravovye otnosheniya vokrug sen'orii vo Francii Starogo por yadka / / Pravo: istoriya, teoriya, praktika. Vypusk 3. Bryansk, 1999. y y p yp y 3. Dokumenty istorii Velikoj francuzskoj revolyucii. T. 1-2. M., 1990- 199 3. Dokumenty istorii Velikoj francuzskoj revolyucii. T. 1-2. M., 1990- 1992. 4. San'yak F. Grazhdanskoe zakonodatel'stvo Francuzskoj revolyucii: 1789- 1804 M., 1928. y j j y 4. San'yak F. Grazhdanskoe zakonodatel'stvo Francuzskoj revolyucii: 1789- 1804. M., 1928. 4. San'yak F. Grazhdanskoe zakonodatel'stvo Francuzskoj revolyucii: 1789- 1804. M., 1928. 5. Tyrsenko A. V. Emmanyuel' ZHozef Sijes i francuzskaya liberal'naya mysl' ego vremeni. M., 2005. 6. Shamp'on E. Franciya nakanune revolyucii po nakazam 1789g. SPb., 19 p y y p g , 7. Archives parlementaires dé 1787 à 1860. Premiѐre série. T. VIII. P.,1875. p y y p g 7. Archives parlementaires dé 1787 à 1860. Premiѐre série. T. VIII. P.,1875 8. Diesbach Ch. de Necker ou la faillité de la vertu. P.,1987. 9. Furet F. Nuit du 4 août // Dictionnaire critique de la Révolution française. P.,1988. 9. Furet F. Nuit du 4 août // Dictionnaire critique de la Révolution française. P.,1988. 29 Вестник Брянского государственного университета. 2020 (4) 10. Goujard Ph. L’abolition de la féodalité dans le district de Neufchâtel (Seine- Inférieure) // Contributions à l’ histoire paysanne de la révolution française.P., 1977. 10. Goujard Ph. L’abolition de la féodalité dans le district de Neufchâtel (Seine- Inférieure) // Contributions à l’ histoire paysanne de la révolution française.P., 1977. ) p y 11. Lefebvre G. La Grande Peur de 1789. P., 1932. Lemarchand G.La Fin du féodalisme dans le pays de Caux. P., 1989. 12. Lemarchand G.La Fin du féodalisme dans le pays de Caux. P., 1989. 13. Leuwers H. Robespierre. P., 2014. 14. Méthivier H. La fin de l’Ancien Régime. P., 1986. 15. Robespierre M. Oeuvres. T.6. P., 1950. 15. Robespierre M. Oeuvres. T.6. P., 1950. 16. Tackett T. La Grande Peur et le complot aristocratique sous la Révolution française // Annales historiques de la Révolution française, 2004, N.1( 335). 16. Tackett T. References La Grande Peur et le complot aristocratique sous la Révolution française // Annales historiques de la Révolution française, 2004, N.1( 335). 17. Tackett T. Par la volonté du peuple: Comment les deputės de 1789 sont devenusrévolutionnaires. P., 1997. 17. Tackett T. Par la volonté du peuple: Comment les deputės de 1789 sont devenusrévolutionnaires. P., 1997. 18. Thouret J.- G. Avis de bons normands à leurs frѐres tous les bons françois de toutes les provinces et de tous les Ordres. P., 1789. 18. Thouret J.- G. Avis de bons normands à leurs frѐres tous les bons françois de toutes les provinces et de tous les Ordres. P., 1789. Вестник Брянского государственного университета. 2020 (4) Об авторе Блуменау Семен Федорович –доктор исторических наук, профессор кафедры всеобщей истории и международных отношений, Брянский государственный уни- верситет имени академика И.Г.Петровского (Россия), E-mail: blumenausf@mail.ru Блуменау Семен Федорович –доктор исторических наук, профессор кафедры всеобщей истории и международных отношений, Брянский государственный уни- верситет имени академика И.Г.Петровского (Россия), E-mail: blumenausf@mail.ru Blumenau Semen Fedorovich - Doctor of History, professor at the chair of world history and international relations, Academician I. G. Petrovsky Bryansk State Univer- sity (Russia), E-mail: blumenausf@mail.ru Blumenau Semen Fedorovich - Doctor of History, professor at the chair of world history and international relations, Academician I. G. Petrovsky Bryansk State Univer- sity (Russia), E-mail: blumenausf@mail.ru 30
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Review of: "A Unified Psychology as Part of a General Social Science"
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Qeios, CC-BY 4.0 · Review, December 12, 2023 Qeios ID: GIXMJT · https://doi.org/10.32388/GIXMJT Review of: "A Unified Psychology as Part of a General Social Science" Tzu-Hua Huang1 1 University of Taipei Potential competing interests: No potential competing interests to declare. In this article, the author discusses numerous terms and presents relevant literature and their own arguments. However, it remains uncertain whether these discussions hold practical academic significance. Perhaps further substantiation is required. Qeios ID: GIXMJT · https://doi.org/10.32388/GIXMJT 1/1
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Outbreak of hepatitis A associated with men who have sex with men (MSM), England, July 2016 to January 2017
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Outbreak description Between July 2016 and January 2017, 37 confirmed cases with either strain 1 or 2 were detected across England as well as Northern Ireland (Figure 1), of which 28 identified as MSM. Of the 37 cases, 24 were Strain 1 and 13 were Strain 2. In addition, 15 probable cases (all MSM), primarily in London, were identified, and typing results are awaited. Infection with the hepatitis A virus (HAV) is most com- monly acquired through ingestion of contaminated food and water, and through faeco-oral contact. In the United Kingdom (UK) hepatitis A is a rare and mainly travel-associated disease, preventable by vaccina- tion [1,2]. Sexually transmitted hepatitis A outbreaks among men who have sex with men (MSM) are well documented [3-6]. We describe an ongoing outbreak in the UK, primarily affecting MSM, caused by two con- currently circulating HAV strains previously not seen in the UK, as well as the intervention strategies that have been instigated to control the outbreak. Cases with the identical strains have been reported in other European countries, prompting the European Centre for Disease Prevention and Control (ECDC) to issue a rapid risk assessment in December 2016 [7]. Strain 1 was first identified by the Virus Reference Department, Public Health England, London, in July 2016. The sequence had not been seen previously in the UK and phylogenetic analysis (Figure 2) showed a clear relation to sequences derived from travellers returning from Central and South America. Strain 1 cases were reported in eight geographically distinct areas in England and Northern Ireland (Figure 3). Of 24 Strain 1 cases, 22 were male, median age 35 years (19–63 years), 19 identified as MSM and eight reported travel within the incubation period, seven of which to Spain (Table). Outbreak of hepatitis A associated with men who have sex with men (MSM), England, July 2016 to January 2017 K Beebeejaun ¹ , S Degala ² , K Balogun ¹ , I Simms ³ , SC Woodhall ³ , E Heinsbroek ⁴ , PD Crook ⁴ , I Kar-Purkayastha ⁵ , J Treacy ⁵ , K Wedgwood ⁶ , K Jordan ⁷ , S Mandal ¹ , SL Ngui ⁸ , M Edelstein ¹ K Wedgwood , K Jordan , S Mandal , SL Ngui , M Edelstein 1. Immunisation, Hepatitis and Blood Safety department, National Infection Service, Public Health England, Colindale, London, United Kingdom , g , g 7. South West Health Protection Team, Public Health England, United Kingdom 8. Virus Reference Department, Public Health England, Colindale, London, United Kingdom Correspondence: Kazim Beebeejaun (kazim.beebeejaun@phe.gov.uk) Article submitted on 27 January 2017 / accepted on 02 February 2017 / published on 02 February 2017 Article submitted on 27 January 2017 / accepted on 02 February 2017 / published on 02 February 2017 or RIVM-HAV16–090 Strain 2 (Event 2) and symptom onset after 31 June 2016 [7]. A probable case was defined as a laboratory-confirmed HAV infection (not yet sequenced) with symptom onset after 31 June 2016, with contact with a confirmed case and/or who identi- fies as MSM. Between July 2016 and January 2017, 37 confirmed cases of hepatitis A with two unique IA genotype strains primarily among men who have sex with men, were reported across eight areas in England and Northern Ireland. Epidemiological and laboratory investigations indicate that these strains may have been imported several times from Spain, with second- ary sexual transmission in the United Kingdom. Local and national public health services are collaborating to control this ongoing outbreak. Rapid communications Rapid communications Citation style for this article: Beebeejaun K, Degala S, Balogun K, Simms I, Woodhall SC, Heinsbroek E, Crook PD, Kar-Purkayastha I, Treacy J, Wedgwood K, Jordan K, Mandal S, Ngui SL, Edelstein M. Outbreak of hepatitis A associated with men who have sex with men (MSM), England, July 2016 to January 2017. Euro Surveill. 2017;22(5):pii=30454. DOI: http://dx.doi.org/10.2807/1560-7917.ES.2017.22.5.30454 www.eurosurveillance.org Article submitted on 27 January 2017 / accepted on 02 February 2017 / published on 02 February 2017 Discussion As at 24 January 2017, 37 HAV infections with two sequences have been identified in eight UK areas, mostly among MSM (median age: 35 years; range: 19-56). HAV infection is most commonly acquired through contaminated food or water. In this outbreak however, epidemiological and laboratory investigations suggest multiple importations from several regions of Spain with secondary sexual transmission within the MSM population in the UK, as nine of the confirmed MSM cases reported travelling to Spain during the incubation period. Ireland, Sweden, Luxembourg and Germany have reported hepatitis A cases with identical viral sequences, some with history of travel to Spain during the incubation period. Spain has reported an increase in male HAV infections, but no further details were available [7]. This outbreak highlights the key role sequencing can play in outbreak detection, as well as the added value of a common European platform to share epidemiological and virological information. Case definition fi d Of note, Strain 2 has mainly been reported in MSM in London to date. Characteristics of concern among cases were noted, including infection in a sex worker with multiple partners, co-infection with sexually transmitted infec- tions (STIs) and use of sex-on-site premises and apps (Grindr, Recon) (Table). Case definition fi d A confirmed case was defined as a laboratory- confirmed HAV infection with the specific outbreak sequence of either VRD_521_2016 Strain 1 (Event 1) www.eurosurveillance.org Figure 1 Probable and confirmed cases of hepatitis A among men who have sex with men, England and Northern Ireland, July 2016– January 2017 (n=52) 0 1 2 3 4 5 6 7 Number of cases MSM (n=43) 35 33 34 36 38 40 31 32 29 30 27 28 37 39 2016 2017 41 42 44 46 48 43 45 47 49 50 51 52 1 Year and ISO week of disease onset Probable (n=15) Strain 2 (n=13) Strain 1 (n=24) ISO: International Organization for Standardization; MSM: men who have sex with men. Probable and confirmed cases of hepatitis A among men who have sex with men, England and Northern Ireland, July 2016– January 2017 (n=52) Year and ISO week of disease onset ISO: International Organization for Standardization; MSM: men who have sex with men. Strain 2 was first notified through the European Union Early Warning and Response System (EWRS) mes- sage from the Netherlands in October 2016 related to two MSM cases at EuroPride 2016, which took place in Amsterdam in July/August 2016. This genotype sequence was detected in 13 cases across six regions in England between November 2016 and January 2017 (Figure 3). Of the 13 cases, 12 were male, median age 39 years (range: 29–78), nine identified as MSM and 11 travelled during the incubation period, of which seven to Spain and two to Germany (Table). Of note, Strain 2 has mainly been reported in MSM in London to date. Characteristics of concern among cases were noted, including infection in a sex worker with multiple partners, co-infection with sexually transmitted infec- tions (STIs) and use of sex-on-site premises and apps (Grindr, Recon) (Table). Strain 2 was first notified through the European Union Early Warning and Response System (EWRS) mes- sage from the Netherlands in October 2016 related to two MSM cases at EuroPride 2016, which took place in Amsterdam in July/August 2016. This genotype sequence was detected in 13 cases across six regions in England between November 2016 and January 2017 (Figure 3). Of the 13 cases, 12 were male, median age 39 years (range: 29–78), nine identified as MSM and 11 travelled during the incubation period, of which seven to Spain and two to Germany (Table). www.eurosurveillance.org Control measures Public Health England (PHE) declared a national inci- dent in December 2016. Local and national laboratory, epidemiology and health protection teams contrib- uted to the response, which comprised: (i) enhanced surveillance for MSM-associated cases through an adapted questionnaire [8], (ii) a joint letter with the British Association for Sexual Health and HIV (BASHH) to all members alerting them to the outbreak and rec- ommending vaccination of at-risk MSM in outbreak areas, according to national guidelines [9,10], test- ing cases for other STIs and partner notification, (iii) disease information and targeted hygiene advice to the public through the National Health Service web portal [11], (iv) liaising with lesbian, gay, bisexual, and transgender (LGBT) and sexual health charities, gay-dating apps and gay venues to raise awareness through social media and health promotion visuals, and (v) giving post-exposure prophylaxis to household and sexual contacts. While the two concurrently circulating strains are virologically distinct, the public health response is intended to address both. Although it has not been possible to establish epidemiological links between all cases within geographical clusters, it is likely that cases are related either through undisclosed sexual contacts or other routes since neither strain is com- monly circulating in England. These missing epidemio- logical links are not unexpected when trying to capture sexual history via short questionnaires, particularly since some cases reported anonymous sex with mul- tiple partners. However, the questionnaires revealed sex-on-premises venues (saunas, clubs) and social networking (dating apps) as potential drivers of the outbreak. While these findings can help focus inter- ventions, they are of particular concern in areas with www.eurosurveillance.org 2 Figure 2 Phylogenetic analysis of virus strains from hepatitis A cases in England and Northern Ireland, July 2016–December 2016 Figure 2 Figure 2 Phylogenetic analysis of virus strains from hepatitis A cases in England and Northern Ireland, July 2016–December 2016 Figure 2 Phylogenetic analysis of virus strains from hepatitis A cases in England and Northern Ireland, July 2016–December 2016 Nucleotide substitution per 100 residues 0 6.3 2 4 6 ▼ ▼ ▼ ♦ ♦ ▼ ● ● ● ▼ ●Spain Travel-associated Non-travel-associated Risk unknown ● ● ● ▼ ● ● ▼ ● ●Gran Canaria , MSM RIVM -HAV16 -090 Event 2 ●Gran Canaria , MSM ●Gran Canaria ♦ ● ♦ ● ● ▼MSM ▼MSM ▼MSM ●Spain ▼MSM ●Spain ▼MSM ▼ ●Spain ▼ ▼ ●Spain ▼MSM ▼MSM ▼MSM ●Spain ▼ ▼ ● RIVM -HAV16 -069 ▼MSM ● VRD_521_2016 Event 1 AV: hepatitis A virus; MSM: men who have sex with men. he tree was constructed in MegAlign (DNAstar) using Clustal Key. ●Spain HAV: hepatitis A virus; MSM: men who have sex with men. www.eurosurveillance.org www.eurosurveillance.org 3 Figure 3 Figure 3 Geographical distribution of hepatitis A cases among men who have sex with men, England and Northern Ireland, July 2016–January 2017 (n=52) Figure 3 Geographical distribution of hepatitis A cases among men who have sex with men, England and Northern Ireland, July 2016–January 2017 (n=52) 3 Geographical distribution of hepatitis A cases among men who have sex with men, England and Nort 2016–January 2017 (n=52) !( !( !( !(!( !( !( !( !( !( !( !( !( !(!( !( !( !( !( !( !( !( !( !( !( !( !( !( !(!( !( !(!( !( !( !( !( !( !( !(!(!( !( !( !( !( !( !( !( !( !( !( South West South East North West East of England East Midlands West Midlands North East Yorkshire and Humber London 80 0 80 40 miles 1:4,000,000 Probable (n=15) Strain 2 (n=13) Strain 1 (n=24) Contains Ordnance Survey data. Crown copyright and database right 2017. Contains National Statistics data. Crown copyright and data Contains Ordnance Survey data. Crown copyright and database right 2017. Contains National Statistics data. Crown copyrigh www.eurosurveillance.org Table Table Characteristics of hepatitis A cases associated with the outbreak, England and Northern Ireland, July 2016–January 2017 (n=52) Table Characteristics of hepatitis A cases associated with the outbreak, England and Northern Ireland, July 2016–January 2017 (n 52) Characteristics of hepatitis A cases associated with the outbreak, England and Northern Ireland, July 2016–January 2017 (n=52) Region Case status (strain) Cases (n) Median age (years) MSM (n) Spain Notable characteristics East Midlands Confirmed (Strain 1) 9 28 6 2 One cluster of three cases of Strain 1 transmitted in a factory through environmental exposure. Confirmed (Strain 2) 3 55 2 2 Probable 0 NA 0 0 Total 12 36 8 4 South West Confirmed (Strain 1) 4 45 3 1 One case operated a private meeting place, used by contacts and multiple anonymous men. Confirmed (Strain 2) 1 NA 0 1 Probable 0 NA 0 0 Total 5 46 3 2 Hampshire Confirmed (Strain 1) 3 35 3 1 Probable case is index case in this area. This case was diagnosed in Spain but never sequenced. Further spread through household and sexual contacts. Confirmed (Strain 2) 0 NA 0 0 Probable 1 NA 1 1 Total 4 32 4 2 North East Confirmed (Strain 1) 3 41 3 1 First identified case with likely importation from Spain. MSM: men who have sex with men; NA: not applicable. MSM: men who have sex with men; NA: not applicable. Authors’ contributions All the authors contributed to the outbreak investigations described here, the presentation of information and the final drafting of the manuscript. Figure 3 Further spread to two cases through household and sexual transmission. Confirmed (Strain 2) 0 NA 0 0 Probable 0 NA 0 0 Total 3 41 3 1 London Confirmed (Strain 1) 2 31 2 0 One Strain 1 case was a sex worker with multiple sexually-transmitted co-infections who reported sex in several gay saunas in London. Three cases reported using apps and websites to meet partners. One Strain 2 case reported 20 sexual contacts within the eight weeks prior to disease onset. Confirmed (Strain 2) 6 35 4 3 Probable 12 34 12 1 Total 20 32 18 4 Yorkshire and Humber Confirmed (Strain 1) 1 NA 0 0 All but one case reported travel; three to Spain and to Germany. One Strain 2 case reported sexual contact with multiple partners at a gay sauna in London. Confirmed (Strain 2) 1 NA 1 1 Probable 1 NA 1 0 Total 3 NA 2 1 North West Confirmed (Strain 1) 0 NA 0 0 Confirmed (Strain 2) 1 NA 1 0 Probable 1 NA 1 0 Total 2 43 2 0 East of England Confirmed (Strain 1) 0 NA 0 0 Confirmed (Strain 2) 1 NA 1 0 Probable 0 NA 0 0 Total 1 NA 1 0 South Midlands Confirmed (Strain 1) 1 NA 1 1 Confirmed (Strain 2) 0 NA 0 0 Probable 0 NA 0 0 Total 1 NA 1 1 Belfast Confirmed (Strain 1) 1 NA 1 1 Confirmed (Strain 2) 0 NA 0 0 Probable 0 NA 0 0 Total 1 NA 1 1 Grand total 52 36 43 16 MSM: men who have sex with men; NA: not applicable www.eurosurveillance.org 5 6. Stene-Johansen K, Tjon G, Schreier E, Bremer V, Bruisten S, Ngui SL, et al. Molecular epidemiological studies show that hepatitis A virus is endemic among active homosexual men in Europe. J Med Virol. 2007;79(4):356-65. .DOI: 10.1002/ jmv.20781 PMID: 17311331 large, active MSM populations, such as London, where several of the recent cases have been reported. This outbreak also highlights the need for HAV aware- ness among MSM and sexual health professionals and the need for health promotion materials that focus on both infection and vaccination. Innovative and evalu- ated communication strategies with targeted messag- ing through social media, apps and venues also need to be readily available to public health agencies. 7. European Centre for Disease Prevention and Control (ECDC). Figure 3 Hepatitis A outbreaks in the EU/EEA mostly affecting men who have sex with men. Stockholm: ECDC. 19 Dec 2016. Available from: http://ecdc.europa.eu/en/publications/Publications/13- 12-2016-RRA-Hepatitis%20A-United%20Kingdom.pdf 8. Public Health England (PHE). Hepatitis A: case questionnaire. London: PHE. 2016. [Accessed 16 Dec 2016]. Available from: https://www.gov.uk/government/publications/ hepatitis-a-case-questionnaire 9. Public Health England (PHE). Green Book, Immunisation against infectious disease: Chapter 17, Hepatitis A. London: PHE. [Accessed 16 Dec 2016]. Available from: https://www.gov.uk/government/publications/ hepatitis-a-the-green-book-chapter-17 Hepatitis A vaccination for MSM in England is currently a risk-based recommendation [9,10]. For the purpose of this investigation, the vaccination status of the cases was not included in the analysis. While some may advo- cate for a universal MSM vaccination policy, it may not be cost-effective or affordable for local governments who commission sexual health services. Vaccine avail- ability also needs to be taken into account as it may impact the ability to vaccinate a large number of indi- viduals in a short timeframe. Enhanced surveillance for HAV in MSM will allow monitoring of the evolving outbreak as well as evaluating intervention impact, and gain a better understanding of HAV transmission in this population. 10. Brook G, Bhagani S, Kulasegaram R, Torkington A, Mutimer D, Hodges E, et al. , Clinical Effectiveness Group British Association for Sexual Health and HIV. United Kingdom National Guideline on the Management of the viral hepatitides A, B and C 2015.Int J STD AIDS. 2016;27(7):501-25. DOI: 10.1177/0956462415624250 PMID: 26745988 11. NHS Choices. Sexual health for gay and bisexual men - Live Well - NHS Choices. Hepatitis A. [Accessed 16 Dec 2016]. Available from: http://www.nhs.uk/Livewell/LGBhealth/Pages/ Sexualhealthgaymen.aspx#hepa Conflict of interest None declared. Acknowledgements We would like to thank all the regional PHE health protection staff involved in the outbreak investigation and management to date. This article is copyright of the authors, 2017. License and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi- cate if changes were made. Acknowledgements References 1. Public Health England. Laboratory reports of hepatitis A infection, and hepatitis C: 2015. Health Protection Report. 2016;10. [Accessed 16 Dec 2016]. Available from: https://www.gov.uk/government/publications/ laboratory-reports-of-hepatitis-a-and-c-2015 2. European Centre for Disease Prevention and Control (ECDC). Hepatitis A virus in the EU/EEA, 1975-2014. ECDC technical report. Stockholm: ECDC. 2016. Available from: http://ecdc. europa.eu/en/publications/Publications/hepatitis-a-virus-EU- EEA-1975-2014.pdf 2. European Centre for Disease Prevention and Control (ECDC). Hepatitis A virus in the EU/EEA, 1975-2014. ECDC technical report. Stockholm: ECDC. 2016. Available from: http://ecdc. europa.eu/en/publications/Publications/hepatitis-a-virus-EU- EEA-1975-2014.pdf 3. Bell A, Ncube F, Hansell A, Davison KL, Young Y, Gilson R, et al. An outbreak of hepatitis A among young men associated with having sex in public venues. Commun Dis Public Health. 2001;4(3):163-70.PMID: 11732354 4. Mindel A, Tedder R. Hepatitis A in homosexuals.Br Med J (Clin Res Ed). 1981;282(6277):1666. .DOI: 10.1136/ bmj.282.6277.1666 PMID: 6786425 4. Mindel A, Tedder R. Hepatitis A in homosexuals.Br Med J (Clin Res Ed). 1981;282(6277):1666. .DOI: 10.1136/ bmj.282.6277.1666 PMID: 6786425 5. Sfetcu O, Irvine N, Ngui SL, Emerson C, McCaughey C, Donaghy P. Hepatitis A outbreak predominantly affecting men who have sex with men in Northern Ireland, October 2008 to July 2009. Euro Surveill. 2011;16(9):19808. Available from: http://www. eurosurveillance.org/ViewArticle.aspx?ArticleId=19808PMID: 21392487 5. Sfetcu O, Irvine N, Ngui SL, Emerson C, McCaughey C, Donaghy P. Hepatitis A outbreak predominantly affecting men who have sex with men in Northern Ireland, October 2008 to July 2009. Euro Surveill. 2011;16(9):19808. Available from: http://www. eurosurveillance.org/ViewArticle.aspx?ArticleId=19808PMID: 21392487 6 www.eurosurveillance.org
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Axion emission from supernova with axion-pion-nucleon contact interaction
˜The œJournal of high energy physics/˜The œjournal of high energy physics
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Published for SISSA by Springer Received: November 4, 2021 Revised: January 25, 2022 Accepted: February 1, 2022 Published: February 17, 2022 Received: November 4, 2021 Revised: January 25, 2022 Accepted: February 1, 2022 Published: February 17, 2022 Open Access, c⃝The Authors. Article funded by SCOAP3. 3 Axion emission from supernovae by hadronic processes 3.2 Nucleon-nucleon bremsstrahlung JHEP02(2022)143 3.3 Pion-pion scattering: π + π →π + a 4 Conclusions and discussion A Axion emissivity from n + p →n + p + a in degenerate limit A Axion emissivity from n + p →n + p + a in degenerate limit 16 Axion emission from supernova with axion-pion-nucleon contact interaction JHEP02(2022)143 Kiwoon Choi,a Hee Jung Kim,a Hyeonseok Seonga and Chang Sub Shinb,a aCenter for Theoretical Physics of the Universe, Institute for Basic Science (IBS), 34126 Daejeon, South Korea bDepartment of Physics and Institute of Quantum Systems (IQS), Chungnam National University, 34134 Daejeon, South Korea E-mail: kchoi@ibs.re.kr, heejungkim@ibs.re.kr, hseong@ibs.re.kr, csshin@cnu.ac.kr Kiwoon Choi,a Hee Jung Kim,a Hyeonseok Seonga and Chang Sub Shinb,a aCenter for Theoretical Physics of the Universe, Institute for Basic Science (IBS), 34126 Daejeon, South Korea bDepartment of Physics and Institute of Quantum Systems (IQS), Chungnam National University, 34134 Daejeon, South Korea E-mail: kchoi@ibs.re.kr, heejungkim@ibs.re.kr, hseong@ibs.re.kr, csshin@cnu.ac.kr Abstract: We examine the axion emission from supernovae with a complete set of relevant axion couplings including the axion-pion-nucleon contact interaction which was ignored in the previous studies. Two processes are affected by the axion-pion-nucleon contact interaction, π−+p →n+a and n+p →n+p+a, and these processes can be the dominant source of axions for some region in the axion parameter space or in astrophysical conditions encountered inside supernovae. We find that the contact interaction can enhance the axion emissivity of π−+ p →n + a by a factor of 2 −4, while the effect on n + p →n + p + a is not significant. We also discuss the relative importance of other pion-induced processes such as π0 + n →n + a and π−+ π0 →π−+ a. Keywords: Beyond Standard Model, Cosmology of Theories beyond the SM ArXiv ePrint: 2110.01972 Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP02(2022)143 Contents 1 Introduction 1 2 Axion couplings to nucleons and pions 3 3 Axion emission from supernovae by hadronic processes 6 3.1 Pion-nucleon scattering 6 3.2 Nucleon-nucleon bremsstrahlung 10 3.3 Pion-pion scattering: π + π →π + a 14 4 Conclusions and discussion 15 A Axion emissivity from n + p →n + p + a in degenerate limit 16 Contents 1 Introduction 1 2 Axion couplings to nucleons and pions 3 3 Axion emission from supernovae by hadronic processes 6 3.1 Pion-nucleon scattering 6 3.2 Nucleon-nucleon bremsstrahlung 10 3.3 Pion-pion scattering: π + π →π + a 14 4 Conclusions and discussion 15 A Axion emissivity from n + p →n + p + a in degenerate limit 16 1 Introduction The axion which was initially introduced as a solution to the strong CP problem [1–4] has turned out to have many interesting phenomenological consequences [5–7]. After recogniz- ing that axions provide a compelling candidate for the dark matter in the Universe [8–10], a lot of efforts have been made to search for axions over the parameter space of the rep- resentative axion models [11–14]. Since the viable parameter region is in the very weakly coupled regime, most of the laboratory experiments searching for axions are ongoing in the direction of the precision measurements using, for example, resonant cavities, nuclear magnetic resonance, light shining through the walls, and polarization of lights in magnetic fields (see [15–17] for comprehensive reviews). A complementary approach which can severely constrain the couplings of light axions is to use astrophysical objects forming a hot and dense environment, e.g., supernovae, stars on the horizontal and red giant branches, neutron stars, and even white dwarfs (see [18] for a review and also [19] for a recent overview). Axions can be produced abundantly from those objects, thereby altering their evolution. One can then derive constraints on the couplings of axions by requiring that the axion emission does not significantly alter the standard evolution scenario which is consistent with the observational data. A core-collapse supernova, e.g., SN1987A, is known to provide stringent constraints on the axion couplings to hadrons, particularly on the couplings to nucleons [20, 21]. The observation of the neutrino flux from SN1987A, which is consistent with the standard scenario [22, 23], suggests that the additional cooling by axion emission from the associated proto-neutron star is constrained as La ≲Lν = O(1 −10) × 1051 erg/ sec, where La and Lν denote the axion and neutrino luminosities around 1 −10 sec after the formation of the proto-neutron star [18]. Among the processes producing axions from supernovae, the nucleon bremsstrahlung process N + N →N + N + a (N = n, p) has been considered as the dominating process – 1 – for many years [24–28]. However, recently it has been noticed that the number density of negatively charged pions inside supernovae can be significantly enhanced by pion-nucleon interactions [29]. Based on this observation, the pion-induced Compton-like process π−+ p →n+a, which was originally studied in [30, 31], has been revisited. Taking into account medium effects, refs. 1 Introduction [32, 33] show that the process dominates over the nucleon-nucleon bremsstrahlung for a wide range of astrophysical conditions encountered inside supernovae.1 Motivated by the importance of the process π−+ p →n + a, in this paper we extend the previous analysis of axion emission from supernovae with a complete set of relevant axion couplings including the axion-pion-nucleon and axion-pion contact interactions which were ignored in the previous studies. Our primary concern is how significantly the contact interactions can affect the axion emissivity. We start with a general axion Lagrangian above the QCD confinement scale, which determines the axion couplings to hadrons below that scale. To highlight the coupling dependence of the axion emissivity more clearly, we compare a new contribution including the effect of contact terms to that from the axion- nucleon couplings only and take the ratio between the two contributions. It is expected that the ratios can lead to cancellation of the uncertainties in nuclear physics and the medium effect. Thus, as a first step towards understanding the contributions of the contact interactions, we consider the tree-level diagrams in the leading order pion-nucleon couplings and the one-pion exchange diagrams for the nucleon-nucleon bremsstrahlung. We also ignore the background matter effect, which should be included in future work. In such an approximation, two processes are affected by the axion-pion-nucleon contact interaction, π−+ p →n + a and n + p →n + p + a. We find that the axion-pion-nucleon contact interaction can enlarge the emission rate of π−+ p →n + a by a factor of 2 −4 depending on the pattern of axion couplings, while the effect on n + p →n + p + a is negligible. We also examine other pion-induced processes such as π0 + n →n + a and π−+ π0 →π−+ a, where the latter process is induced by the axion-pion contact interaction. We then find that π0 + n →n + a can be as important as π−+ p →n + a, again depending on the pattern of axion couplings, while π−+π0 →π−+a is negligible compared to π−+p →n+a over the entire axion parameter space for astrophysical conditions encountered inside proto-neutron stars. JHEP02(2022)143 This paper is organized as follows. 1The medium effects also modify the axion-nucleon couplings. The modification is expected to be an O(1) effect in general, while it could result in ∼10-times enhancement of the axion-neutron coupling in the KSVZ model because the accidental cancellation of that coupling in vacuum is spoiled [34]. Here we presume the values of axion couplings in vacuum for our numerical estimation. 3Here for simplicity we ignore the effects of flavor mixings. 1 Introduction In section 2, we introduce the relevant axion couplings to nucleons and pions in the context of a generic axion model and discuss the model dependence of couplings for a simple class of axion models. In section 3, we inves- tigate the axion emission from supernovae by a variety of pion-induced processes and the nucleon-nucleon bremsstrahlung processes, with a complete set of relevant axion couplings. Section 4 is a summary and conclusion. 1The medium effects also modify the axion-nucleon couplings. The modification is expected to be an O(1) effect in general, while it could result in ∼10-times enhancement of the axion-neutron coupling in the KSVZ model because the accidental cancellation of that coupling in vacuum is spoiled [34]. Here we presume the values of axion couplings in vacuum for our numerical estimation. – 2 – Cu,d for string-theoretic axions are of the order of αGUT/2π [36]. 3 2For string-theoretic axions that arise from the zero modes of higher-dimensional p-form gauge fie there is no UV completion with a linearly realized U(1)PQ. It has been noted that the tree-level values 2 Axion couplings to nucleons and pions In this section, we briefly discuss the axion couplings to nucleons and pions for generic axions whose couplings are constrained only by the (approximate) global U(1) Peccei- Quinn (PQ) symmetry [1–4]. Without loss of generality, at scales below the axion decay constant fa, one can always choose a field basis for which only the axion field transforms under the PQ symmetry as U(1)PQ : a →a + constant, (2.1) (2.1) while all other fields are invariant [35]. In such a field basis, the axion couplings at low energy scales around µ = O(1) GeV include while all other fields are invariant [35]. In such a field basis, the axion couplings at low energy scales around µ = O(1) GeV include JHEP02(2022)143 Leff= cG g2 s 32π2 a fa Ga µν ˜Gaµν + ∂µa 2fa  Cu¯uγµγ5u + Cd ¯dγµγ5d  , (2.2) (2.2) where the axion decay constant fa defines the axion field range as a ∼= a + 2πfa, Ga µν are the gluon field strength, and u and d are the up and down quarks. Here cG is an integer- valued parameter describing the U(1)PQ breaking by the QCD anomaly, while Cu and Cd are continuous real-valued parameters describing the U(1)PQ-preserving axion couplings to the light quarks renormalized at µ = O(1) GeV. For axion models which have a UV completion with a linearly realized U(1)PQ, the low energy parameters cG and Cu,d in eq. (2.2) are determined mainly by the U(1)PQ charges defined in the UV model.2 As an illustrative example, let us consider axion models in which the first generation quark masses are generated by the following Yukawa couplings:3 LYukawa = λu  σ Λ nu Q1uc 1Hu + λd  σ Λ nd Q1dc 1Hd + h.c., (2.3) (2.3) where σ is a PQ-charged gauge-singlet scalar field whose vacuum expectation value deter- mines the axion decay constant as ⟨σ⟩= 1 √ 2faeia/fa, (2.4) (2.4) Qi and uc i, dc i (i = 1, 2, 3) denote the three generations of the left-handed SU(2)L-doublet quarks and the left-handed SU(2)L-singlet antiquarks, respectively, Hu and Hd are SU(2)L- doublet Higgs fields, and finally Λ is a cutoffscale of the model. Cu,d for string-theoretic axions are of the order of αGUT/2π [36]. 3 ere for simplicity we ignore the effects of flavor mixings. 2 Axion couplings to nucleons and pions To derive the low energy axion couplings in this model, we first make the following axion-dependent field redefinition at a scale around fa: Qi and uc i, dc i (i = 1, 2, 3) denote the three generations of the left-handed SU(2)L-doublet quarks and the left-handed SU(2)L-singlet antiquarks, respectively, Hu and Hd are SU(2)L- doublet Higgs fields, and finally Λ is a cutoffscale of the model. To derive the low energy axion couplings in this model, we first make the following axion-dependent field redefinition at a scale around fa: Φ →eiqΦa/faΦ (Φ = ψ, Hu,d), (2.5) (2.5) and subsequently integrate out all massive fields heavier than µ = O(1) GeV, where qΦ is the PQ charge of Φ (in the normalization convention with qσ = 1) for the linearly 2For string-theoretic axions that arise from the zero modes of higher-dimensional p-form gauge field, there is no UV completion with a linearly realized U(1)PQ. It has been noted that the tree-level values of Cu,d for string-theoretic axions are of the order of αGUT/2π [36]. – 3 – realized U(1)PQ, and ψ stands for all chiral fermions in the model. Then the axion-gluon coupling cG, which arises as a consequence of the axion-dependent field redefinition of ψ, corresponds to the coefficient of the U(1)PQ-SU(3)c-SU(3)c anomaly, while the couplings Cu,d to the light quarks are determined by (i) a contribution from the axion-dependent field- redefinition of {Q1, uc 1, dc 1}, (ii) the tree-level threshold correction from the axion mixing with the Z boson which is induced by the field redefinition of Hu,d, and finally (iii) the radiative corrections caused by the gauge and Yukawa couplings in the model [36]. Putting these together, one finds cG = 2 X ψ qψTr(T 2 c (ψ)), Cu = −nu −(qHu + qHd) cos2 β + ∆Cu, Cd = −nd −(qHu + qHd) sin2 β + ∆Cd, (2.6) JHEP02(2022)143 (2.6) where Tc(ψ) is the color charge of ψ, tan β = ⟨Hu⟩/⟨Hd⟩, and the radiative corrections ∆Cu,d = O(10−2 −10−3) can be safely ignored if the tree level values of Cu,d are of order unity [36]. The above results indicate that a variety of different patterns of cG and Cu,d are possible even within the framework of relatively simple axion models. Let us present explicitly the parameter values for some examples. 2 Axion couplings to nucleons and pions In the KSVZ model [11, 12], Hu = (iσ2Hd)∗and all SM fields are neutral under the linearly realized U(1)PQ, and therefore nu = nd = qHu = qHd = 0. The model also involves a heavy PQ-charged exotic quark Q generating the U(1)PQ-SU(3)c-SU(3)c with cG = 1. The resulting couplings of the KSVZ axion at µ = O(1) GeV are given by KSVZ : cG = 1, Cu = ∆Cu = O(10−2), Cd = ∆Cd = O(10−2), (2.7) (2.7) where ∆Cu,d are induced mostly by the axion-gluon coupling cG causing a running of Cu,d over the scales from the mass of the exotic quark Q to µ = O(1) GeV [36]. On the other hand, the minimal DFSZ model [13, 14] has nu = nd = 0, qHu = qHd = −1 and all chiral fermions in the SM model have qψ = 1/2, which result in DFSZ : cG = 6, Cu = 2 cos2 β + ∆Cu, Cd = 2 sin2 β + ∆Cd (2.8) (2.8) with ∆Cu,d = O(10−3) which are smaller than those of the KSVZ model because in the DFSZ model the running of Cu,d starts from a lower scale around the top quark mass [36]. It is an interesting possibility that U(1)PQ plays the role of a flavor symmetry which explains the fermion mass hierarchies [37–39]. In such a case, nu,d can be non-zero integers and the model can have a more diverse pattern of cG and Cu,d. Note that, while nu,d in the Yukawa couplings (eq. (2.3)) are required to be non-negative, the sign in front of nu,d in eq. (2.6) can be flipped by replacing σ in eq. (2.3) with σ∗. One can further generalize the model by introducing additional U(1)PQ-charged Higgs doublet, and then Cu,d receive additional contribution depending on the vacuum expectation value of the added Higgs field. With this observation, in the following we regard Cu and Cd as real-valued free parameters, with ∆Cu,d = O(10−3) which are smaller than those of the KSVZ model because in the DFSZ model the running of Cu,d starts from a lower scale around the top quark mass [36]. It is an interesting possibility that U(1)PQ plays the role of a flavor symmetry which explains the fermion mass hierarchies [37–39]. In such a case, nu,d can be non-zero integers and the model can have a more diverse pattern of cG and Cu,d. 2 Axion couplings to nucleons and pions Note that, while nu,d in the Yukawa couplings (eq. (2.3)) are required to be non-negative, the sign in front of nu,d in eq. (2.6) can be flipped by replacing σ in eq. (2.3) with σ∗. One can further generalize the model by introducing additional U(1)PQ-charged Higgs doublet, and then Cu,d receive additional contribution depending on the vacuum expectation value of the added Higgs field. With this observation, in the following we regard Cu and Cd as real-valued free parameters, – 4 – and cG as an integer-valued additional free parameter, without specifying the underlying UV model. From the couplings in eq. (2.2) defined at µ = O(1) GeV, we can derive the axion couplings to nucleons and pions which are relevant for the axion emission from supernova. Including the conventional pion-nucleon couplings, the interactions are given by [6, 40] Lint = gA 2fπ  ∂µπ0(¯pγµγ5p −¯nγµγ5n) + √ 2∂µπ+¯pγµγ5n + √ 2∂µπ−¯nγµγ5p  +∂µa 2fa  Cap¯pγµγ5p + Can¯nγµγ5n + CaπN fπ (iπ+¯pγµn −iπ−¯nγµp)  +∂µa 2fa Caπ fπ  π0π+∂µπ−+ π0π−∂µπ+ −2π+π−∂µπ0 , (2 JHEP02(2022)143 +∂µa 2fa Caπ fπ  π0π+∂µπ−+ π0π−∂µπ+ −2π+π−∂µπ0 , (2.9) (2.9) where fπ = 92.4 MeV is the pion decay constant and where fπ = 92.4 MeV is the pion decay constant and Cap −Can = gA  Cu −Cd + mu −md mu + md  cG  , Cap + Can = g0  Cu + Cd −cG  , CaπN = Cap −Can √ 2gA , Caπ = 2(Cap −Can) 3gA (2.10) (2.10) with the nucleon matrix elements of the light quark axial vector currents given by gA = ∆u −∆d ≃1.2723(23), g0 = ∆u + ∆d ≃0.521(53), (2.11) (2.11) where sµ∆q = ⟨N|¯qγµγ5q|N⟩(q = u, d) for the nucleon spin four vector sµ. Here the numerical value of g0 is chosen for the axion-quark couplings Cu,d renormalized at µ = 2 GeV in the MS scheme [41], and the small contributions from the axion couplings to the heavier quarks Q = {s, c, b, t} are ignored. The above results show that the entire axion couplings to nucleons and pions, including the axion-pion-nucleon contact interaction CaπN and the axion-pion contact interaction Caπ, are determined by the two free parameters Can and Cap. 2 Axion couplings to nucleons and pions An interesting feature of these parameters is that in some axion models they can have a hierarchical pattern such as |Cap| ≫|Can| or |Cap −Can| ≫|Cap + Can| without fine tuning of any continuous parameter in the underlying UV model. For instance, including the radiative corrections induced by the axion-gluon coupling cG, the KSVZ and string-theoretic axions have |Cu,d| = O(10−2cG) [36], which results in |Cap| ≃0.48|cG| ≫|Can| = O(10−2|cG|) (2.12) (2.12) for the nucleon matrix elements in eq. (2.11) and the light quark mass ratio mu/md = 0.48(3). Also, for the axion couplings in eq. (2.6), the anomaly coefficient cG and the tree level value of Cu + Cd are all quantized parameters. Then, for a model with U(1)PQ- charges yielding (2.13) cG = −(nu + nd + qHu + qHd) = O(1) , (2.13) – 5 – the model predicts Cap −Can = O(1), Cap + Can = g0(∆Cu + ∆Cd) ≲O(10−2) . (2.14) (2.14) At any rate, axions generically have the axion-pion-nucleon contact interaction given by CaπN = (Cap −Can)/ √ 2gA (gA ≃1.27) and the axion-pion contact interaction Caπ = 2(Cap −Can)/3gA. On the other hand, these contact interactions were not taken into account in the previous studies of axion emission from supernovae. In section 3, we will examine the effects of those contact interactions on the axion emission rates to see how important they can be. JHEP02(2022)143 3 Axion emission from supernovae by hadronic processes In this section, we examine the axion production by hadron collisions inside a newly born proto-neutron star. We consider three types of processes, the pion-nucleon scattering π + N →N + a (N = n, p), the nucleon-nucleon bremsstrahlung N + N →N + N + a, and the pion-pion scattering π + π →π + a. The relative importance of each process depends on the pattern of axion couplings, as well as on the density and temperature of the corresponding astrophysical environment. Our prime goal is to examine the effects of the two contact interactions, the axion-pion-nucleon contact coupling CaπN and the axion-pion contact coupling Caπ in eq. (2.9), which were not taken into account before except for the nucleon-nucleon bremsstrahlung [42]. We will examine this question in a simple approximation keeping only the leading order in pion-nucleon couplings and ignoring medium effects. Accordingly, we can find a simple form of the coupling dependence in that approximation, and it shows the relative importance of the contribution from each coupling at a rough estimate. 4The squared matrix element depends on two independent angles, each from the Mandelstam variables s and u. We approximate these Mandelstam variables as s = (pp + pπ−)2 ≃m2 p + m2 π−+ 2mpEπ−and u = (pp −pa)2 ≃m2 p −2mpEa. 3.1 Pion-nucleon scattering Let us first discuss the pion-nucleon scattering process π + N →N + a. For T ∼40 MeV and the nucleon mass density ρ ∼1014 g/cm3 encountered inside a proto-neutron star, the pion and nucleon number densities roughly obey [29] nπ0 nπ−∼nπ+ nπ0 ∼np nn = O(0.1) . (3.1) (3.1) It is then expected that π−+ p →n + a and π0 + n →n + a are the dominating process depending upon the involved axion couplings. The Feynman diagrams for these processes are depicted in figure 1 and figure 2, showing that at leading order in pion-nucleon couplings π0+n →n+a involves only the axion-neutron coupling Can, while π−+p →n+a depends on three axion couplings, Cap, Can, and the axion-pion-nucleon contact interaction CaπN. Recently, the process π−+ p →n + a has been argued to be the dominating process to produce axions for a wide range of astrophysical conditions encountered inside super- novae [32, 33]. The axion emissivity (the energy loss induced by axion emissions per unit – 6 – p n ⇡− a n p n ⇡− a p p n ⇡− a <latexit sha1_base64="AMtjGePCnHY1LwtQUDwYLj/szeE=">AB7Xi cdVDLSgMxFM3UV62vqks3wSK4GmbamVp3hYK4rGAf0A4lk6ZtbCYZkoxQhv6DGxeKuPV/3Pk3ZtoKnrgwuGce7n3njBmVGnH+bBya+sbm1v57cLO7t7+Q fHwqK1EIjFpYcGE7IZIEUY5aWmqGenGkqAoZKQThuZ37knUlHBb/UsJkGExpyOKEbaSO3GIEV8PiWHLtS8f1LD2akWvP9jHhu2a1B13YWKIEVmoPie3 8ocBIRrjFDSvVcJ9ZBiqSmJF5oZ8oEiM8RWPSM5SjiKgXVw7h2dGcKRkKa4hgv1+0SKIqVmUWg6I6Qn6reXiX95vUSPakFKeZxowvFy0ShUAuYvQ6H VBKs2cwQhCU1t0I8QRJhbQIqmBC+PoX/k3bZdqu2d+OV6lerOPLgBJyCc+C1AH16AJWgCDO/AnsCzJaxH68V6XbmrNXMfgB6+0TCv6Peg=</lat exit>Can <latexit sha1_base64="x6zU2sLpLc4gqASfde6XAGQgUE=">AB7XicdVDLSgMxFM3UV62vqks3wSK4GmbamVp3hYK4rGAf0A4lk6ZtbCYZkoxQhv 6DGxeKuPV/3Pk3ZtoKnrgwuGce7n3njBmVGnH+bBya+sbm1v57cLO7t7+QfHwqK1EIjFpYcGE7IZIEUY5aWmqGenGkqAoZKQThuZ37knUlHBb/UsJkGExpyOKEbaSO3GIEXxfFAsOXal4vuXHsxIteb7GfHcsluDru0sUAIrNAfF9/5Q4CQiXGOGlOq5TqyDFElNMSPzQj9RJEZ4isakZyhHEVFBurh2Ds+MoQjI U1xDRfq94kURUrNotB0RkhP1G8vE/yeoke1YKU8jRhOPlolHCoBYwex0OqSRYs5khCEtqboV4giTC2gRUMCF8fQr/J+2y7VZt78Yr1a9WceTBCTgF58AF6AOrkETtAGd+ABPIFnS1iP1ov1umzNWauZY/AD1tsnDgiPfA=</latexit>Cap <latexit sha1_base64="srE0jL1iUjRQ1uK7OgX7tw62FE=">AB8XicdVDLSgNBEOz1GeMr6tHLYBA8Lbt5GW+BgHiSCOaByRJmJ5NkyOzsMjMrhCV/4cWD Il79G2/+jbNJBUtaCiqunu8iPOlHacD2tldW19YzOzld3e2d3bzx0ctlQYS0KbJOSh7PhYUc4EbWqmOe1EkuLA57TtT+qp376nUrFQ3OpRL0AjwQbMoK1ke7q/QT3IoauZ/1c3rGLxXL5oRSUqmWykpuQW3ilzbmSMPSzT6ufeICRxQIUmHCvVdZ1IewmWmhFOZ9lerGiEyQSPaNdQgQOqvGR+8QydGmWAhqE0JTSaq98nEhwo NQ180xlgPVa/vVT8y+vGelj1EiaiWFNBFouGMUc6ROn7aMAkJZpPDcFEMnMrImMsMdEmpKwJ4etT9D9pFWy3YpduSvna5TKODBzDCZyBC+dQgytoQBMICHiAJ3i2lPVovVivi9YVazlzBD9gvX0CfhOQ1w=</latexit>Ca⇡N Figure 1. Diagrams for π−+ p →n + a from the axion couplings in eq. (2.9). n n ⇡0 a n n n ⇡0 a n <latexit sha1_base64="VaPSw8fVwi4e9H1eHLbfb7/87w=">AB7XicdVDLSgMxFM3UV62vqks3wSK4Gmba mVp3hW7cWcE+oB1KJk3b2EwyJBmhDP0HNy4Ucev/uPNvzLQVPTAhcM593LvPWHMqNKO82Hl1tY3Nrfy24Wd3b39g+LhUVuJRGLSwoIJ2Q2RIoxy0tJUM9KNJUFRyEgnDYyv3NPpKC3+pZTIjTkdUYy0kdqNQYr4fFAsOXal4vu XHsxIteb7GfHcsluDru0sUAIrNAfF9/5Q4CQiXGOGlOq5TqyDFElNMSPzQj9RJEZ4isakZyhHEVFBurh2Ds+MoQjIU1xDRfq94kURUrNotB0RkhP1G8vE/yeoke1YKU8jRhOPlolHCoBYwex0OqSRYs5khCEtqboV4giTC2gRUMCF 8fQr/J+2y7VZt78Yr1a9XceTBCTgF58AF6AOrkATtAGd+ABPIFnS1iP1ov1umzNWauZY/AD1tsnDbOPgw=</latexit>Can <latexit sha1_base64="VaPSw8fVwi4e9H1eHLbfb7/87w=">AB7XicdVDLSgMxFM3UV62vqks3wSK4GmbamVp3hW7cWcE+oB1KJk3b2EwyJBmhDP0HNy4Ucev/uPNvzLQVPTAhcM593LvPWHMqNKO82Hl1tY3Nrfy24Wd3b39 g+LhUVuJRGLSwoIJ2Q2RIoxy0tJUM9KNJUFRyEgnDYyv3NPpKC3+pZTIjTkdUYy0kdqNQYr4fFAsOXal4vuXHsxIteb7GfHcsluDru0sUAIrNAfF9/5Q4CQiXGOGlOq5TqyDFElNMSPzQj9RJEZ4isakZyhHEVFBurh2Ds+MoQjIU1xDRfq94kURUrNotB0RkhP1G8vE/yeoke1YKU8jRhOPlolHCoBYwex0OqSRYs5khCEtqboV4giTC2gRUMCF8fQr/J+2y7VZt78Yr1a9XceTBCTgF58AF6AOrkATtAGd+ABPIFnS1iP1ov1umzNWauZY/AD1tsnDbOPgw=</ latexit>Can Figure 2. Diagrams of axion production for the process π0 + n →n + a. p n ⇡− a n p n ⇡− a p p n ⇡− a <latexit sha1_base64="AMtjGePCnHY1LwtQUDwYLj/szeE=">AB7Xi cdVDLSgMxFM3UV62vqks3wSK4GmbamVp3hYK4rGAf0A4lk6ZtbCYZkoxQhv6DGxeKuPV/3Pk3ZtoKnrgwuGce7n3njBmVGnH+bBya+sbm1v57cLO7t7+Q fHwqK1EIjFpYcGE7IZIEUY5aWmqGenGkqAoZKQThuZ37knUlHBb/UsJkGExpyOKEbaSO3GIEV8PiWHLtS8f1LD2akWvP9jHhu2a1B13YWKIEVmoPie3 8ocBIRrjFDSvVcJ9ZBiqSmJF5oZ8oEiM8RWPSM5SjiKgXVw7h2dGcKRkKa4hgv1+0SKIqVmUWg6I6Qn6reXiX95vUSPakFKeZxowvFy0ShUAuYvQ6H VBKs2cwQhCU1t0I8QRJhbQIqmBC+PoX/k3bZdqu2d+OV6lerOPLgBJyCc+C1AH16AJWgCDO/AnsCzJaxH68V6XbmrNXMfgB6+0TCv6Peg=</lat exit>Can <latexit sha1_base64="x6zU2sLpLc4gqASfde6XAGQgUE=">AB7XicdVDLSgMxFM3UV62vqks3wSK4GmbamVp3hYK4rGAf0A4lk6ZtbCYZkoxQhv 6DGxeKuPV/3Pk3ZtoKnrgwuGce7n3njBmVGnH+bBya+sbm1v57cLO7t7+QfHwqK1EIjFpYcGE7IZIEUY5aWmqGenGkqAoZKQThuZ37knUlHBb/UsJkGExpyOKEbaSO3GIEXxfFAsOXal4vuXHsxIteb7GfHcsluDru0sUAIrNAfF9/5Q4CQiXGOGlOq5TqyDFElNMSPzQj9RJEZ4isakZyhHEVFBurh2Ds+MoQjI U1xDRfq94kURUrNotB0RkhP1G8vE/yeoke1YKU8jRhOPlolHCoBYwex0OqSRYs5khCEtqboV4giTC2gRUMCF8fQr/J+2y7VZt78Yr1a9WceTBCTgF58AF6AOrkETtAGd+ABPIFnS1iP1ov1umzNWauZY/AD1tsnDgiPfA=</latexit>Cap <latexit sha1_base64="srE0jL1iUjRQ1uK7OgX7tw62FE=">AB8XicdVDLSgNBEOz1GeMr6tHLYBA8Lbt5GW+BgHiSCOaByRJmJ5NkyOzsMjMrhCV/4cWD Il79G2/+jbNJBUtaCiqunu8iPOlHacD2tldW19YzOzld3e2d3bzx0ctlQYS0KbJOSh7PhYUc4EbWqmOe1EkuLA57TtT+qp376nUrFQ3OpRL0AjwQbMoK1ke7q/QT3IoauZ/1c3rGLxXL5oRSUqmWykpuQW3ilzbmSMPSzT6ufeICRxQIUmHCvVdZ1IewmWmhFOZ9lerGiEyQSPaNdQgQOqvGR+8QydGmWAhqE0JTSaq98nEhwo NQ180xlgPVa/vVT8y+vGelj1EiaiWFNBFouGMUc6ROn7aMAkJZpPDcFEMnMrImMsMdEmpKwJ4etT9D9pFWy3YpduSvna5TKODBzDCZyBC+dQgytoQBMICHiAJ3i2lPVovVivi9YVazlzBD9gvX0CfhOQ1w=</latexit>Ca⇡N Figure 1. Diagrams for π−+ p →n + a from the axion couplings in eq. (2.9). n n ⇡0 a n n n ⇡0 a n <latexit sha1_base64="VaPSw8fVwi4e9H1eHLbfb7/87w=">AB7XicdVDLSgMxFM3UV62vqks3wSK4Gmba mVp3hW7cWcE+oB1KJk3b2EwyJBmhDP0HNy4Ucev/uPNvzLQVPTAhcM593LvPWHMqNKO82Hl1tY3Nrfy24Wd3b39g+LhUVuJRGLSwoIJ2Q2RIoxy0tJUM9KNJUFRyEgnDYyv3NPpKC3+pZTIjTkdUYy0kdqNQYr4fFAsOXal4vu XHsxIteb7GfHcsluDru0sUAIrNAfF9/5Q4CQiXGOGlOq5TqyDFElNMSPzQj9RJEZ4isakZyhHEVFBurh2Ds+MoQjIU1xDRfq94kURUrNotB0RkhP1G8vE/yeoke1YKU8jRhOPlolHCoBYwex0OqSRYs5khCEtqboV4giTC2gRUMCF 8fQr/J+2y7VZt78Yr1a9XceTBCTgF58AF6AOrkATtAGd+ABPIFnS1iP1ov1umzNWauZY/AD1tsnDbOPgw=</latexit>Can <latexit sha1_base64="VaPSw8fVwi4e9H1eHLbfb7/87w=">AB7XicdVDLSgMxFM3UV62vqks3wSK4GmbamVp3hW7cWcE+oB1KJk3b2EwyJBmhDP0HNy4Ucev/uPNvzLQVPTAhcM593LvPWHMqNKO82Hl1tY3Nrfy24Wd3b39 g+LhUVuJRGLSwoIJ2Q2RIoxy0tJUM9KNJUFRyEgnDYyv3NPpKC3+pZTIjTkdUYy0kdqNQYr4fFAsOXal4vuXHsxIteb7GfHcsluDru0sUAIrNAfF9/5Q4CQiXGOGlOq5TqyDFElNMSPzQj9RJEZ4isakZyhHEVFBurh2Ds+MoQjIU1xDRfq94kURUrNotB0RkhP1G8vE/yeoke1YKU8jRhOPlolHCoBYwex0OqSRYs5khCEtqboV4giTC2gRUMCF8fQr/J+2y7VZt78Yr1a9XceTBCTgF58AF6AOrkATtAGd+ABPIFnS1iP1ov1umzNWauZY/AD1tsnDbOPgw=</ latexit>Can Figure 2. Diagrams of axion production for the process π0 + n →n + a. n n ⇡0 a n <latexit sha1_base64="VaPSw8fVwi4e9H1eHLbfb7/87w=">AB7XicdVDLSgMxFM3UV62vqks3wSK4GmbamVp3hW7cWcE+oB1KJk3b2EwyJBmhDP0HNy4Ucev/uPNvzLQVPTAhcM593LvPWHMqNKO82Hl1tY3Nrfy24Wd3b39 g+LhUVuJRGLSwoIJ2Q2RIoxy0tJUM9KNJUFRyEgnDYyv3NPpKC3+pZTIjTkdUYy0kdqNQYr4fFAsOXal4vuXHsxIteb7GfHcsluDru0sUAIrNAfF9/5Q4CQiXGOGlOq5TqyDFElNMSPzQj9RJEZ4isakZyhHEVFBurh2Ds+MoQjIU1xDRfq94kURUrNotB0RkhP1G8vE/yeoke1YKU8jRhOPlolHCoBYwex0OqSRYs5khCEtqboV4giTC2gRUMCF8fQr/J+2y7VZt78Yr1a9XceTBCTgF58AF6AOrkATtAGd+ABPIFnS1iP1ov1umzNWauZY/AD1tsnDbOPgw=</ latexit>Can JHEP02(2022)143 Figure 2. Diagrams of axion production for the process π0 + n →n + a. 3.1 Pion-nucleon scattering As already noticed, CaπN is not an independent parameter, but is determined as CaπN = √ 2C−/gA (see eq. (2.10)). Then the third term can be interpreted as a higher order term as it is suppressed compared to other terms by additional power of Eπ/mN. However, our numerical estimation gives Cpπ− a ≃0.02 (2C2 + + C2 −) + 0.04 C2 aπN + 0.01 CaπNC−for typical parameter values, e.g., T = 40 MeV, |pπ| ≃√3mπ−T ≃130 MeV. Note that the third term is comparable to the others. The relative importance of each term coincides with the final axion emissivity in eq. (3.7) up to a small enhancement by the phase space integration. We remark that the above expression of Cpπ− a corresponds to the leading order result (in 1/mN) for which the three axion coupling combinations, i.e., 2C2 + + C2 −, CaπNC−and C2 aπN, are pretended to be independent parameters. As already noticed, CaπN is not an independent parameter, but is determined as CaπN = √ 2C−/gA (see eq. (2.10)). Then the third term can be interpreted as a higher order term as it is suppressed compared to other terms by additional power of Eπ/mN. However, our numerical estimation gives Cpπ− a ≃0.02 (2C2 + + C2 −) + 0.04 C2 aπN + 0.01 CaπNC−for typical parameter values, e.g., T = 40 MeV, |pπ| ≃√3mπ−T ≃130 MeV. Note that the third term is comparable to the others. The relative importance of each term coincides with the final axion emissivity in eq. (3.7) up to a small enhancement by the phase space integration. JHEP02(2022)143 For |pp| ≫|pπ,a|, the axion emissivity can be further approximated as Qpπ− a ≃zpzπ− f2af2π s m7 NT 11 128π10 Z dxp   x2 pex2 p (ex2p + zn)(ex2p + zp)   Z dxπ x2 πϵπCpπ− a eϵπ−yπ −zπ− ! , (3.6) where zi = e(µi−mi)/T are the fugacities, ϵπ = Eπ/T, yπ = mπ−/T, xπ = |pπ|/T, and xp = |pp|/√2mNT. The emissivity eq. (3.6) depends on many astrophysical parameters which are related to each other by the equation of state, e.g., the temperature T and the chemical potentials µi (i = n, p, π−). To parameterize the astrophysical condition in terms of T and the total mass density ρ, we use the fugacities obtained in [29] and numerically calculate the integral in eq. (3.6) around T ∼40 MeV and ρ ∼1014 g/cm3. 5The emissivity of π−+ p →n + a obtained in [32] for CaπN = 0 is bigger than ours by a factor ∼2. As the analysis of [32] takes into account leading order medium effects, while ours does not, it is likely that this difference originates from medium effects. 3.1 Pion-nucleon scattering volume and second) of this process is given by volume and second) of this process is given by Qpπ− a = Z Y α=π,p,n,a d3pα (2π)32Eα " (2π)4δ(4)(pπ + pp −pn −pa) × fπ(pπ)fp(pp)(1 −fn(pn)) X sp,sn |Mπ−+p→n+a|2 Ea # , (3.2) Qpπ− a = Z Y α=π,p,n,a d3pα (2π)32Eα " (2π)4δ(4)(pπ + pp −pn −pa) × fπ(pπ)fp(pp)(1 −fn(pn)) X sp,sn |Mπ−+p→n+a|2 Ea # , (3.2) (3.2) where pα = (Eα, pα) are the particle four-momenta, fα(pα) are the Fermi-Dirac or Bose- Einstein distribution function, and sN (N = p, n) denotes the nucleon spin. Although the integrand has angular dependence after applying the energy-momentum conservation, the nucleon distribution functions can be approximated to be independent of those angles in the non-relativistic limit. Then, the squared matrix element can be first integrated over the relative angle of pp with respect to pa, while the integration over the solid angle of pa amounts to a factor of 4π.4 Taking the non-relativistic limit for the initial proton and integrating over the relative angle between pπ−and pa, we find Z dΩπ− X sp,sn |Mπ−+p→n+a|2 = 8πm4 N f2af2π Cpπ− a , (3.3) (3.3) where mN is the nucleon mass and Cpπ− a is a dimensionless quantity which can be expanded in powers of 1/mN as Cpπ− a ≃2 3g2 A |pπ| mN 2  2C2 + + C2 −  +  Eπ mN 2 C2 aπN + √ 2gA  Eπ mN 3 1 −1 3 |pπ| Eπ 2! CaπNC−, (3.4) (3.4) 4The squared matrix element depends on two independent angles, each from the Mandelstam variables s and u. We approximate these Mandelstam variables as s = (pp + pπ−)2 ≃m2 p + m2 π−+ 2mpEπ−and u = (pp −pa)2 ≃m2 p −2mpEa. 4The squared matrix element depends on two independent angles, each from the Mandelstam variables s and u. We approximate these Mandelstam variables as s = (pp + pπ−)2 ≃m2 p + m2 π−+ 2mpEπ−and u = (pp −pa)2 ≃m2 p −2mpEa. – 7 – – 7 – where where C± = 1 2 (Cap ± Can) , Eπ = q m2 π−+ |pπ|2. (3.5) (3.5) We remark that the above expression of Cpπ− a corresponds to the leading order result (in 1/mN) for which the three axion coupling combinations, i.e., 2C2 + + C2 −, CaπNC−and C2 aπN, are pretended to be independent parameters. 3.1 Pion-nucleon scattering We then find Qpπ− a erg · cm−3 s−1 ≃1.4 × 1033 T 7.2 40 ρ1.1 14 109 GeV fa !2  2C2 + + C2 −  + 2.3 × 1033 T 6.6 40 ρ1.1 14 109 GeV fa !2 C2 aπN + 1.2 × 1033 T 7.5 40 ρ1.1 14 109 GeV fa !2 CaπNC−, (3.7) (3.7) where T40 ≡T/(40 MeV) and ρ14 ≡ρ/(1014 g/cm3). We stress that the above approxima- tion is valid only for a narrow range of T and ρ, i.e., for T40 ∈[0.9, 1.1] and ρ14 ∈[1, 3], which is enough for our purpose to examine the effect of the axion-pion-nucleon contact interaction for an ambient condition inside supernovae.5 Obviously the second and third terms in the r.h.s. of eq. (3.7) represent the con- tributions to the axion emissivity from the axion-pion-nucleon contact interaction CaπN. – 8 – 36 38 40 42 44 46 48 50 1032 1033 1034 1035 36 38 40 42 44 46 48 50 1032 1033 1034 1035 Figure 3. Axion emissivities of π−+p →n+a for the KSVZ, DFSZ, and a model with |C−| ≫|C+|. All models are assumed to have fa9 ≡(fa/cG)/109 GeV = 1. The solid curves represent the total emissivity including the effect of the contact interaction CaπN, while the dashed curves are the emissivity without including the contribution from CaπN. 36 38 40 42 44 46 48 50 1032 1033 1034 1035 36 38 40 42 44 46 48 50 1032 1033 1034 1035 JHEP02(2022)143 Figure 3. Axion emissivities of π−+p →n+a for the KSVZ, DFSZ, and a model with |C−| ≫|C+|. All models are assumed to have fa9 ≡(fa/cG)/109 GeV = 1. The solid curves represent the total emissivity including the effect of the contact interaction CaπN, while the dashed curves are the emissivity without including the contribution from CaπN. Because it is expected that the ratio is less sensitive to the uncertainty in nuclear physics, we take the ratio between each term as ∆Qpπ− a, C2 aπN ∆Qpπ− a, (2C2 ++C2 −) ≃2.0 C2 − 2C2 + + C2 − ! , ∆Qpπ− a, CaπNC− ∆Qpπ− a, (2C2 ++C2 −) ≃0.9 C2 − 2C2 + + C2 − ! . (3.8) (3.8) Here we use the relation CaπN = √ 2C−/gA in eq. (2.10). Therefore, the contributions from the contact interactions enhance the axion emissivity by O(1) in general. 6A simple way to realize such a case is to introduce PQ-charged exotic quarks in the minimal DFSZ model, which generate ∆cG = −4. 3.1 Pion-nucleon scattering (3.10) (3.10) Using again the fugacities obtained in [29], Qnπ0 a can be further approximated as Qnπ0 a erg · cm−3 s−1 ≃1.5 × 1033 T 7.5 40 ρ1.0 14 109 GeV fa !2 C2 an (3.11) (3.11) for T40 ∈[0.9, 1.1] and ρ14 ∈[1, 3]. This shows that Qnπ0 a can be comparable to Qpπ− a for T ∼40 MeV and ρ ∼1014 g/cm3, unless |Can| ≪|Cap|. It is also straightforward to confirm that the other pion-nucleon scattering processes, i.e. π0+p →p+a and π++n →p+a, give subleading contribution relative to π−+p →n+a and π0 + n →n + a for T ∼40 MeV and ρ ∼1014 g/cm3. For instance, for the process π0 + p →p + a, we find Qpπ0 a erg · cm−3 s−1 ≃2.7 × 1032 T 10.3 40 ρ0.56 14 109 GeV fa !2 C2 ap ≃zp zn C2 ap C2an Qnπ0 a erg · cm−3 s−1 , (3.12) (3.12) where the fugacities of [29] are used for the last expression. This shows that for T40 ∈ [0.9, 1.1] and ρ14 ∈[1, 3], Qpπ0 a < Qpπ− a over the entire axion parameter space. 3.1 Pion-nucleon scattering We highlight in figure 3 how much CaπN enhances the axion emissivity for three benchmark axion models with fa9 ≡(fa/cG)/109 GeV = 1; the KSVZ model of eq. (2.7) (red), the DFSZ model of eq. (2.8) with tan β = 5 (blue), and a model (green) to realize |C−| ≫|C+| ≃0 by satisfying the condition eq. (2.13) for the PQ charges. For the third model, we choose cG = 2, nu = nd = 0, qHu = qHd = −1, tan β = 5 for the model parameters in eq. (2.6), which result in Cap ≃−Can ≃−1.62.6 Note that in our convention, the axion decay constant fa is defined by the axion field range a ∼= a + 2πfa, and the axion-gluon coupling is given by cG/fa for an integer-valued parameter cG. We show that the contact interaction can enhance the axion emissivity by a factor 2−4, depending on the pattern of axion couplings, and this conclusion will not change significantly when we include the corrections, e.g., the medium effects [32, 33]. Since zp/zn ∼zπ0/zπ−= O(0.1) inside proto-neutron star [29], the process π0 + n → n + a shown in figure 2 can be as important as π−+ p →n + a. Taking the same approach as eq. (3.6), the axion emissivity of π0 + n →n + a can be approximated as Qnπ0 a ≃1 2 znzπ0 f2af2π s m7 NT 11 128π10 Z ∞ 0 dxn x2 nex2 n (ex2n + zn)2 Z ∞ 0 dxπ x2 πϵπ0 Cnπ0 a eϵπ0−yπ0 −zπ0 ! , (3.9) (3.9) 6A simple way to realize such a case is to introduce PQ-charged exotic quarks in the minimal DFSZ model, which generate ∆cG = −4. – 9 – ⇡− n p p n a ⇡− n a p p n ⇡0 ⇡− a ⇡− <latexit sha1_base64="MWedM/cqbMYIRq3ISXcQY5KYvr w=">ACzXicjVHLTsJAFD3UF+ILdemkZi4IkVIYUlk40oxETACIW0ZcEJpm3ZqQhC3/oBb/S3jH+hfeGcoiS6ITtP2zLn3 nJl7rx24PBKG8ZHSVlbX1jfSm5mt7Z3dvez+QTPy49BhDcd3/fDWtiLmco81Bcuw1CZo1tl7XsU3GWw8sjLjv3YhJwLpj a+jxAXcsQdRdrTe1OgHXL2e9bM7IFysl0yzpCpSL5hyYJVMv5A21ckhW3c+o4M+fDiIMQaDB0HYhYWInjYKMBAQ18WUuJAQ V3GTKkjSmLUYZF7Ii+Q9q1E9ajvfSMlNqhU1x6Q1LqOCGNT3khYXmaruKxcpbsMu+p8pR3m9DfTrzGxArcE/uXbpH5X52s RWCAiqBU02BYmR1TuISq67Im+s/qhLkEBAncZ/iIWFHKRd91pUmUrXL3loq/qkyJSv3TpIb40vekga8mK+HDTP8gUzX7gu 5arnyajTOMIxTmeZVRxgToa5O3hBa94060WHvUnuapWirRHOLX0p6/ASK9k2A=</latexit>Ca⇡N <latexit sha1_base64="MWedM/cqbMYIRq3ISXcQY5KYvrw=">ACzXicjVHLTsJAFD3UF+ILdemkZi4IkVIYUlk40ox ETACIW0ZcEJpm3ZqQhC3/oBb/S3jH+hfeGcoiS6ITtP2zLn3nJl7rx24PBKG8ZHSVlbX1jfSm5mt7Z3dvez+QTPy49BhDcd3/fDWtiLmco81Bcuw1CZo1tl7XsU3GWw8sjLjv3YhJwLpja+jxAXcsQdRdrTe1OgHXL2e9bM7IFysl0yzpCpSL5hyYJVMv 5A21ckhW3c+o4M+fDiIMQaDB0HYhYWInjYKMBAQ18WUuJAQV3GTKkjSmLUYZF7Ii+Q9q1E9ajvfSMlNqhU1x6Q1LqOCGNT3khYXmaruKxcpbsMu+p8pR3m9DfTrzGxArcE/uXbpH5X52sRWCAiqBU02BYmR1TuISq67Im+s/qhLkEBAncZ/iIWFHKRd9 1pUmUrXL3loq/qkyJSv3TpIb40vekga8mK+HDTP8gUzX7gu5arnyajTOMIxTmeZVRxgToa5O3hBa94060WHvUnuapWirRHOLX0p6/ASK9k2A=</latexit>Ca⇡N <latexit sha1_base64="Fwvzno9xW64xZx6yK7PgafKY=">ACy3icjVHLTsJAFD3UF+ILdemkZi4IkWbwpLIxo0J JvJIgJC2DihtE07NUFk6Q+41f8y/oH+hXeGkuiC6DRtz5x7zp259zqhx2NhGB8ZbW19Y3Mru53b2d3bP8gfHjXjIlc1nADL4jajh0zj/usIbjwWDuMmD1xPNZyxjUZbz2wKOaBfyemIetN7JHPh9y1BVHtWn9md0M+7+cLRvGyYlqWqStQvrQWwDItvVQ0 1CogXfUg/4uBgjgIsED4EYQ82Yno6KMFASFwPM+IiQlzFGebIkTchFSOFTeyYviPadVLWp73MGSu3S6d49Ebk1HFGnoB0EWF5mq7icos2VW5ZyqnvNuU/k6a0KswD2xf/mWyv/6ZC0CQ1RUDZxqChUjq3PTLInqiry5/qMqQRlC4iQeUDwi7Crns+6 8sSqdtlbW8U/lVKycu+m2gRf8pY04OU9dWgeVEsWcXSrVmoXqWjzuIEpzineZRxTXqaKg5vuAVb9qNFmuP2tNCqmVSzF+Le35G8vNkt4=</latexit>Ca⇡ Figure 4. Other diagrams involving the contact interactions. ⇡− n a p p n <latexit sha1_base64="MWedM/cqbMYIRq3ISXcQY5KYvr w=">ACzXicjVHLTsJAFD3UF+ILdemkZi4IkVIYUlk40oxETACIW0ZcEJpm3ZqQhC3/oBb/S3jH+hfeGcoiS6ITtP2zLn3 nJl7rx24PBKG8ZHSVlbX1jfSm5mt7Z3dvez+QTPy49BhDcd3/fDWtiLmco81Bcuw1CZo1tl7XsU3GWw8sjLjv3YhJwLpj a+jxAXcsQdRdrTe1OgHXL2e9bM7IFysl0yzpCpSL5hyYJVMv5A21ckhW3c+o4M+fDiIMQaDB0HYhYWInjYKMBAQ18WUuJAQ V3GTKkjSmLUYZF7Ii+Q9q1E9ajvfSMlNqhU1x6Q1LqOCGNT3khYXmaruKxcpbsMu+p8pR3m9DfTrzGxArcE/uXbpH5X52s RWCAiqBU02BYmR1TuISq67Im+s/qhLkEBAncZ/iIWFHKRd91pUmUrXL3loq/qkyJSv3TpIb40vekga8mK+HDTP8gUzX7gu 5arnyajTOMIxTmeZVRxgToa5O3hBa94060WHvUnuapWirRHOLX0p6/ASK9k2A=</latexit>Ca⇡N ⇡− n p p n a <latexit sha1_base64="MWedM/cqbMYIRq3ISXcQY5KYvrw=">ACzXicjVHLTsJAFD3UF+ILdemkZi4IkVIYUlk40ox ETACIW0ZcEJpm3ZqQhC3/oBb/S3jH+hfeGcoiS6ITtP2zLn3nJl7rx24PBKG8ZHSVlbX1jfSm5mt7Z3dvez+QTPy49BhDcd3/fDWtiLmco81Bcuw1CZo1tl7XsU3GWw8sjLjv3YhJwLpja+jxAXcsQdRdrTe1OgHXL2e9bM7IFysl0yzpCpSL5hyYJVMv 5A21ckhW3c+o4M+fDiIMQaDB0HYhYWInjYKMBAQ18WUuJAQV3GTKkjSmLUYZF7Ii+Q9q1E9ajvfSMlNqhU1x6Q1LqOCGNT3khYXmaruKxcpbsMu+p8pR3m9DfTrzGxArcE/uXbpH5X52sRWCAiqBU02BYmR1TuISq67Im+s/qhLkEBAncZ/iIWFHKRd9 1pUmUrXL3loq/qkyJSv3TpIb40vekga8mK+HDTP8gUzX7gu5arnyajTOMIxTmeZVRxgToa5O3hBa94060WHvUnuapWirRHOLX0p6/ASK9k2A=</latexit>Ca⇡N ⇡0 ⇡− a ⇡− <latexit sha1_base64="Fwvzno9xW64xZx6yK7PgafKY=">ACy3icjVHLTsJAFD3UF+ILdemkZi4IkWbwpLIxo0J JvJIgJC2DihtE07NUFk6Q+41f8y/oH+hXeGkuiC6DRtz5x7zp259zqhx2NhGB8ZbW19Y3Mru53b2d3bP8gfHjXjIlc1nADL4jajh0zj/usIbjwWDuMmD1xPNZyxjUZbz2wKOaBfyemIetN7JHPh9y1BVHtWn9md0M+7+cLRvGyYlqWqStQvrQWwDItvVQ0 1CogXfUg/4uBgjgIsED4EYQ82Yno6KMFASFwPM+IiQlzFGebIkTchFSOFTeyYviPadVLWp73MGSu3S6d49Ebk1HFGnoB0EWF5mq7icos2VW5ZyqnvNuU/k6a0KswD2xf/mWyv/6ZC0CQ1RUDZxqChUjq3PTLInqiry5/qMqQRlC4iQeUDwi7Crns+6 8sSqdtlbW8U/lVKycu+m2gRf8pY04OU9dWgeVEsWcXSrVmoXqWjzuIEpzineZRxTXqaKg5vuAVb9qNFmuP2tNCqmVSzF+Le35G8vNkt4=</latexit>Ca⇡ a Figure 4. Other diagrams involving the contact interactions. JHEP02(2022)143 JHEP02(2022)143 where ϵπ0 = Eπ0/T, yπ0 = mπ0/T, and Cnπ0 a ≃4 3g2 A |pπ| mN 2 C2 an. 3.2 Nucleon-nucleon bremsstrahlung For many years, the nucleon-nucleon bremsstrahlung has been considered to be the dom- inating process for axion emission from supernovae. Although a recent study indicates that the axion emissivity of the bremsstrahlung process is sensitive to the corrections to the one-pion exchange as well as the medium effects [28], here we do a simpler analysis ignoring these corrections since we are mainly concerned with a relative importance of the axion-pion-nucleon contact interaction CaπN compared to the other axion-nucleon inter- actions. In [42], the same analysis has been done for the nucleon-nucleon bremsstrahlung with the contact interaction. It shows that the contribution from the contact interaction is negligible at the squared matrix element level. In this subsection, we examine the con- tribution from the contact interaction to the final axion emissivity including the phase – 10 – space integration, and confirm that it is still negligible for the environmental parameters of SN 1987A.7 Among the three possible nucleon-nucleon bremsstrahlung processes, n+n →n+n+a, n+p →n+p+a, and p+p →p+p+a, at leading order in pion-nucleon couplings only the second process is affected by CaπN through the first two diagrams of figure 4. The axion emissivity of the three bremsstrahlung processes is given by QI a = Z Y α=N1,N2, N3,N4, a d3pα (2π)32Eα " (2π)4δ(4)(p1 + p2 −p3 −p4 −pa) (3.13) (3.13) JHEP02(2022)143 × f1(p1)f2(p2)(1 −f3(p3))(1 −f4(p4)) X spins SI|MI|2Ea # (I = nn, np, pp), where p1,2 = (E1,2, p1,2) and p3,4 = (E3,4, p3,4) denote the initial and final nucleon four- momenta, pa = (Ea, pa) is the axion four-momentum, and SI is a symmetry factor for identical particles in the initial and final states, i.e., Snn = Spp = 1/4 and Snp = 1. In the supernova environments, |pa| ∼T ≪|pN| ∼max √mNT, pF  where pF is the nucleon Fermi momentum. Therefore we take the following approximation p1 + p2 ≃p3 + p4 , (3.14) (3.14) which simplifies the kinematics significantly. With this approximation and also at leading order in 1/mN, the squared matrix elements averaged over the axion momentum direction are given by * X spins |Mnn|2 + = 16 3 g4 A f4π m4 N f2a C2 an |k|4 (|k|2 + m2π)2 + |l|4 (|l|2 + m2π)2 +(1 −β) |k|2|l|2 (|k|2 + m2π)(|l|2 + m2π) ! 7In appendix A, we estimate the axion emissivity in the degenerate limit by applying the analytic method presented in [27]. In that estimation, the contact interaction seems to contribute to the axion emissivity in the same order of magnitude, but the environmental parameters given in [29, 32] turn out to be not degenerate enough to apply the method. 3.2 Nucleon-nucleon bremsstrahlung , (3.15) * X spins |Mpp|2 + = 16 3 g4 A f4π m4 N f2a C2 ap |k|4 (|k|2 + m2π)2 + |l|4 (|l|2 + m2π)2 +(1 −β) |k|2|l|2 (|k|2 + m2π)(|l|2 + m2π) ! , (3.16) * X spins |Mnp|2 + = 16 3 g2 A f4π m4 N f2a " g2 A ( (4C2 + + 2C2 −) |k|4 (|k|2 + m2π)2 + (C2 + + C2 −) |l|4 (|l|2 + m2π)2 −2  (C2 + + C2 −) −(3C2 + + C2 −) β 3  |k|2|l|2 (|k|2 + m2π)(|l|2 + m2π) ) +3C2 aπN |pa|2|k|2 (|k|2 + m2π)2 # , (3.17) 7 * X spins |Mnn|2 + = 16 3 g4 A f4π m4 N f2a C2 an |k|4 (|k|2 + m2π)2 + |l|4 (|l|2 + m2π)2 +(1 −β) |k|2|l|2 (|k|2 + m2π)(|l|2 + m2π) ! , (3.15) * X spins |Mpp|2 + = 16 3 g4 A f4π m4 N f2a C2 ap |k|4 (|k|2 + m2π)2 + |l|4 (|l|2 + m2π)2 | |2| |2 ! (3.15) +(1 −β) |k|2|l|2 (|k|2 + m2π)(|l|2 + m2π) ! , (3.16) (3.16) ! * X spins |Mnp|2 + = 16 3 g2 A f4π m4 N f2a " g2 A ( (4C2 + + 2C2 −) |k|4 (|k|2 + m2π)2 + (C2 + + C2 −) |l|4 (|l|2 + m2π)2 −2  (C2 + + C2 −) −(3C2 + + C2 −) β 3  |k|2|l|2 (|k|2 + m2π)(|l|2 + m2π) ) +3C2 aπN |pa|2|k|2 (|k|2 + m2)2 # , (3.17) ! = 16 3 g2 A f4π m4 N f2a " g2 A ( (4C2 + + 2C2 −) |k|4 (|k|2 + m2π)2 + (C2 + + C2 −) |l|4 (|l|2 + m2π)2 −2  (C2 + + C2 −) −(3C2 + + C2 −) β 3  |k|2|l|2 (|k|2 + m2π)(|l|2 + m2π) ) # 2 πN |pa|2|k|2 (|k|2 + m2π)2 # , (3.17) (3.17) 7In appendix A, we estimate the axion emissivity in the degenerate limit by applying the analytic method presented in [27]. In that estimation, the contact interaction seems to contribute to the axion emissivity in the same order of magnitude, but the environmental parameters given in [29, 32] turn out to be not degenerate enough to apply the method. 3.2 Nucleon-nucleon bremsstrahlung – 11 – – 11 – where k ≡p1 −p3, l ≡p1 −p4, β ≡3  k · l |k| |l| 2 . (3.18) (3.18) For the neutron-proton bremsstrahlung, we define the momentum exchanges as k = pi n−pf p and l = pi n −pf n. While the squared matrix elements of n + n →n + n + a and p + p →p + p + a are the same as the previous results [28], the squared matrix element of n+p →n+p+a includes an additional contribution from the contact interaction CaπN. We remark that we have only displayed the leading-order contribution (in 1/mN) for each coupling term in the angle- averaged squared matrix elements. Then, compared to other terms, the term induced by CaπN in eq. (3.17) is intrinsically higher order as it is suppressed by |pa|2/|pN|2 ∼T/mN for |k| ∼|l| ∼|pN|. This indicates that the contribution from CaπN to the axion emissivity of n+p →n+p+a is likely to be negligible as pointed out in [42]. If the typical values into the kinetic parameters are taken, e.g., T = 40 MeV, β ≃1.3 (non-degenerate limit), |k| ∼ |l| ∼|pN| ≃√3mNT ≃340 MeV, |pa| ≃Ea ≃|pN|2/(2mN) ≃60 MeV, we could see a numerical estimate of the square brackets in eq. (3.17), [ · · · ] ≃6.6 C2 ++2.2 C2 −+0.07 C2 aπN. The estimation predicts a relative importance of each term, which is shown in eq. (3.22), although there appears some enhancement of the contribution from the contact interaction after the phase space integration. JHEP02(2022)143 The axion emissivity in eq. (3.13) can be simplified by taking non-relativistic limit for nucleons together with the approximation eq. (3.14). 8Our numerical results agree well with the analytic results of the previous works [25, 27]; for the contri- butions from C±, the agreement is at the level of O(10)% discrepancy in both degenerate (zn/p ≫1) and non-degenerate (zn/p ≪1) limits. We also confirm that the contribution from CaπN agrees well with an analytic result in the degenerate limit [27]. See appendix A. 3.2 Nucleon-nucleon bremsstrahlung Following [25, 26], we can write the axion emissivities in a form which allows a numerical calculation of the phase space integration: QI a ≃ s mNT 13 29π16 Z ∞ 0 du+ Z ∞ 0 du− Z 1 −1 dγ+− Z u− 0 du3c Z 4π dΩ3c √u+u−u3c(u−−u3c)2 × f1f2(1 −f3)(1 −f4) X spins SI D |MI|2E p4c=−p3c, Ea=2T (u−−u3c) , (3.19) h QI a ≃ s mNT 13 29π16 Z ∞ 0 du+ Z ∞ 0 du− Z 1 −1 dγ+− Z u− 0 du3c Z 4π dΩ3c √u+u−u3c(u−−u3c)2 × f1f2(1 −f3)(1 −f4) X spins SI D |MI|2E p4c=−p3c, Ea=2T (u−−u3c) , (3.19) (3.19) where ui ≡ p2 i 2mNT , p± ≡p1 ± p2 2 , pjc ≡pj −p+, γkl ≡pk · pl |pk||pl|. (3.20) ui ≡ p2 i 2mNT , p± ≡p1 ± p2 2 , pjc ≡pj −p+, γkl ≡pk · pl |pk||pl|. (3.20) (3.20) Again, we use the fugacities of nucleons from [29] to numerically calculate the above axio Again, we use the fugacities of nucleons from [29] to numerically calculate the above axion – 12 – emissivities, which results in8 emissivities, which results in8 emissivities, which results in8 Qnn a erg · cm−3 s−1 ≃3.5 × 1033 T 3.9 40 ρ2.1 14 109 GeV fa 2 C2 an, (3.21) Qnp a erg · cm−3 s−1 ≃7.5 × 1033 T 6.9 40 ρ1.5 14 109 GeV fa 2 C2 + + 2.5 × 1033 T 6.9 40 ρ1.5 14 109 GeV fa 2 C2 − (3.22) + 2.7 × 1032 T 7.9 40 ρ1.5 14 109 GeV fa 2 C2 aπN, Qpp a erg · cm−3 s−1 ≃9.9 × 1031 T 9.9 40 ρ0.92 14 109 GeV fa 2 C2 ap (3.23) (3.21) (3.22) JHEP02(2022)143 Qpp a erg · cm−3 s−1 ≃9.9 × 1031 T 9.9 40 ρ0.92 14 109 GeV fa 2 C2 ap (3.23) (3.23) for T40 ∈[0.9, 1.1] and ρ14 ∈[1, 3]. for T40 ∈[0.9, 1.1] and ρ14 ∈[1, 3]. for T40 ∈[0.9, 1.1] and ρ14 ∈[1, 3]. for T40 ∈[0.9, 1.1] and ρ14 ∈[1, 3]. for T40 ∈[0.9, 1.1] and ρ14 ∈[1, 3]. – 13 – 36 38 40 42 44 46 48 50 1031 1032 1033 1034 1035 36 38 40 42 44 46 48 50 1031 1032 1033 1034 1035 Figure 5. The axion emissivity Qnp a for the three benchmark models considered in figure 3. 3.2 Nucleon-nucleon bremsstrahlung The solid (dotted) curves correspond to the total value of Qnp a (the piece induced only by CaπN). 36 38 40 42 44 46 48 50 1031 1032 1033 1034 1035 36 38 40 42 44 46 48 50 1031 1032 1033 1034 1035 JHEP02(2022)143 Figure 5. The axion emissivity Qnp a for the three benchmark models considered in figure 3. The solid (dotted) curves correspond to the total value of Qnp a (the piece induced only by CaπN). The above result shows that, as anticipated from the structure of the squared matrix element, the contribution to Qnp a from CaπN is indeed about one order of magnitude smaller than the contribution from C−for astrophysical environments with T ∼40 MeV and ρ ∼ 1014 g/cm3. Using the relation CaπN = √ 2C−/gA, the ratios between the contribution from the contact interaction and the other terms become, respectively, ∆Qnp a, C2 aπN ∆Qnp a, C2 + ≃0.04 C2 aπN C2 + ! , ∆Qnp a, C2 aπN ∆Qnp a, C2 − ≃0.1. (3.24) ∆Qnp a, C2 aπN ∆Qnp a, C2 + ≃0.04 C2 aπN C2 + ! , ∆Qnp a, C2 aπN ∆Qnp a, C2 − ≃0.1. (3.24) (3.24) As in the case of Qpπ− a , these ratios are expected to be less sensitive to the corrections beyond the one-pion exchange and the medium effects, so the effect of the contact inter- action CaπN on the nucleon-nucleon bremsstrahlung is negligible. In figure 5, we compare the total value of the axion emissivity Qnp a (solid curves) with the piece ∆Qnp a, C2 aπN (dotted curves) induced only by CaπN for the three benchmark models considered in figure 3. The result shows that the contribution from CaπN is negligible for 30 ≲T/MeV ≲50 and 1 ≲ρ/(1014 g/cm3) ≲3, which is expected to be true for even wider range of T and ρ. Note that Qnp a is comparable to (or even larger than) Qnn a , although the proton number density is significantly smaller than the neutron number density. This is partly due to the symmetry factor S compensating the small proton fraction.9 3.3 Pion-pion scattering: π + π →π + a 3.3 Pion-pion scattering: π + π →π + a 3.3 Pion-pion scattering: π + π →π + a 3.3 Pion-pion scattering: π + π →π + a Let us finally consider the possible consequence of the axion-pion contact interaction Caπ in eq. (2.9). Axions can be produced by this coupling through the pion-pion scattering process π−+ π0 →π−+ a (see the third diagram in figure 4). The corresponding emissivity can be simplified without any kinematic approximation as follows: Qπ−π0 a = 9 212π7 C2 aπ f2πf2a zπ−zπ0T 9 Z dxindx0dΩin π−dΩπ0 x2 in q y2π + x2 in x2 0 q y2π + x2 0 x2 a Eout/T × pπ0 · pa T 2 2 1 e √ y2π+x2 in−yπ −zπ− 1 e √ y2π+x2 0−yπ −zπ0 eEout/T−yπ eEout/T−yπ −zπ−, (3.25) Qπ−π0 a = 9 212π7 C2 aπ f2πf2a zπ−zπ0T 9 Z dxindx0dΩin π−dΩπ0 x2 in q y2π + x2 in x2 0 q y2π + x2 0 x2 a Eout/T × pπ0 · pa T 2 2 1 e √ y2π+x2 in−yπ −zπ− 1 e √ y2π+x2 0−yπ −zπ0 eEout/T−yπ eEout/T−yπ −zπ−, (3.25) (3.25) 9The relative importance of the neutron-proton bremsstrahlung is discussed within the framework of the neutrino emission through the nucleon-nucleon bremsstrahlung [43]. 9The relative importance of the neutron-proton bremsstrahlung is discussed within the framework of t neutrino emission through the nucleon-nucleon bremsstrahlung [43]. 9The relative importance of the neutron-proton bremsstrahlung is discussed within the framework of the neutrino emission through the nucleon-nucleon bremsstrahlung [43]. – 14 – where xin = |pin π−|/T for the incoming π−, x0 = |pπ0|/T, xa = |pa|/T, yπ = mπ/T, and finally Eout = q m2π + (pin π−+ pπ0 −pa)2 is the energy of the outgoing π−. Like the emissivity of other processes, we use the fugacities of pions from [29] and calculate the integral in eq. (3.25) numerically to find Qπ−π0 a erg · cm−3 s−1 = 3.6 × 1031 T 10.1 40 ρ1.1 14 109 GeV fa !2 C2 aπ. (3.26) (3.26) The above result shows that the axion emissivity of the pion-pion scattering π−+ π0 → π−+a is negligible compared to that of π−+p →n+a for T ∼40 MeV and ρ ∼1014 g/cm3 and Caπ = 2(Cap −Can)/(3gA) (see eq. (2.10)). JHEP02(2022)143 4 Conclusions and discussion In this paper, we have studied the axion emission from supernovae with a complete set of relevant axion couplings including the axion-pion-nucleon contact interaction CaπN and the axion-pion contact interaction Caπ in eq. (2.9). A recent study suggests that the abun- dance of negatively charged pions inside supernovae is significantly enhanced by the strong interactions [29], indicating that the pion-induced process π−+p →a+n is the dominating process for a wide range of astrophysical conditions encountered inside supernovae [32, 33]. We thus examined how this pion-induced process is affected by CaπN. We also examined the effect of CaπN on the nucleon-nucleon bremsstrahlung which has been considered as the dominating process for many years. Since we are mainly concerned with the role of the two previously ignored couplings CaπN and Caπ, we have focused on the axion coupling dependence of the axion emissivity within a simple approximation to keep only the leading order in pion-nucleon couplings, which also ignores medium effects. In such an approximation, we could show the axion coupling dependence more explicitly and examine the ignored couplings for three processes. Two processes, π−+ p →a + n and n + p →n + p + a, are affected by CaπN, and the pion-pion scattering, π + π →π + a, is affected by Caπ. We found that CaπN can enhance the axion emissivity of π−+ p →a + n by a factor of 2 −4, depending on the pattern of axion couplings determined by the underlying axion model, while there is no substantial effect on n + p →n + p + a. Although it is independent of CaπN, we have also examined the axion emissivity of π0 + n →n + a and find that it can be comparable to the emissivity of π−+ p →a + n over a wide range of axion parameter space. For the axion-pion contact interaction Caπ, we find that the corresponding axion emissivity is always negligible compared to that of π−+ p →a + n for ambient conditions encountered inside supernovae. Let us make final remarks on the approximation we made. For the matrix elements, the higher-order diagrams could give comparable contributions due to the strong interaction. Moreover, the medium effects significantly change the axion emissivity, particularly for the nucleon-nucleon bremsstrahlung [28, 33]. 4 Conclusions and discussion However, even including these effects, the relative contribution of the axion-pion-nucleon contact interaction CaπN to the axion emissivity – 15 – would remain similar because it is likely that the ratio ∆QI a, CaπN /QI a is less sensitive to the corrections than the emissivity itself, where QI a is the axion emissivity of the I-th process, and ∆QI a, CaπN is the part of QI a induced by CaπN. One of the purposes of this work is to call attention to the possible importance of the contact interactions which have been neglected so far. It would be interesting to perform the analysis taking into account more precise matrix elements and medium effects with a complete set of axion couplings. We will investigate this issue in a self-consistent way for both the pion-nucleon scattering and the nucleon-nucleon bremsstrahlung in future works. Acknowledgments JHEP02(2022)143 This work was supported by IBS under the project code, IBS-R018-D1. We are grateful to S. Yun for helpful discussions, especially on the nucleon-nucleon bremsstrahlung process in the early stage of this project. This work was supported by IBS under the project code, IBS-R018-D1. We are grateful to S. Yun for helpful discussions, especially on the nucleon-nucleon bremsstrahlung process in the early stage of this project. A Axion emissivity from n + p →n + p + a in degenerate limit A Axion emissivity from n + p →n + p + a in degenerate limit A Axion emissivity from n + p →n + p + a in degenerate limit In the degenerate regime, the axion emissivity for the nucleon-nucleon bremsstrahlung can be derived in an analytical way because the phase space is highly constrained [27]. We can apply the method to include the contribution from the contact term, which is a higher order in the expansion with respect to |pa|/|pN| ∼ p T/mN (see eq. (3.17)). Although our system belongs to the non-degenerate regime, the analytical estimation provides some insights for the numerical results. Moreover, the analytical result is applicable for the degenerate system like a neutron star. (∆Qnp a )C2 aπN (Qnp a )CaπN=0 = 123π2 124g2 A T 2 p2 F,n y x2 C2 aπN  H  2xy x + y  −H  2xy y −x   ×   C2 −+ C2 +  F(y) −2  C2 −+ C2 +  G(y) +  C2 −+ 2C2 +   1 −y x  F  2xy y −x  +  1 + y x  F  2xy x + y  −1 , ≃ C2 aπN C2 −+ 3C2 + 7.0 T pF,n !2 , = (Cap −Can)2 (Cap −Can)2 + 3 (Cap + Can)2 7.7 T pF,n !2 , (A.1) + pF,n ! = (Cap −Can)2 (Cap −Can)2 + 3 (Cap + Can)2 7.7 T pF,n !2 , (A.1) (A.1) where x ≡mπ/(2pF,n), y ≡mπ/(2pF,p), and where x ≡mπ/(2pF,n), y ≡mπ/(2pF,p), and F(u) ≡1 2  3 − 1 1 + u2 −3u cot−1 u  , G(v) ≡1 −v cot−1 v, H(w) ≡− w 1 + w2 + cot−1 w. – 16 – 0 5 10 15 20 25 30 10-2 10-1 100 101 Figure 6. The ratio of the axion emissivity contributed from the contact interaction, i.e. CaπN coupling, to that from the other couplings, Cap and Can. We compare the numerical result (red dots) with an analytic estimation (dashed). For benchmark parameters, we assume µp −mp ≃ 0.25(µn −mn) and Cap = −Can = 1/2. 0 5 10 15 20 25 30 10-2 10-1 100 101 JHEP02(2022)143 Figure 6. The ratio of the axion emissivity contributed from the contact interaction, i.e. CaπN coupling, to that from the other couplings, Cap and Can. A Axion emissivity from n + p →n + p + a in degenerate limit We compare the numerical result (red dots) with an analytic estimation (dashed). For benchmark parameters, we assume µp −mp ≃ 0.25(µn −mn) and Cap = −Can = 1/2. For the second equality of eq. (A.1), we keep the leading order with respect to x and y. Given the parameter values in the main text, e.g. T ≃40 MeV and pF,n ≃200 −300 MeV, eq. (A.1) leads to the wrong interpretation that the new contribution from the contact interaction is comparable to the other contributions from the Cap and Can couplings. The discrepancy between the analytic estimation in eq. (A.1) and the numerical results in eq. (3.22) is caused by applying the formula derived in the degenerate limit to the non- degenerate system [32]. 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Some aspects of associations and interactions between biomedical variables
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Abstract The objective of this text is to investigate the association between biomedical variables using Fisher’s exact test as non- parametric tests. In common applications of Fisher’s exact test, one can find the associations between categorical variables through calculation the probability of significance. For the purpose of illustration, represented different levels of categorical variables, several contingency tables were simulated for the investigation the associations as well as interactions between biomedical variables. The results of Fisher’s exact test indicated some discrepancy between statistical and biological issues, which may indicate that some statistical result may not match biological features. The results presented in 13 Figures showed plots of descriptive variables (gender and disease incidence). The association pattern is more obvious when the lines representing the sex in the plots are not parallel; these imply that there is an interaction effect between gender and diseases incidence. Whereas, when the lines were parallel, these indicate no interaction. It was concluded that the non-significant biometric association between biomedical variables need precise biological understanding of the mode of action, and that further investigations are needed. Keywords: Biomedical variables; Association; Interaction; Alrawi Rakhad Abdulrazak 1, *, Abdulrahman Jubran Mohamed 2, Al-Rawi Rafal Abdulrazaq 3 and Al-Rawi Raafat Abdulrazak 4 Alrawi Rakhad Abdulrazak 1, *, Abdulrahman Jubran Mohamed 2, Al-Rawi Rafal Abdulrazaq 3 and Al-Rawi Raafat Abdulrazak 4 1 Grand Canyon University, Arizona, USA. 2 Center of Research and Educational Studies, Ministry of Education, Iraq. 3 Deptartment of Pathology, Hawler Medical University, Erbil, Kurdistan, Iraq. 4 ArlövKronetorpsvägen 2 (Burlöv Center), Malmo, Swede. P bli i hi R i d 12 J 2020 i d 25 J 2020 d 27 J 2020 1 Grand Canyon University, Arizona, USA. 2 Center of Research and Educational Studies, Ministry of Education, Iraq. 3 Deptartment of Pathology, Hawler Medical University, Erbil, Kurdistan, Iraq. 4 ArlövKronetorpsvägen 2 (Burlöv Center), Malmo, Swede. 1 Grand Canyon University, Arizona, USA. 2 Center of Research and Educational Studies, Ministry of Education, Iraq. 3 Deptartment of Pathology, Hawler Medical University, Erbil, Kurdistan, Iraq. 4 ArlövKronetorpsvägen 2 (Burlöv Center), Malmo, Swede. 1 Grand Canyon University, Arizona, USA. Article DOI: https://doi.org/10.30574/wjarr.2020.5.1.0018 Article DOI: https://doi.org/10.30574/wjarr.2020.5.1.0018 Copyright © 2020 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons E-mail address: Copyright © 2020 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 (RESEARCH ARTICLE) World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 World Journal of Advanced Research and Reviews e-ISSN: 2581-9615, Cross Ref DOI: 10.30574/wjarr Journal homepage: https://www.wjarr.com (RESEARCH ARTICLE) Some aspects of associations and interactions between biomedical variables Alrawi Rakhad Abdulrazak 1, *, Abdulrahman Jubran Mohamed 2, Al-Rawi Rafal Abdulrazaq 3 and Al-Rawi Raafat Abdulrazak 4 1 Grand Canyon University, Arizona, USA. 2 Center of Research and Educational Studies, Ministry of Education, Iraq. 3 Deptartment of Pathology, Hawler Medical University, Erbil, Kurdistan, Iraq. 4 ArlövKronetorpsvägen 2 (Burlöv Center), Malmo, Swede. Publication history: Received on 12 January 2020; revised on 25 January 2020; accepted on 27 January 2020 Article DOI: https://doi.org/10.30574/wjarr.2020.5.1.0018 Abstract The objective of this text is to investigate the association between biomedical variables using Fisher’s exact test as non- parametric tests. In common applications of Fisher’s exact test, one can find the associations between categorical variables through calculation the probability of significance. For the purpose of illustration, represented different levels of categorical variables several contingency tables were simulated for the investigation the associations as well as The association pattern is more obvious when the lines representing the sex in the plots are not parallel; these imply that there is an interaction effect between gender and diseases incidence. Whereas, when the lines were parallel, these indicate no interaction. It was concluded that the non-significant biometric association between biomedical variables need precise biological understanding of the mode of action, and that further investigations are needed. Keywords: Biomedical variables; Association; Interaction; 1. Introduction and justification Medicine is the science and practice of establishing the diagnosis, prognosis, treatment, and prevention of diseases. Medicine is general term includes biomedical science, biomedical research, medical technology, and medical statistics. Medical statistics deals with applications of statistics to medicine and clinical research. It is the science of collecting, World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 World Journal of Advanced Research and Reviews e-ISSN: 2581-9615, Cross Ref DOI: 10.30574/wjarr Journal homepage: https://www.wjarr.com (RESEARCH ARTICLE) World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 World Journal of Advanced Research and Reviews e-ISSN: 2581-9615, Cross Ref DOI: 10.30574/wjarr Journal homepage: https://www.wjarr.com (RESEARCH ARTICLE) Some aspects of associations and interactions between biomedical variables Alrawi Rakhad Abdulrazak 1, *, Abdulrahman Jubran Mohamed 2, Al-Rawi Rafal Abdulrazaq 3 and Al-Rawi Raafat Abdulrazak 4 1 Grand Canyon University, Arizona, USA. 2 Center of Research and Educational Studies, Ministry of Education, Iraq. 3 Deptartment of Pathology, Hawler Medical University, Erbil, Kurdistan, Iraq. 4 ArlövKronetorpsvägen 2 (Burlöv Center), Malmo, Swede. Publication history: Received on 12 January 2020; revised on 25 January 2020; accepted on 27 January 2020 Article DOI: https://doi.org/10.30574/wjarr.2020.5.1.0018 Abstract The objective of this text is to investigate the association between biomedical variables using Fisher’s exact test as non- parametric tests. In common applications of Fisher’s exact test, one can find the associations between categorical variables through calculation the probability of significance. For the purpose of illustration, represented different levels of categorical variables several contingency tables were simulated for the investigation the associations as well as World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 World Journal of Advanced Research and Reviews e-ISSN: 2581-9615, Cross Ref DOI: 10.30574/wjarr Journal homepage: https://www.wjarr.com (RESEARCH ARTICLE) Some aspects of associations and interactions between biomedical variables Alrawi Rakhad Abdulrazak 1, *, Abdulrahman Jubran Mohamed 2, Al-Rawi Rafal Abdulrazaq 3 and Al-Rawi Raafat Abdulrazak 4 1 Grand Canyon University, Arizona, USA. 2 Center of Research and Educational Studies, Ministry of Education, Iraq. 3 Deptartment of Pathology, Hawler Medical University, Erbil, Kurdistan, Iraq. 4 ArlövKronetorpsvägen 2 (Burlöv Center), Malmo, Swede. Publication history: Received on 12 January 2020; revised on 25 January 2020; accepted on 27 January 2020 Article DOI: https://doi.org/10.30574/wjarr.2020.5.1.0018 Abstract The objective of this text is to investigate the association between biomedical variables using Fisher’s exact test as non- parametric tests. In common applications of Fisher’s exact test, one can find the associations between categorical variables through calculation the probability of significance. For the purpose of illustration, represented different levels of categorical variables several contingency tables were simulated for the investigation the associations as well as World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 World Journal of Advanced Research and Reviews e-ISSN: 2581-9615, Cross Ref DOI: 10.30574/wjarr Journal homepage: https://www.wjarr.com (RESEARCH ARTICLE) Some aspects of associations and interactions between biomedical variables Alrawi Rakhad Abdulrazak 1, *, Abdulrahman Jubran Mohamed 2, Al-Rawi Rafal Abdulrazaq 3 and Al-Rawi Raafat Abdulrazak 4 1 Grand Canyon University, Arizona, USA. 2 Center of Research and Educational Studies, Ministry of Education, Iraq. 3 Deptartment of Pathology, Hawler Medical University, Erbil, Kurdistan, Iraq. 4 ArlövKronetorpsvägen 2 (Burlöv Center), Malmo, Swede. Publication history: Received on 12 January 2020; revised on 25 January 2020; accepted on 27 January 2020 Article DOI: https://doi.org/10.30574/wjarr.2020.5.1.0018 Abstract The objective of this text is to investigate the association between biomedical variables using Fisher’s exact test as non- parametric tests. In common applications of Fisher’s exact test, one can find the associations between categorical variables through calculation the probability of significance. For the purpose of illustration, represented different levels of categorical variables, several contingency tables were simulated for the investigation the associations as well as interactions between biomedical variables. The results of Fisher’s exact test indicated some discrepancy between statistical and biological issues, which may indicate that some statistical result may not match biological features. The results presented in 13 Figures showed plots of descriptive variables (gender and disease incidence). Copyright © 2020 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. 1.1. Biological variables Identification and description of variables used in biomedical research is a necessary component. Research question, objectives and rational are the steps in any research work. Depending on the data collected, researchers decide what the statistical methods to be used for analysis [3]. Researchers have to be familiar with the variety of variables involved for result presentation. Information collected about a sample of patients comprises characteristics which vary among the patients, such as age, sex, body temperature, blood glucose level, blood group, blood pressure, pulse rate, severity of disease (mild, moderate, severe), and sensitive/resistance to diseases are some examples for biological variables. A basic distinction among these variables is their quantitative or qualitative (categorical) measurements. Quantitative variables are those which can be measured quantitatively (numerically), such as blood pressure, glucose level, as well as other biochemical variables namely {glutathione-S-transferase activity (GST), super oxide dismutase activity (SOD), glutathione reductase activity (GR), catalase activity (Cat), total antioxidant activity (TAC) and malondialdehyde (MDA), Creatinine (Creat), Alanine Aminotransferase (ALT), AST, Gamma-Glutamyl transferase}. In statistics, these variables represent continuous variables. Furthermore, discrete variables (discontinuous variable usually counts) can take only few numbers of values, such number of children in a family, number of patients visited a clinic per day. On the other hand, qualitative (categorical) variables may have two or more categories such as sex (male/female), alive/dead, blood group (A/B/O), genotypes (AA/Aa/aa), and stages of a disease (stage I to stage IV). Usually, for data analysis, categories are coded as numerical values (e.g., 1 for male, 2 for female). Qualitative variables are summarized through counting (frequency) and percentage. Data for two or more categorical variables may be presented in the form of a contingency table showing count, frequency and percentages. Furthermore, it is possible to convert a measurement variable to categorical ones, dividing individuals up into a two or more classes based on ranges of the variable. Many researchers convert quantitative continuous variables to categorical ones using “Cut-points”. Instead of presenting the mean fasting glucose level, one may prefer to present the proportion of diabetics in population using a fasting glucose level of 110 mg/dL as the cut-point to categorize the subjects as diabetic/ non-diabetic. 1.2. Statistical analysis of data It’s important for all biologists to learn and understand statistics. The type of descriptive and analytical measures to be used in data summarization and analysis, all depend on the type of variables. Type of the variables decides the method of statistical analyses, parametric or non-parametric. Parametric methods, such as Analysis of Variance (ANOVA), Pearson’s correlation, and regression, require the assumption that the data follow a normal distribution. Non- parametric methods make no assumptions about the distribution of the data [3]. Chi-square test (or Fisher’s exact test if the numbers are very small) is the most often used method to compare categorical data. One of the first steps in deciding which statistical test to use is determining what kinds of variables the experiment has. What kind of variables they are, and what is the null hypothesis. A statistical hypothesis test is a method of making decisions using experimental data. A result is called statistically significant if it is unlikely to have occurred by chance. In other words, there is a biological reason for the significance, or just like to say that the differences caused by biological factor(s). These decisions are always made using null-hypothesis tests. For example, if researcher wants to investigate the pulse rate in male and female, the null hypothesis, H0, will be pulse rate of male and female are same. Statistical significance is a possible finding of the test, declared when the observed sample is unlikely to have occurred by chance, if the null hypothesis were true. One characteristic of the test is either to reject or accept the null hypothesis (Alternative hypothesis will not be discussed as it is out of the scope of this article). A calculated value is compared to tabulated values given in all statistical books. The fact that the null hypothesis is assumed to be true from the start of the experiment; if there is a lack of evidence against it. P-value is the probability, assuming the null hypothesis is true, of observing a result at least as extreme as the test statistic. The null hypothesis, H0, which be used in constructing its test statistic. Furthermore, p<0.05 or p<0.01 refer to the probability to reject null hypothesis when it is in fact true. This called α, the probability of type I error (rejecting a null hypothesis when it is in fact true). 1.1. Biological variables Other example if researchers are studying the relationship between levels of HDL (the "good cholesterol") and blood pressure, you could measure the HDL level, then divide people into two groups, "low HDL" (less than 40 mg/dl) and "normal HDL" (40 or more mg/dl) and compare the mean blood pressures of the two groups. Converting measurement variables to categorical variables, if you split into two groups, "categorizing" in general is common in medicine. For example, in a population one measured the variable systolic blood pressure in a sample of 40 males and 40 females, and the null hypothesis is “Males and females have the same systolic blood pressure”. One would compare the mean blood pressure in males and females with a two-sample t- test (parametric test). But if the variable is converted to hypertension status (hypertensive/normal), as qualitative (categorical) variable, then researcher can compare the hypertension frequencies in males and females with a Chi- square test or Fisher’s exact test (non-parametric test). However, statistical analysis of quantitative continuous variables give more accurate and precise compared for categorizing variables. That’s to say that by categorizing measurement variables, researchers may have made an obvious, highly significant relationship between variables become completely non-significant [3, 4]. 1. Introduction and justification Medicine is the science and practice of establishing the diagnosis, prognosis, treatment, and prevention of diseases. Medicine is general term includes biomedical science, biomedical research, medical technology, and medical statistics. Medical statistics deals with applications of statistics to medicine and clinical research. It is the science of collecting, summarizing, presenting and interpreting data in medical practice, as well as using them to estimate the magnitude of associations and test hypotheses. Medical research, experiment design, and statistics are to be unified in order to get better understanding of the results [1].During our statistical analysis, to investigate comparison of the proportions of categorized variables (the prevalence level of Helicobacter pylori: low, moderate or high) of gastritis in males and females using a Fisher's exact test of independence, some results were found not realistic. The results of Fisher’s exact test did not show significant associations between gender and disease incidence for data without interaction, although both sexes have similar linear trends indicating the magnitude and direction of each sex value depend on the value of the other variable, which should come up with a significant association between gender and diseases incidence. Such contradictory between statistical and biological outcomes may indicate that some statistical result may not match biological background. It was reported that “Fisher's test is said to be exact, but some say it provides an exact answer to the wrong question” [2]. Alrawi et al. / World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 1.3. Association and interaction It is very important to understand relationship between variables to draw the right conclusion from a statistical analysis. The relationship between biomedical variables determines how the right conclusions are reached. Without an understanding of this, one may fall into many pitfalls that accompany statistical analysis and infer wrong results from the data. There are several different kinds of relationships between variables. Before drawing a conclusion, one should first understand how one variable changes with the other. How the variables are related, is the relationship linear or quadratic or inverse or logarithmic or something else? There could be a number of indirect consequences and deducing cause and effect can be challenging. Correlation between variables can be positive or negative. Positive correlation implies an increase of one quantity causes an increase in the other whereas in negative correlation, an increase in one variable will cause a decrease in the other. Association between two variables means the values of one variable relate in some way to the values of the other. Association is usually measured by correlation for two continuous variables and by cross tabulation and a Chi-square test or Fisher’s exact test for categorical variables. Essentially, association means the values of one variable generally co-occur with certain values of the other. The direction of association would always depend on the variables that you have whether both variables are moving in one direction or either of the variables are going in opposite directions. On the other hand, an interaction between two variables means the effect of one of those variables on other variable is not constant (the effect differs at different values of the other) [3 - 6]. 2. Methodology Due to some results in statistical analysis of my experimental data were found to be not realistic using a Fisher's exact test of independence, as we were not satisfied, the authors were ambition to learn more and more about the test and why some results did not sound realistic. Therefore, we took the initiative to simulate some contingency tables for statistical analysis to mimic the real medical data. Many 2x2 and 2x3 contingency tables have been formed and built up to figure out the association between biomedical variables. It was focused on gender and disease incidence as a sample and model for the present study. The null hypothesis (Ho) of this study assumed that males and females have similar susceptibility or resistance to a particular disease. Statistical Analysis was done by using GraphPad Software statistical package computer software. Fisher’s exact test was used to calculate the significant differences between studied variables. A probability value of less than 0.05 was considered statistically significant (probability of rejection of null hypothesis when it’s in fact true). Simulated data were plotted on Microsoft Excel Worksheet. The patterns of the plots were moved to Microsoft Word [2]. 3. Results and discussion The results come up with 13 Figures showing plots of the descriptive statistics (gender and number of disease incidence) as medical models manipulated in this study to investigate the associations of a 2x2 and a 2x3 contingency tables using Fisher's exact test. The probability values are presented in each plot. The plots are obvious when the lines representing the sex in the plot are not parallel; this implies there is an interaction effect between gender and diseases incidence. When the lines are parallel these indicate there were no interaction (no statistical significant interaction). The patterns of the plots were presented and discussed in 5 scenarios, as follow: 1.2. Statistical analysis of data When researchers reject null hypothesis, this indicate that there is a biological effect, which cause of the variation (difference) between variables [3]. 96 Alrawi et al. / World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 Alrawi et al. / World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 Alrawi et al. / World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 Figure 1 A plot of a 2x2 contengency table of disease incidence and gender (Similar trend, no interaction, P = 0.6158) Figure 2 A plot of a 2x3 contengency table of disease incidence and gender (Similar trend, no interaction, P = 0.9336) Figure 1 A plot of a 2x2 contengency table of disease incidence and gender (Similar trend, no interaction, P = 0.6158) Figure 1 A plot of a 2x2 contengency table of disease incidence and gender (Similar trend, no interaction, P = 0.6158) Figure 1 A plot of a 2x2 contengency table of disease incidence and gender (Similar trend, no interaction, P = 0.6158) Figure 1 A plot of a 2x2 contengency table of disease incidence and gender Figure 1 A plot of a 2x2 contengency table of disease incidence and gender (Similar trend, no interaction, P = 0.6158) Figure 2 A plot of a 2x3 contengency table of disease incidence and gender (Similar trend, no interaction, P = 0.9336) d i N i ti b t bi di l i bl b t i t ti i t ith (Similar trend, no interaction, P = 0.6158) Figure 2 A plot of a 2x3 contengency table of disease incidence and gender (Similar trend, no interaction, P = 0.9336) (Similar trend, no interaction, P = 0.6158) Figure 2 A plot of a 2x3 contengency table of disease incidence and gender Figure 2 A plot of a 2x3 contengency table of disease incidence and gender (Si il d i i P 0 9336) (Similar trend, no interaction, P = 0.9336) 3.2. Second scenario: No associations between biomedical variables, but interactions exist without changing rank of sex: 3.1. First scenario: No associations and no interactions between biomedical variables: Figure 1 and 2 showplots of disese incedence and gender. Both plots shows similar trends of sex to various disease incidence. Incidence of disease 1 was less that that of disease 2 in both sexes (Figure 1), and that disese 3 was more frequent compared to diseases 1 and 2 in both sexes (Figure 2).The probability values obtained from Fisher’s exact test were equal 0.6158and 0.9336, respectively.These values considered the association between gender and diseases incidence to be not statistically significant. With deep look at the plots, it can be seen that both sexes have similar linear trends (no interaction between gender and diseases incidence indicating additive pattern). Nevertheless, this kind of interaction refers to a situation where both the magnitude and direction of each variable's value can depend on the value of the other variable. Males show higher disease incidence compared to females. This norm of reaction shows lines that are parallel indicating each gender is responding to different diseases in a same way. The discrepancy between statistical and biological outcomes may indicate that some time statistical result may not match biological issue. This statistical result may cause misleading conclusion, which medical researchers should be aware. Biostatistician and biologists must discuss such phenomena. 97 3.2. Second scenario: No associations between biomedical variables, but interactions exist without changing k f 3.2. Second scenario: No associations between biomedical variables, but interactions exist without changing rank of sex: Figures 3, and 4 show probability values of more than 0.05, obtained from Fisher's exact test for a 2x2 contingency table. Similarly, Figures 5, 6, and 7 show show probability values of more than 0.05, obtained from Fisher's exact test for a 2x3 contingency table. These probability values considered the association between gender and diseases incidence to be not statistically significant. It can be seen that both sexes have different trends for the the number of disease incidence cases, indicating marginal interaction between gender and diseases incidence, as the rank of male was higher than female, where the lines of males and females are not parallel, Such phenomena may suggest that males are little bit more sensitive to disease 1 more than females, but both of them have almost similar resistance for disease 2. In other words, this interaction may refer to departure from an additive effects model on a particular scale. These characteristic indicated that such discrepancy between statistical non significant association between gender and disease incidence (reflected by number of infected people), may have biological background and cause, which should be thoroughly investigated. Therfore, medical rsearchers can interpret their results depending on their background, and the real variables under their investigations. Once again, the contradictory between statistical and biological issues may indicate that cautions should be taken by researchers when presenting, discussing their findings as well as the conclusion and recommendations. 98 Alrawi et al. / World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 Alrawi et al. / World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 Figure 3 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.5282). Figure 3 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.5282). Figure 4 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.3962). Figure 4 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.3962). Figure 4 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.3962). rank, P = 0.3962). Figure 5 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.1596) Figure 5 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.1596) Figure 5 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.1596) 99 Alrawi et al. / World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 Figure 6 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.8366) Figure 6 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.8366) Figure 7 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.7177) Figure 6 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.8366) Figure 7 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.7177) Figure 7 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.7177) Figure 7 Number of diseases incidence of the male and female cases (Different trends, interaction with out change in rank, P = 0.7177) 3.3. Third scenario: No associations between biomedical variables, but interactions exist with changing rank f 3.3. Alrawi et al. / World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 Third scenario: No associations between biomedical variables, but interactions exist with changing rank of sex: Figures 8 shows probability value of P = 0.5136, obtained from Fisher's exact test for a 2x2 contingency table. Similarly, Figures 9 shows probability value of P = 0.3906obtained from Fisher's exact test for a 2x3 contingency table. These probability values considered the association between gender and diseases incidence to be not statistically significant. It can be seen that both sexes have different trends for the the number of disease incidence cases, indicating interaction between gender and diseases incidence. The interaction is obvious on the plot because the lines of males and females are not parallel. Ranks of male and female were changed. Such phenomena may suggest that males are more sensitive to disease 1 compared to females in Figure 8 and vise versa to disease 2. Similarly, males are more sensitive to disease 1 and 2 compared to females (Figure 9), but not to disease 3. These characteristic indicated that such discrepancy have biological background and cause, which should be thoroughly investigated. Once again, the contradictory between statistical and biological issues may indicate that researchers beware when presenting and discussing their results. 100 Alrawi et al. / World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 Figure 8 Number of diseases incidence of the male and female cases (Different trends, interaction exist and change in rank, P = 0.5136). Figure 8 Number of diseases incidence of the male and female cases (Different trends, interaction exist and change in rank, P = 0.5136). Figure 9 Number of diseases incidence of the male and female cases (Different trends, interaction exist and change in rank, P = 0.3906) Figure 9 Number of diseases incidence of the male and female cases (Different trends, interaction exist and change in rank, P = 0.3906) .4. Fourth scenario: Both associations and interactions exist between biomedical variables Figures 10, 11, 12, and 13 show probability values of less than 0.05, obtained from Fisher's exact test for a 2x2 and 2x3 contingency tables. These probability values considered the association between gender and diseases incidence to be highly statistical significant. Alrawi et al. / World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 Furthermore, it can be seen that both sexes have different trends for the the number of disease incidence cases, as well as change in the rank of both males and females for diseases incedence, indicating strong and severe interaction between gender and diseases incidence. The interaction is obvious on the plots because the lines of males and females are not parallel. Such phenomena may suggest that males are more sensitive to some diseases compared to females and that females are more sensirive to other diseases compared to males. These characteristic reflect different biological background and cause, which should be thoroughly investigated. Figures 10, 11, 12, and 13 show probability values of less than 0.05, obtained from Fisher's exact test for a 2x2 and 2x3 contingency tables. These probability values considered the association between gender and diseases incidence to be highly statistical significant. Furthermore, it can be seen that both sexes have different trends for the the number of disease incidence cases, as well as change in the rank of both males and females for diseases incedence, indicating strong and severe interaction between gender and diseases incidence. The interaction is obvious on the plots because the lines of males and females are not parallel. Such phenomena may suggest that males are more sensitive to some diseases compared to females and that females are more sensirive to other diseases compared to males. These characteristic reflect different biological background and cause, which should be thoroughly investigated. 101 Alrawi et al. / World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 Figure 10 Number of diseases incidence of the male and female cases (Different trend, interaction with change in rank, P = 0.0014). Figure 10 Number of diseases incidence of the male and female cases (Different trend, interaction with change in rank, P = 0.0014). rank, P = 0.0014). Figure 11 Number of diseases incidence of the male and female cases (Different trend, interaction with extreme change in rank, P = 0.0029). Figure 11 Number of diseases incidence of the male and female cases (Different trend, interaction with extreme change in rank, P = 0.0029). Figure 11 Number of diseases incidence of the male and female cases (Different trend, interaction with extreme change in rank, P = 0.0029). change in rank, P = 0.0029). 5. Conclusion and suggestion It is concluded that integrated multidisciplinary team (statisticians, biologists, physicists, and pathologists) should work together in medical research activities to have more efficient and tangible impact on better interpreting and understanding statistical results. 4. Final remark During many attempts of various data combinations of a 2x2 and 2x3 contingency tables, results of Fisher’s exact test did not show significant associations between gender and disease incidence for data without interaction. It is surprise, although both sexes have similar linear trends indicating additive pattern, and refers to a situation where both the magnitude and direction of each variable's value can depend on the value of the other variable, which should come up with a significant association between gender and diseases incidence. In other words, this indicating each sex is responding to disease incidence in a similar way, indicating that both sexes have similar linear trends and should have biological background and cause. Once again, the discrepancy between statistical and biological outcomes may indicate During many attempts of various data combinations of a 2x2 and 2x3 contingency tables, results of Fisher’s exact test did not show significant associations between gender and disease incidence for data without interaction. It is surprise, although both sexes have similar linear trends indicating additive pattern, and refers to a situation where both the magnitude and direction of each variable's value can depend on the value of the other variable, which should come up with a significant association between gender and diseases incidence. In other words, this indicating each sex is responding to disease incidence in a similar way, indicating that both sexes have similar linear trends and should have biological background and cause. Once again, the discrepancy between statistical and biological outcomes may indicate that some time statistical result may not match biological issue. The scenario that showed “associations exist and no interactions between biomedical variables” phenomena must be discussed by biostatistician and medical researchers. Alrawi et al. / World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 Figure 12 Number of diseases incidence of the male and female cases (Different trend, interaction with extreme change in rank, P = 0.0074). Figure 12 Number of diseases incidence of the male and female cases (Different trend, interaction with extreme change in rank, P = 0.0074). Figure 12 Number of diseases incidence of the male and female cases (Different trend, interaction with extreme change in rank, P = 0.0074). 102 Alrawi et al. / World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 Figure 13 Number of diseases incidence of the male and female cases (Different trend, interaction with extreme change in rank, P = 0.0023). Figure 13 Number of diseases incidence of the male and female cases (Different trend, interaction with extreme change in rank, P = 0.0023). Disclosure of conflict of interest The authors declare that they have no conflicts of interest. The authors declare that they have no conflicts of interest. Acknowledgments The authors express their thanks to Prof. Dr. Abdulrazaq Abdulhamid for his help in statistical manipulation. Acknowledgments must be inserted here. Disclosure of conflict of interest [3] Barbara I and Susan D. (2018). Introductory Statistics. Rice University. Houston, Texas 77005. OpenStax.org. [4] Shreemathi SM, Ashma DM and Sachit G. (2017). Types of biological variables. J Thorac Dis, 9(6), 1730–1733. [4] Shreemathi SM, Ashma DM and Sachit G. (2017). Types of biological variables. J Thorac Dis, References [1] Rafal A. (2017). Is medical research at the right track? Global Journal of Medical Research. K Interdisciplinary, 16(6), 1-7. [1] Rafal A. (2017). Is medical research at the right track? Global Journal of Medical Research. K Interdisciplinary, 16(6), 1-7. 2] GraphPad Software, Inc. (2018). 7825 Fay Avenue, Suite 230, La Jolla, CA 92037 USA. [3] Barbara I and Susan D. (2018). Introductory Statistics. Rice University. Houston, Texas 77005. OpenStax.org. [3] Barbara I and Susan D. (2018). Introductory Statistics. Rice University. Houston, Texas 77005. OpenStax.org. [4] Shreemathi SM, Ashma DM and Sachit G. (2017). Types of biological variables. J Thorac Dis, 9(6), 1730–1733. 103 Alrawi et al. / World Journal of Advanced Research and Reviews, 2020, 05(01), 095–104 [5] Wikipedia, The free encyclopedia. (2019). Association (Statistics). [5] Wikipedia, The free encyclopedia. (2019). Association (Statistics). [6] KarenG. (2019). The Difference between Interaction and Association. Alrawi RA, Abdulrahman JM, Al-Rawi RA and Al-Rawi RA. (2020). Some aspects of associations and interactions between biomedical variables.World Journal of Advanced Research and Reviews, 5(1), 95-104. How to cite this article Alrawi RA, Abdulrahman JM, Al-Rawi RA and Al-Rawi RA. (2020). Some aspects of associations and interactions between biomedical variables.World Journal of Advanced Research and Reviews, 5(1), 95-104. Alrawi RA, Abdulrahman JM, Al-Rawi RA and Al-Rawi RA. (2020). Some aspects of associations and interactions between biomedical variables.World Journal of Advanced Research and Reviews, 5(1), 95-104. 104
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BAD sensitizes breast cancer cells to docetaxel with increased mitotic arrest and necroptosis
Scientific reports
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Jasdeep Mann1, Ning Yang1, Rachel Montpetit1, Raven Kirschenman1, Hélène Lemieux2,3 & Ing Swie Goping1,4* Breast cancer patients are commonly treated with taxane (e.g. docetaxel) chemotherapy, despite poor outcomes and eventual disease relapse. We previously identified the Bcl-2-associated death promoter (BAD) as a prognostic indicator of good outcome in taxane-treated breast cancer patients. We also demonstrated that BAD expression in human breast carcinoma cells generated larger tumors in mouse xenograft models. These paradoxical results suggest that BAD-expressing tumors are differentially sensitive to taxane treatment. We validated this here and show that docetaxel therapy preferentially reduced growth of BAD-expressing xenograft tumors. We next explored the cellular mechanism whereby BAD sensitizes cells to docetaxel. Taxanes are microtubule inhibiting agents that cause cell cycle arrest in mitosis whereupon the cells either die in mitosis or aberrantly exit (mitotic slippage) and survive as polyploid cells. In response to docetaxel, BAD-expressing cells had lengthened mitotic arrest with a higher proportion of cells undergoing death in mitosis with decreased mitotic slippage. Death in mitosis was non-apoptotic and not dependent on Bcl-XL interaction or caspase activation. Instead, cell death was necroptotic, and dependent on ROS. These results suggest that BAD is prognostic for favourable outcome in response to taxane chemotherapy by enhancing necroptotic cell death and inhibiting the production of potentially chemoresistant polyploid cells. Triple-negative breast cancer patients receive taxane chemotherapy, such as docetaxel (Taxotere®), as standard first-line treatment despite an overall poor prognosis, high rate of relapse, and adverse effects1. While multiple causes of cellular taxane resistance are known, these have not yet provided clinical markers to guide taxane ther- apy decisions2–4. Understanding the molecular mechanisms that mediate outcome to taxane therapy may iden- tify predictive biomarkers and novel therapeutic targets. The Bcl-2 family member BAD (Bcl-2-associated death promoter) is a prognostic indicator for good clinical outcome of taxane-treated breast cancer patients5. BAD modulates breast cancer cell proliferation and tumor progression by regulating cell cycle progression6,7. Thus, understanding how BAD better predicts patient outcome could aid in understanding docetaxel chemoresistance. Taxanes are anti-mitotic drugs that perturb microtubule dynamics, leading to chronic activation of the spindle assembly checkpoint and inhibition of the anaphase promoting complex that delays the degradation of cyclin B1 and inhibits mitotic exit8. Ideally, this aberrant mitotic arrest initiates cell death in mitosis by facilitating the accumulation of a caspase-dependent death signal9. www.nature.com/scientificreports www.nature.com/scientificreports BAD sensitizes breast cancer cells to docetaxel with increased mitotic arrest and necroptosis p Jasdeep Mann1, Ning Yang1, Rachel Montpetit1, Raven Kirschenman1, Hélène Lemieux2,3 & Ing Swie Goping1,4* Jasdeep Mann1, Ning Yang1, Rachel Montpetit1, Raven Kirschenman1, Hélène Lemieux2,3 & Ing Swie Goping1,4* Scientific Reports | (2020) 10:355 | https://doi.org/10.1038/s41598-019-57282-1 Jasdeep Mann1, Ning Yang1, Rachel Montpetit1, Raven Kirschenman1, Hélène Lemieux2,3 & Ing Swie Goping1,4* Often, however, cells degrade sufficient cyclin B1 prior to full activation of apoptotic caspases, and cells slip out of mitosis in the absence of cytokinesis and enter G1 as polyploid cells. These polyploid cells have differential fates of G1 arrest, post-mitotic death, or continued cell cycle progression10,11. The survival and expansion of these polyploid cells is postulated to generate aggressive clones that are resistant to therapy12,13. y Programmed necrosis, termed necroptosis, is a form of caspase-independent cell death that is activated in response to many anticancer drugs14. Cells can activate necroptosis in the absence of functional apoptosis15. The cell morphology of necroptotic death is similar to that of necrosis, as it includes loss of plasma membrane integrity, mitochondrial dysfunction, oxidative stress, and absence of nuclear fragmentation16. Recent evidence suggests necroptosis can be exploited for cancer therapy, in particular, for apoptosis-resistant cancers17. Taxane treatment can promote necroptosis, although the mechanism remains unclear18. 1Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada. 2Department of Medicine, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada. 3Faculty Saint-Jean, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada. 4Department of Oncology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada. *email: igoping@ualberta.ca Scientific Reports | (2020) 10:355 | https://doi.org/10.1038/s41598-019-57282-1 www.nature.com/scientificreports/ 1 7 14 21 28 35 42 49 0 50 100 150 200 250 300 350 Time (Days) Tumor Volume (mm3) Vector + Vehicle Vector + Dtxl BAD + Vehicle BAD + Dtxl * * * Vector BAD a b c d e 1 2 3 4 5 0 20 40 60 80 Time (Days) % Cell Death Vector BAD * * * * ** Vehicle Docetaxel 0 20 40 60 80 100 120 0 25 50 75 100 Time (Days) Percent survival Vector + Vehicle Vector + Dtxl BAD + Vehicle BAD + Dtxl Vector + Vehicle Vector + Dtxl BAD + Vehicle BAD + Dtxl 0 150 300 450 600 Tumor Volume (mm3) * * * Docetaxel-treated Figure 1. BAD increases sensitivity to docetaxel. (a) MDA-MB-231 cells expressing vector or BAD were treated with 125 nM docetaxel for 5 days. Cells were stained with Annexin V-647 and PI and analyzed via flow cytometry daily. Cell death in control group were subtracted from the docetaxel treated group. Annexin V+/PI+ population is depicted. Student’s t-test; n = 3. (b) MDA-MB-231 cells expressing vector or BAD were injected into the mammary fat pads of Taconic nude mice. Jasdeep Mann1, Ning Yang1, Rachel Montpetit1, Raven Kirschenman1, Hélène Lemieux2,3 & Ing Swie Goping1,4* Red arrows indicate docetaxel or vehicle injection time points. Tumor volume was measured weekly. One-way ANOVA with Dunnett’s post-hoc test; Vector + vehicle = 8, vector + docetaxel = 6, BAD + vehicle = 7, BAD + docetaxel = 7. (c) Scatter plot of tumor volume at day 49. (d) Representative images of tumors in the mammary gland of nude mice. Arrows indicate tumor location. (e) Kaplan-Meier survival curve of mice treated with vehicle or docetaxel. a 1 2 3 4 5 0 20 40 60 80 Time (Days) % Cell Death Vector BAD * * * * ** Docetaxel-treated 1 7 14 21 28 35 42 49 0 50 100 150 200 250 300 350 Time (Days) Tumor Volume (mm3) Vector + Vehicle Vector + Dtxl BAD + Vehicle BAD + Dtxl * * * b 1 7 14 21 28 35 42 49 0 50 100 150 200 250 300 350 Time (Days) Tumor Volume (mm3) Vector + Vehicle Vector + Dtxl BAD + Vehicle BAD + Dtxl * * * b b a Vector BAD d Vehicle Docetaxel Vector BAD c d Vector + Vehicle Vector + Dtxl BAD + Vehicle BAD + Dtxl 0 150 300 450 600 Tumor Volume (mm3) * * * Docetaxel treated Vector d Vehicle Docetaxel e 0 20 40 60 80 100 120 0 25 50 75 100 Time (Days) Percent survival Vector + Vehicle Vector + Dtxl BAD + Vehicle BAD + Dtxl d e e c Vector Figure 1. BAD increases sensitivity to docetaxel. (a) MDA-MB-231 cells expressing vector or BAD were treated with 125 nM docetaxel for 5 days. Cells were stained with Annexin V-647 and PI and analyzed via flow cytometry daily. Cell death in control group were subtracted from the docetaxel treated group. Annexin V+/PI+ population is depicted. Student’s t-test; n = 3. (b) MDA-MB-231 cells expressing vector or BAD were injected into the mammary fat pads of Taconic nude mice. Red arrows indicate docetaxel or vehicle injection time points. Tumor volume was measured weekly. One-way ANOVA with Dunnett’s post-hoc test; Vector + vehicle = 8, vector + docetaxel = 6, BAD + vehicle = 7, BAD + docetaxel = 7. (c) Scatter plot of tumor volume at day 49. (d) Representative images of tumors in the mammary gland of nude mice. Arrows indicate tumor location. Jasdeep Mann1, Ning Yang1, Rachel Montpetit1, Raven Kirschenman1, Hélène Lemieux2,3 & Ing Swie Goping1,4* (e) Kaplan-Meier survival curve of mice treated with vehicle or docetaxel. In the current study, we demonstrated that BAD increases sensitivity of breast cancer tumor xenografts to docetaxel treatment in vivo. BAD-expressing cells prolonged mitotic arrest, and enhanced cell death. Cell death was not dependent on caspases or Bcl-XL indicating a non-apoptotic pathway. Instead, cell death had morpholog- ical hallmarks of necrosis and was dependent on reactive oxygen species (ROS) and the necroptotic kinase MLKL. Thus, we identified a novel role for BAD in enhancing necroptosis during taxane-induced mitotic arrest. Our results provide a potential cellular mechanism wherein BAD is prognostic for clinical docetaxel chemotherapy. Results BAD i BAD increases sensitivity of breast cancer cells to docetaxel. We had previously shown BAD- dependent taxane sensitivity in the breast carcinoma cell lines MCF-7 (luminal B), SKBR-3 (HER2) and MDA-MB-468 (TNBC)5. To study the structure/function relationship of BAD in docetaxel-treated breast cancer cells, we utilized MDA-MB-231 cells stably expressing ectopic BAD7. We performed a longitudinal cell death assay over 5 days of docetaxel treatment to determine docetaxel sensitivity (Fig. 1a). At the earliest measured time point BAD protected cells from docetaxel-induced cell death. This was transient, however, as with increasing time BAD sensitized cells to docetaxel-toxicity. To examine the in vivo relevance of these effects, we performed orthotopic mammary fat pad xenografts in nude mice. Mice were treated with docetaxel on the days indicated by the red arrows (Fig. 1b) and tumor volume was measured. Similar to what we had reported previously, BAD tumors grew significantly larger than vector tumors due to increased cell proliferation and survival signalling7. Tumor growth of BAD expressing cells was significantly decreased in response to docetaxel treatment (Fig. 1c,d). On the other hand, there was no change in tumor size in docetaxel-treated vector control tumors. Additionally, overall survival of mice with BAD tumors treated with docetaxel was increased relative to untreated BAD tumors (Fig. 1e). Altogether, these results indicate BAD expression increases tumor volume, however, these cells are more sensitive to docetaxel treatment with enhanced cell death and decreased tumor size. BAD increases length in docetaxel-mediated mitotic arrest to promote cell death over mitotic slippage. Docetaxel binds to tubulin and disrupts microtubule dynamics, induces prolonged mitotic arrest, Scientific Reports | (2020) 10:355 | https://doi.org/10.1038/s41598-019-57282-1 www.nature.com/scientificreports/ Figure 2. BAD increases length in mitotic arrest with docetaxel treatment. (a) Representative cell fates of MDA-MB-231 cells expressing vector or BAD treated with 125 nM docetaxel for 72 hours. Scale bar = 20 μM. (b) Immunofluorescence images taken 48 hours after docetaxel treatment and stained with DAPI and α-Tubulin. Scale bar = 20 μM. (c) Cell fates of cells treated with docetaxel for 72 hours. Each horizontal line represents an individual cell. Line endpoint represents the time at which the indicated cell fate occurred. (d) Scatter-plot representation of individual cell which either slipped or died in mitosis, and the corresponding hours of mitotic arrest. Median and interquartile range are shown. Mann-Whitney statistical test. (e) Length of mitotic arrest for all cell fates. Mann-Whitney statistical test. Results BAD i (f) Cells were treated with 125 nM docetaxel for 16, 24, or 48 hours. DMSO control treatment was for 48 hours. Right: Quantification of cyclin B1 levels normalized to tubulin. Student’s t-test; n = 3. (g) Cell cycle phases of docetaxel treated cells were analyzed daily for 5 days with PI staining and flow cytometry. Student’s t-test; n = 3. Figure 2. BAD increases length in mitotic arrest with docetaxel treatment. (a) Representative cell fates of MDA-MB-231 cells expressing vector or BAD treated with 125 nM docetaxel for 72 hours. Scale bar = 20 μM. (b) Immunofluorescence images taken 48 hours after docetaxel treatment and stained with DAPI and α-Tubulin. Scale bar = 20 μM. (c) Cell fates of cells treated with docetaxel for 72 hours. Each horizontal line represents an individual cell. Line endpoint represents the time at which the indicated cell fate occurred. (d) Scatter-plot representation of individual cell which either slipped or died in mitosis, and the corresponding hours of mitotic arrest. Median and interquartile range are shown. Mann-Whitney statistical test. (e) Length of mitotic arrest for all cell fates. Mann-Whitney statistical test. (f) Cells were treated with 125 nM docetaxel for 16, 24, or 48 hours. DMSO control treatment was for 48 hours. Right: Quantification of cyclin B1 levels normalized to tubulin. Student’s t-test; n = 3. (g) Cell cycle phases of docetaxel treated cells were analyzed daily for 5 days with PI staining and flow cytometry. Student’s t-test; n = 3. and can lead to cell death in mitosis19. Cells that do not die in mitosis ‘slip’ out of mitosis and re-enter G1 with- out cytokinesis. This phenomenon is referred to as ‘mitotic slippage’20. To understand how BAD sensitized cells to docetaxel, we used time-lapse live imaging to characterize cell morphology in response to docetaxel. Representative cell fates of docetaxel-treated cells are shown (Fig. 2a). We defined death in mitosis as cells with mitotic morphology that eventually ceased all cell movement. This cell death had a non-apoptotic morphology, indicated by cell swelling, granulation of the cytoplasm, and lack of cellular blebbing. Mitotic slipped cells were defined as cells with mitotic morphology that then transitioned to become flat adherent cells. These slipped cells remained viable and maintained subcellular movements. Mitotic slippage at imaging endpoint was confirmed by DAPI-staining, which revealed large multinucleated cells (Fig. Results BAD i Vimentin antibody was used as a negative control. Right: The flow throughs (FT) from the IPs were retained and subjected to western blot. (d) Annexin V+/PI+ staining and flow cytometry analysis of 125 nM docetaxel treated cells after 5 days. One-way ANOVA with Dunnett’s post-hoc test; n = 5. mitotic arrest upon docetaxel treatment in association with inhibited cyclin B1 degradation. This may allow the cells to accumulate a greater cell death signal, favouring cell death in mitosis versus mitotic slippage. These data therefore suggest BAD sensitizes cells to docetaxel by facilitating mitotic arrest-dependent non-apoptotic cell death. The BAD and Bcl-XL interaction is dispensable for docetaxel cell death. The anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2, and Bcl-XL regulate death in mitosis and mitotic slippage. Mcl-1 degradation is required for death in mitosis25. Additionally, phosphorylation of Bcl-2, and Bcl-XL by CDK1 upon taxane treatment inactivates these anti-apoptotic proteins and enables mitotic death26,27, whereas lack of Bcl-XL phos- phorylation is associated with mitotic slippage and cell survival26. Thus, we examined Mcl-1, Bcl-2, and Bcl-XL after docetaxel addition to determine if BAD cells elevated death in mitosis due to increased degradation or phosphorylation of these anti-apoptotic Bcl-2 family members. We found no difference in Mcl-1 and Bcl-2 lev- els between vector and BAD cells (Fig. 3a). However, Bcl-XL levels were higher with more phosphorylation on Ser62 in BAD cells compared to vector cells (Fig. 3a), confirming a higher proportion of BAD-expressing cells with active CDK1 in mitotic arrest. Furthermore, phosphorylation of Ser62 attenuates the anti-apoptotic activ- ity of Bcl-XL by inhibiting binding to BAX28, suggesting cells are sensitized to undergo apoptosis. Since BAD binds to Bcl-XL to displace BAX and stimulate cell death29, we tested whether the BAD and Bcl-XL interaction was necessary for docetaxel-mediated cell death. Immunoprecipitation of BAD protein at 24 hours of docetaxel treatment revealed a strong interaction between BAD and phospho-Bcl-XL (Ser62) (Fig. 3b). Surprisingly, we did not see a Bcl-XL and BAX interaction under any conditions (Fig. 3c), which was inconsistent with an apoptotic “primed to die” phenotype. Therefore, we functionally tested the requirement of the BAD and Bcl-XL interaction for docetaxel-mediated cell death using a cell line expressing BAD-L114A that abrogates BAD:Bcl-XL binding30. The BAD L114A mutant did not attenuate docetaxel cell death, indicating the BAD and Bcl-XL interaction is not required for cell death (Fig. 3d). Results BAD i 2b), typical of cells that have undergone mitotic exit in the absence of cytokinesis21.f To quantify differences in cell fates in vector versus BAD cells, we generated cell fate maps over 72 hours of drug treatment (Fig. 2c). The bar length indicates the time each cell spent in mitotic arrest, and the bar color indicates the subsequent cell fate until the experimental endpoint. Cells that entered mitosis (Fig. 2c) either remained in mitosis (blue), died in mitosis (green) or slipped out of mitotic arrest (purple). The average length a cell spent in mitotic arrest that culminated in death in mitosis was longer than for the cells that underwent slip- page, (Fig. 2d), supporting a model of competing pathways between cell death and mitotic exit22,23. Additionally, BAD cells showed significantly longer times in mitotic arrest than vector control cells, irrespective of cell fate (Fig. 2e). Degradation of cyclin B1 is critical for determining cell fate, as the premature attenuation of cyclin B1 prior to accumulation of a sufficient cell death signal ultimately leads to mitotic slippage24. BAD cells retained higher levels of cyclin B1, suggesting that enhanced cyclin B1 stability lengthened mitotic arrest thus enabling the cells to accumulate a cell death signal (Fig. 2f). In support of this, DNA content-based cell cycle analysis revealed a greater proportion of BAD cells in mitotic arrest (G2/M; days 1–2), that then is followed by a higher amount of cell death (sub-G1 population; days 4–5) (Fig. 2g). Results BAD i Dtxl (hrs) Vector cells BAD cells - 16 24 48 - 16 24 48 Mcl-1 Bcl-2 Bcl-XL pBcl-XL-S62 Tubulin 0 16 24 48 0 16 24 48 0.0 0.5 1.0 1.5 2.0 pBcl-XL-Ser62/Bcl-XL (Normalized to Tubulin) Docetaxel (Hours) Vector BAD * * Input Vector BAD _ + _ + IP: BAD _ + _ + IP: GST _ + _ + Dtxl, 24 hrs BAD Bcl-XL pBcl-XL-S62 Vector BAD Vector BAD a b 0 16 24 48 0 16 24 48 0.0 0.5 1.0 1.5 2.0 pBcl-XL-Ser62/Bcl-XL (Normalized to Tubulin) Docetaxel (Hours) Vector BAD * * Input Vector BAD _ + _ + IP: BAD _ + _ + IP: GST _ + _ + Dtxl, 24 hrs BAD Bcl-XL pBcl-XL-S62 Vector BAD Vector BAD b Dtxl (hrs) Vector cells BAD cells - 16 24 48 - 16 24 48 Mcl-1 Bcl-2 Bcl-XL pBcl-XL-S62 Tubulin a b b a d Vector BAD L114A 0 10 20 30 40 50 % Cell Death ** * Input Vimentin-IP FT Bcl-XL-IP FT - - - - - - + + + + + + Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD Dtxl Bcl-XL BAD BAX c - - - - - - + + + + + + Dtxl Input Vimentin-IP FT Bcl-XL-IP FT Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD Bcl-XL BAD BAX - - - - - - + + + + + + Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD Input IP: Vimentin IP: Bcl-XL Dtxl Bcl-XL BAD BAX d % Cell Death d c - - - - - - + + + + + + Dtxl Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD Bcl-XL BAD BAX Input IP: Vimentin IP: Bcl-XL c Figure 3. BAD and Bcl-XL binding is not required for docetaxel killing. (a) Left: MDA-MB-231 cells stably expressing vector or BAD were treated with 125 nM docetaxel for the indicated time points. Control is DMSO for 48 hours. Right: Quantification of protein band density for pBcl-XL-Ser62 over total Bcl-XL. Student’s t- test; n = 3. (b) Immunoprecipitation (IP) of BAD antibody after 24 hours of 125 nM docetaxel treatment. GST antibody was used as a negative control. (c) Immunoprecipitation with Bcl-XL antibody after 125 nM docetaxel for 5 days. Results BAD i Taken together, these results reveal BAD increases length in Scientific Reports | (2020) 10:355 | https://doi.org/10.1038/s41598-019-57282-1 www.nature.com/scientificreports/ Dtxl (hrs) Vector cells BAD cells - 16 24 48 - 16 24 48 Mcl-1 Bcl-2 Bcl-XL pBcl-XL-S62 Tubulin 0 16 24 48 0 16 24 48 0.0 0.5 1.0 1.5 2.0 pBcl-XL-Ser62/Bcl-XL (Normalized to Tubulin) Docetaxel (Hours) Vector BAD * * Input Vector BAD _ + _ + IP: BAD _ + _ + IP: GST _ + _ + Dtxl, 24 hrs BAD Bcl-XL pBcl-XL-S62 Vector BAD Vector BAD a c b - - - - - - + + + + + + Dtxl Input Vimentin-IP FT Bcl-XL-IP FT Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD Bcl-XL BAD BAX - - - - - - + + + + + + Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD Input IP: Vimentin IP: Bcl-XL Dtxl Bcl-XL BAD BAX d Vector BAD L114A 0 10 20 30 40 50 % Cell Death ** * Figure 3. BAD and Bcl-XL binding is not required for docetaxel killing. (a) Left: MDA-MB-231 cells stably expressing vector or BAD were treated with 125 nM docetaxel for the indicated time points. Control is DMSO for 48 hours. Right: Quantification of protein band density for pBcl-XL-Ser62 over total Bcl-XL. Student’s t- test; n = 3. (b) Immunoprecipitation (IP) of BAD antibody after 24 hours of 125 nM docetaxel treatment. GST antibody was used as a negative control. (c) Immunoprecipitation with Bcl-XL antibody after 125 nM docetaxel for 5 days. Vimentin antibody was used as a negative control. Right: The flow throughs (FT) from the IPs were retained and subjected to western blot. (d) Annexin V+/PI+ staining and flow cytometry analysis of 125 nM docetaxel treated cells after 5 days. One-way ANOVA with Dunnett’s post-hoc test; n = 5. Results BAD i Thus, these results indicate that BAD-potentiation of docetaxel sensitivity is not dependent on binding to Bcl-XL. Docetaxel can induce necroptotic cell death. The observation that Bcl-XL does not regulate the ability of BAD to enhance docetaxel-induced cell death, suggests that BAD modulates a non-apoptotic mechanism. To assess apoptosis, we examined the apoptotic markers of cleaved caspase-3, cleaved PARP and BAX activation. Docetaxel treatment induced minimal caspase cleavage and detectable PARP cleavage that was similar between vector and BAD-expressing cells (Fig. 4a). BAX activation was not induced by docetaxel treatment (Fig. 4b). On the other hand, these markers were positive for apoptosis induced by a known apoptotic inducer, staurosporine (Supplementary Fig. 1)31. Therefore, despite having a competent apoptotic machinery, apoptosis is minimally activated with docetaxel. Instead, the majority of cells appeared necrotic with compromised plasma membranes Scientific Reports | (2020) 10:355 | https://doi.org/10.1038/s41598-019-57282-1 www.nature.com/scientificreports/ 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 - - Vector Cells BAD Cells Vector Cells BAD Cells Days Days Control Docetaxel BAD BAD Cleaved Caspase-3 Cleaved Caspase-3 Cleaved PARP Cleaved PARP Tubulin Tubulin - - + + Vector Input BAX - - + + - - + + BAD Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD IP: 6A7 BAX IP: Vimentin a b - - + + Dtxl Vector BAD Vector BAD Cleaved Caspase-3 Dtxl r o tc e V D A B PI Annexin V le x a t e c o D Time (days) 1 5 0 2 3 4 1 2 3 4 5 0 5 10 15 Time (Days) % Annexin-V +/PI - Vector BAD 0 1 2 3 4 5 0 1 2 3 4 5 0.0 0.5 1.0 Cleaved Caspase-3 (Normalized to Tubulin) Docetaxel (Days) Vector BAD 0 1 2 3 4 5 0 1 2 3 4 5 0.0 0.5 1.0 1.5 2.0 2.5 Cleaved PARP (Normalized to Tubulin) Docetaxel (Days) Vector BAD c P = 0.0664 P = 0.0666 Figure 4. Docetaxel is killing the cells through a non-apoptotic mechanism. (a) MDA-MB-231 cells stably expressing vector or BAD were treated with DMSO control (left) or 125 nM docetaxel (middle) and subjected to western blot. Right: Protein band quantification was performed. Student’s t-test; no statistical significance. Results BAD i (b) Cells were treated with 125 nM docetaxel for 3 days prior to immunoprecipitation (IP) with 6A7 BAX antibody. Vimentin antibody was used as a negative control. Input lanes were also probed with cleaved caspase-3 antibody (right). (c) Cells were treated with 125 nM docetaxel for 5 days and stained with Annexin V-647 and PI daily and analyzed via flow cytometry. The Annexin V+/PI− population is graphed. Student’s t-test; no statistical significance between vector and BAD. Right: Dot plots from flow cytometric analysis. Annexin V positive cells are on the x-axis, PI positive cells are on the y-axis. Time, in days, is increasing to the right. 0 1 2 3 4 5 0 1 2 3 4 5 0.0 0.5 1.0 Cleaved Caspase-3 (Normalized to Tubulin) Docetaxel (Days) Vector BAD P = 0.0664 P = 0.0666 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 - - Vector Cells BAD Cells Vector Cells BAD Cells Days Days Control Docetaxel BAD BAD Cleaved Caspase-3 Cleaved Caspase-3 Cleaved PARP Cleaved PARP Tubulin Tubulin - - + + Vector Input BAX - - + + - - + + BAD Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD IP: 6A7 BAX IP: Vimentin a b - - + + Dtxl Vector BAD Vector BAD Cleaved Caspase-3 Dtxl 0 1 2 3 4 5 0 1 2 3 4 5 0.0 0.5 1.0 Cleaved Caspase-3 (Normalized to Tubulin) Docetaxel (Days) Vector BAD 0 1 2 3 4 5 0 1 2 3 4 5 0.0 0.5 1.0 1.5 2.0 2.5 Cleaved PARP (Normalized to Tubulin) Docetaxel (Days) Vector BAD P = 0.0664 P = 0.0666 1 2 3 4 5 1 2 3 4 5 Vector Cells BAD Cells Days Control BAD Cleaved Caspase-3 C Cleaved PARP Tubulin a a - - + + Vector Input BAX - - + + - - + + BAD Vector BAD Vector BAD Vector BAD Vector BAD Vector BAD IP: 6A7 BAX IP: Vimentin b C Dtxl b r o tc e V D A B PI Annexin V le x a t e c o D Time (days) 1 5 0 2 3 4 1 2 3 4 5 0 5 10 15 Time (Days) % Annexin-V +/PI - Vector BAD c c c Figure 4. Results BAD i AMP-activated protein kinase (AMPK), a cell energy sensor, boosts mitochondrial respiration during low energy status in mitosis and inhibits ROS-mediated Scientific Reports | (2020) 10:355 | https://doi.org/10.1038/s41598-019-57282-1 www.nature.com/scientificreports/ 0 10 20 30 40 % Dead Cells Vector BAD Dtxl ZVAD NSA - - - + - - + + - + + - + + + * * ** **** * * **** Figure 5. Docetaxel is killing the cells through a necroptotic mechanism. MDA-MB-231 cells stably expressing vector or BAD were treated with 125 nM docetaxel (Dtxl), 20 μM Z-VAD-FMK (ZVAD), or 5 μM necrosulfonamide (NSA) for 5 days. Cells were stained with Annexin V-647 and PI and analyzed via flow cytometry. The Annexin V+/PI+ population is represented in a bar graph. Two-way ANOVA with Tukey’s post-hoc test; n = 3. 0 10 20 30 40 % Dead Cells Vector BAD Dtxl ZVAD NSA - - - + - - + + - + + - + + + * * ** **** * * **** Figure 5. Docetaxel is killing the cells through a necroptotic mechanism. MDA-MB-231 cells stably expressing vector or BAD were treated with 125 nM docetaxel (Dtxl), 20 μM Z-VAD-FMK (ZVAD), or 5 μM necrosulfonamide (NSA) for 5 days. Cells were stained with Annexin V-647 and PI and analyzed via flow cytometry. The Annexin V+/PI+ population is represented in a bar graph. Two-way ANOVA with Tukey’s 0 10 20 30 40 % Dead Cells Vector BAD Dtxl ZVAD NSA - - - + - - + + - + + - + + + * * ** **** * * **** 0 10 20 30 40 % Dead Cells Vector BAD Dtxl ZVAD NSA - - - + - - + + - + + - + + + * * ** **** * * **** Figure 5. Docetaxel is killing the cells through a necroptotic mechanism. MDA-MB-231 cells stably expressing vector or BAD were treated with 125 nM docetaxel (Dtxl), 20 μM Z-VAD-FMK (ZVAD), or 5 μM necrosulfonamide (NSA) for 5 days. Cells were stained with Annexin V-647 and PI and analyzed via flow cytometry. The Annexin V+/PI+ population is represented in a bar graph. Two-way ANOVA with Tukey’s post-hoc test; n = 3. Results BAD i a b Routine Leak ET 0 20 40 60 80 100 Oxygen flux (pmol.s.million cells) Vector BAD Vector + Dtxl BAD + Dtxl *** *** * P = 0.051 AMPK Tubulin pAMPK -T172 Dtxl (hrs) Vector cells BAD cells - 16 24 48 - 16 24 48 0 16 24 48 0 16 24 48 0 1 2 3 pAMPK/AMPK Levels (Normalized to Tubulin Docetaxel (Hours) Vector BAD * n.s. Figure 6. BAD-mediated mitotic arrest is downstream of AMPK. (a) MDA-MB-231 cells stably expressing vector or BAD were treated with 125 nM docetaxel for 48 hours prior to high-resolution respirometry. One- way ANOVA with Dunnett’s post-hoc test; n = 3. (b) Cells were treated with 125 nM docetaxel for 16, 24, or 48 hours. DMSO control treatment was for 48 hours. Right: Quantification of pAMPK-Thr172 levels over AMPK total protein were normalized to tubulin. One-way ANOVA with Dunnett’s post-hoc test; n = 3. a Routine Leak ET 0 20 40 60 80 100 Oxygen flux (pmol.s.million cells) Vector BAD Vector + Dtxl BAD + Dtxl *** *** * P = 0.051 b AMPK Tubulin pAMPK -T172 Dtxl (hrs) Vector cells BAD cells - 16 24 48 - 16 24 48 0 16 24 48 0 16 24 48 0 1 2 3 pAMPK/AMPK Levels (Normalized to Tubulin Docetaxel (Hours) Vector BAD * n.s. b b Figure 6. BAD-mediated mitotic arrest is downstream of AMPK. (a) MDA-MB-231 cells stably expressing vector or BAD were treated with 125 nM docetaxel for 48 hours prior to high-resolution respirometry. One- way ANOVA with Dunnett’s post-hoc test; n = 3. (b) Cells were treated with 125 nM docetaxel for 16, 24, or 48 hours. DMSO control treatment was for 48 hours. Right: Quantification of pAMPK-Thr172 levels over AMPK total protein were normalized to tubulin. One-way ANOVA with Dunnett’s post-hoc test; n = 3. necroptosis36,37. We therefore measured whether BAD expression altered oxygen consumption rate and AMPK activation upon docetaxel treatment. As expected, BAD expression increased oxygen consumption7 and this was unaffected by docetaxel treatment (Fig. 6a). Correspondingly, levels of active phosphorylated AMPK did not change in response to docetaxel. On the other hand, in vector control cells, docetaxel elevated the oxygen flux to reach similar levels to BAD-expressing cells (Fig. 6a). In line with this, AMPK activation also increased in response to docetaxel, suggesting that in vector cells, AMPK could attenuate necroptosis (Fig. 6b). Results BAD i Docetaxel is killing the cells through a non-apoptotic mechanism. (a) MDA-MB-231 cells stably expressing vector or BAD were treated with DMSO control (left) or 125 nM docetaxel (middle) and subjected to western blot. Right: Protein band quantification was performed. Student’s t-test; no statistical significance. (b) Cells were treated with 125 nM docetaxel for 3 days prior to immunoprecipitation (IP) with 6A7 BAX antibody. Vimentin antibody was used as a negative control. Input lanes were also probed with cleaved caspase-3 antibody (right). (c) Cells were treated with 125 nM docetaxel for 5 days and stained with Annexin V-647 and PI daily and analyzed via flow cytometry. The Annexin V+/PI− population is graphed. Student’s t-test; no statistical significance between vector and BAD. Right: Dot plots from flow cytometric analysis. Annexin V positive cells are on the x-axis, PI positive cells are on the y-axis. Time, in days, is increasing to the right. (Annexin-V positive/PI positive) (Fig. 1a). In contrast, apoptotic inducers trigger initial phosphatidylserine externalization followed by secondary necrosis (Annexin-V positive/PI negative) (Fig. 4c and Supplementary Fig. 1). Thus, docetaxel caused concurrent plasma membrane damage and phosphatidylserine exposure, consist- ent with necrotic-like cell death. Necroptosis is a regulated form of necrotic cell death that compensates when the apoptotic pathway is blocked32. The necrosome is the core executioner complex that consists of RIP3 oligomers and MLKL and is acti- vated in a RIP1-dependent and independent manner32,33. Docetaxel has previously been shown to initiate both necrosis and apoptosis in MDA-MB-231 cells34, so we assessed whether necroptosis was triggered in our studies. Inhibition of caspases did not attenuate cell death (Fig. 5), consistent with necroptotic signaling. Inhibition of necroptosis with the MLKL inhibitor necrosulfonamide35 significantly decreased cell death in BAD cells only, indicating that BAD stimulates docetaxel-induced necroptosis. The addition of both a pan-caspase inhibitor, z-VAD-FMK, and necrosulfonamide significantly reduced cell death in both vector and BAD cells, confirming one mechanism of cell death might be activated when the other is compromised. Therefore, BAD stimulates docetaxel-induced cell death through necroptosis. Oxidative phosphorylation regulates docetaxel cell death. We have previously shown that BAD increases mitochondrial metabolism to promote cell survival7. Results BAD i Therefore, these data suggest BAD maintains adequate energy levels during mitotic arrest to circumvent AMPK activation and facilitate increased necroptosis. BAD requires reactive oxygen species for docetaxel induced cell death. BAD promotes cell sur- vival and mitochondrial metabolism by stimulating complex I activity of the electron transport chain7, which is a main site of cellular ROS production38. Reactive oxygen species (ROS) are metabolic by-products of mito- chondrial respiration and are required for maintaining redox-homeostasis of cancer cells39. Additionally, ROS has been shown to facilitate necroptosis40,41. BAD-expressing cells significantly increased ROS levels in response to docetaxel relative to vector control cells (Fig. 7a). To determine if BAD-enhanced necroptosis was dependent on ROS, we measured cell death in the presence of the ROS scavenger N-acetyl cysteine (NAC). Cell death was significantly attenuated only in docetaxel-treated BAD cells with NAC (Fig. 7b). We further examined whether ROS-dependent cell death in BAD cells was dependent on complex I. Inhibition of complex I by rotenone did not attenuate docetaxel cell death in BAD cells (Fig. 7c). Thus, these data suggest BAD confers docetaxel cell death that is dependent on ROS but does not require complex I activity. In conclusion, BAD prolongs docetaxel-mediated mitotic arrest to promote ROS-dependent necroptotic cell death (Fig. 7d). Scientific Reports | (2020) 10:355 | https://doi.org/10.1038/s41598-019-57282-1 www.nature.com/scientificreports/ a b Mitotic Arrest Necroptosis Time in Mitotic Arrest BAD Mitotic Slippage Docetaxel d ROS Cancer Cell Cyclin B1 Degradation n.s. Control Rotenone Docetaxel Rot + Dtxl 0 20 40 60 80 % Cell Death Control NAC Docetaxel Dtxl + NAC 0 20 40 60 % Cell Death Vector BAD * * P = 0.076 Vector BAD 0 2 4 6 I F M ni e s a e r c n I dlo F o D ( cetaxel/Cont lo r ) * c Figure 7. BAD requires ROS for docetaxel cell death. (a) MDA-MB-231 cells stably expressing vector or BAD were treated with 125 nM docetaxel or DMSO control for 48 hours prior to staining with CM-H2DCFDA to measure reactive oxygen species (ROS). Mean fluorescence intensity (MFI) was measured via flow cytometry. Fold increase of docetaxel/control is graphed. Student’s t-test; n = 3. (b) MDA-MB-231 cells stably expressing vector or BAD were treated with 125 nM docetaxel or 10 mM N-acetyl cysteine (NAC) for 5 days. Annexin V+/ PI+ staining flow cytometry analysis of cells is graphed. Results BAD i Two-way ANOVA with Tukey’s post-hoc test; n = 3. (c) MDA-MB-231 cells expressing BAD were treated with 125 nM docetaxel or 50 nM rotenone for 5 days. Annexin V+/PI+ staining flow cytometry analysis of cells is graphed. Student’s t-test; n = 3. (d) Schematic representation of docetaxel-mediated cell death. BAD increases length in mitotic arrest by inhibiting cyclin B1 degradation, leading to a ROS-dependent necroptotic cell death. a Vector BAD 0 2 4 6 I F M ni e s a e r c n I dlo F o D ( cetaxel/Cont lo r ) * a b n.s. Control Rotenone Docetaxel Rot + Dtxl 0 20 40 60 80 % Cell Death Control NAC Docetaxel Dtxl + NAC 0 20 40 60 % Cell Death Vector BAD * * P = 0.076 Vector BAD 0 2 4 6 I F M ni e s a e r c n I dlo F o D ( cetaxel/Cont lo r ) * c b Control NAC Docetaxel Dtxl + NAC 0 20 40 60 % Cell Death Vector BAD * * P = 0.076 n.s. Control Rotenone Docetaxel Rot + Dtxl 0 20 40 60 80 % Cell Death c b c Mitotic Arrest Necroptosis Time in Mitotic Arrest BAD Mitotic Slippage Docetaxel d ROS Cancer Cell Cyclin B1 Degradation Mitotic Arrest Necroptosis Time in Mitotic Arrest BAD Docetaxel d ROS Cancer Cell Cyclin B1 Degradation d Cyclin B1 Degradation Necroptosis Mitotic Arrest Mitotic Slippage Mitotic Slippage Figure 7. BAD requires ROS for docetaxel cell death. (a) MDA-MB-231 cells stably expressing vector or BAD were treated with 125 nM docetaxel or DMSO control for 48 hours prior to staining with CM-H2DCFDA to measure reactive oxygen species (ROS). Mean fluorescence intensity (MFI) was measured via flow cytometry. Fold increase of docetaxel/control is graphed. Student’s t-test; n = 3. (b) MDA-MB-231 cells stably expressing vector or BAD were treated with 125 nM docetaxel or 10 mM N-acetyl cysteine (NAC) for 5 days. Annexin V+/ PI+ staining flow cytometry analysis of cells is graphed. Two-way ANOVA with Tukey’s post-hoc test; n = 3. (c) MDA-MB-231 cells expressing BAD were treated with 125 nM docetaxel or 50 nM rotenone for 5 days. Annexin V+/PI+ staining flow cytometry analysis of cells is graphed. Student’s t-test; n = 3. (d) Schematic representation of docetaxel-mediated cell death. Results BAD i BAD increases length in mitotic arrest by inhibiting cyclin B1 degradation, leading to a ROS-dependent necroptotic cell death. Scientific Reports | (2020) 10:355 | https://doi.org/10.1038/s41598-019-57282-1 www.nature.com/scientificreports/ previous reports by others with MDA-MB-231 cells49. Therefore, apoptosis is not a major cell death pathway for docetaxel in these triple-negative breast carcinoma cells. Recently, necroptosis activation has been shown to over- come chemotherapy resistance50. Aldehyde dehydrogenase inhibitors kill ovarian cancer stem cells by activating necroptosis, in part, by the induction of mitochondrial uncoupling proteins and reduction in mitochondrial oxidative phosphorylation51. In line with this, another group showed that docetaxel-resistant prostate cancer cells induced a shift from glycolysis to oxidative phosphorylation to confer a survival advantage52. In our model system, we saw a similar increase in mitochondrial metabolism in vector cells exposed to docetaxel, suggesting this was associated with a higher resistance to cell death than BAD cells. g BAD-enhanced docetaxel-mediated cell death was dependent on ROS. Impaired oxidative phosphorylation can lead to loss of inner transmembrane potential, reduction of ATP, and production of mitochondrial ROS53. However, oxidative phosphorylation was not impaired in docetaxel-treated BAD cells. Additionally, ROS levels peak in G2 and mitosis and can cause oxidative damage54. BAD increased length in docetaxel-mediated mitotic arrest, suggesting that increased ROS were a result of extended mitosis. Chemotherapeutic agents generate ROS in cancer cells to push ROS levels over a threshold to induce cell death39. Additionally, ROS can activate necropto- sis in certain cell types55. Therefore, we hypothesize BAD-mediated prolonged mitotic arrest alters redox homeo- stasis during taxane treatment to cause cell death.f g Docetaxel has been a standard chemotherapeutic regimen both alone and in combination for many differ- ent cancers56. Although docetaxel prolongs overall and disease-free survival, a significant number of patients eventually acquire chemoresistance and succumb to their disease. Polyploidization is a key factor in resistance and relapse of docetaxel therapy, and can arise following docetaxel-mediated mitotic arrest57. Polyploid cells are large, multinucleated cells that aberrantly exited mitosis without undergoing cytokinesis, through a process known as ‘mitotic slippage’12. Polyploid cells are characterized by excessive centrosomes, with unstable chromo- somes, and are highly resistant to chemotherapy13. Slipped polyploid cells can continue to cycle, or emerge from chemotherapy-induced senescence with stem cell-like features and display a more aggressive phenotype58,59. In support of this, prostate cancer cells treated with docetaxel generated a population of ‘slipped’ cells, of which a small percentage survived and gave rise to a chemoresistant and cancer stem cell positive population60. We observed that BAD promotes death in mitosis over mitotic slippage. Materials and Methods Cell culture and reagents. MDA-MB-231 cells were from ATCC. Cells were cultured in RPMI 1640 medium (Life Technologies) with 10% FBS as previously described5. Ectopic BAD expression and stable cell line generation was as before7. Cell lines were routinely tested for mycoplasma contamination and passaged a maxi- mum of 25 times. Z-VAD-FMK was from R&D systems, and necrosulfonamide was from Calbiochem. Cell culture and reagents. Flow cytometry analysis of cell death and cell cycle. Cells were incubated with 125 nM docetaxel (Sigma-Aldrich) or 2.5 μM staurosporine (Sigma-Aldrich) for the indicated times. Cells were harvested and washed twice with 1 X PBS prior to incubation with Annexin-V 647 (Invitrogen) in 1 X Annexin V binding buffer (1:20 dilution) for 15 minutes at room temperature in the dark, according to the manufacturer’s instructions. Cells were spun down to remove Annexin-V prior to addition of 20 μg/ml propidium iodide for 5 minutes (Life Technologies). Fluorescence was measured on the FL-4 and FL-2 channels with the BD Accuri™ C6 flow cytom- eter. To measure cell cycle, cells were fixed overnight in 70% ethanol and permeabilized with 0.25% Triton X-100 (Sigma-Aldrich) following addition of 10 μg/ml RNase A (Sigma-Aldrich) and 20 μg/ml PI. Mouse studies. Animal procedures were performed in accordance with the guidelines and regulations set forth by the Canadian Council on Animal Care and approved by the University of Alberta Health Sciences 2 Animal Care and Use Committee (Protocol # AUP00000386). 3 × 106 cells were injected into the left mam- mary gland (#4) of nude mice (CrTac:NCr-Foxn1nu, Taconic) in 100 μl of a 1:1 matrigel/media mix. RPMI 1640 medium with no supplemental serum or antibiotic and Corning® Matrigel® Basement Membrane Matrix was used. Docetaxel (10 mg/kg) was administered via intraperitoneal injection on days as indicated. Animals were monitored weekly and sacrificed when tumors reached 20 mm in diameter. Tumors were collected and tumor volume (mm3) was calculated with the formula of (length × width × height)/2. Hematoxylin and eosin staining was performed as previously described7. Protein analysis. Protocols for immunoblotting and co-immunoprecipitation (IP) were as previ- ously described5. Antibodies were from the following sources: Cyclin B1, CDK1, phospho-histone H3 Ser10, pAMPK-Thr172, AMPK, Mcl-1, Bcl-2, Bcl-XL, pBcl-XL-Ser62 and cleaved PARP were from Cell Signaling Technologies. BAD, tubulin and BAX conformational antibody clone 6A7 were from Sigma-Aldrich. www.nature.com/scientificreports/ BAD cells had a significantly longer mitotic arrest phase and underwent significantly more mitotic necroptosis. These data suggest that this longer mitotic arrest allowed the cell death signal to accrue leading to a cell fate of death over cyclin B1 degradation-mediated slippage23. Thus, in response to taxane chemotherapy, BAD high patient tumors may undergo greater cell death with less mitotic slippage, and decreased capacity to evolve chemoresistance and cancer stem-like properties, in line with the increased overall survival of these patients. p In summary, BAD increases cell death in response to docetaxel treatment. We have shown BAD expres- sion increases length in mitotic arrest, with more death in mitosis and less mitotic slippage and polyploidy. Additionally, this cell death is necroptotic and dependent on reactive oxygen species. Understanding the mecha- nism of docetaxel cell death will aid in understanding BAD’s function as a prognostic biomarker in breast cancer. This may guide future studies examining whether BAD predicts patient response to taxane therapy and may suggest non-taxane chemotherapy for breast cancer patients with low BAD levels. Discussion h In the current study, we show that BAD sensitizes cells to docetaxel treatment in vitro and in vivo. Docetaxel treat- ment did not significantly trigger apoptotic cell death and instead displayed necrotic cell death morphologies. Inhibition of the necroptosis executioner, MLKL, attenuated cell death, indicating that BAD enhanced docetaxel toxicity via necroptosis. BAD enhanced death in mitosis in association with increased length in mitotic arrest and elevated cyclin B1. Thus, BAD enhances docetaxel sensitivity by facilitating longer mitotic arrest and activating necroptotic cell death in mitosis. p Our previous results reveal high BAD protein levels are associated with a 3.7-fold increased probability of overall survival of primary breast cancer patients treated with taxane5. BAD stimulates cell cycle progression leading to increased breast cancer cell number and tumor growth7. Similarly, ectopic BAD expression increased prostate cancer cell number and tumor growth42. The present data validated BAD-dependent tumor growth in a mouse model and demonstrated that this increased tumor growth is sensitive to docetaxel. Docetaxel is an anti-mitotic drug that targets actively proliferating cells by stabilizing microtubules and inducing cell death43. Thus, docetaxel may target BAD cells more effectively due to their increased proliferation. h y gf y p Necrosis is an unregulated form of cell death, while necroptosis is programmed necrosis characterized by the activation of the RIP3-dependent pathway44. Necroptosis is activated in response to many anticancer drugs and contributes to their cytotoxicity45. TNF was the first documented inducer of the necroptotic pathway46. In response to TNF, overactivation of PARP1 by ROS-mediated DNA damage was shown to cause necrosis47. More recently, TNF-induced necroptosis and PARP-1-mediated necrosis have been established as two distinct path- ways to programmed necrosis48. Our results showed no difference in PARP cleavage between vector and BAD cells in response to docetaxel, suggesting that BAD did not specifically stimulate PARP-mediated necroptosis. Inhibiting apoptotic caspases with zVAD-FMK alone did not inhibit docetaxel-induced cell death, similar to Scientific Reports | (2020) 10:355 | https://doi.org/10.1038/s41598-019-57282-1 www.nature.com/scientificreports/ Data availabilityh y The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. Received: 19 June 2019; Accepted: 2 December 2019; Published: xx xx xxxx Immunofluorescence. e. Immunofluorescence was performed as previously described7. Reactive oxygen species. Cells were grown in 125 nM docetaxel or DMSO control for 48 hours prior to wash in Hank’s Balanced Salt Solution (ThermoFisher) followed by staining in 1.5 μM chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (Invitrogen) for 45 minutes at 37 °C in the dark. Mean fluorescence intensity (MFI) was measured in the FL-1 channel using flow cytometry. High-resolution respirometry. High-resolution respirometry was performed as previously described61. Briefly, respiration of intact cells (1 × 106 cells/mL) was measured in RPMI 1640 culture medium (10% fetal calf serum) under cellular routine conditions (ROUTINE). After inhibition of ATP-synthase with 2 μg/mL oligomy- cin, respiration declined to the resting or leak-compensating state (LEAK state). 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RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis, Science (80-.) https://doi.org/10.1126/science.1172308 (2009). p g ( ) 42. Smith, A.J., Karpova, Y., D’Agostino, R., Willingham, R. M. & Kulik, G. Expression of the Bcl-2 protein BAD promotes prostate cancer growth, PLoS One., https://doi.org/10.1371/journal.pone.0006224 (2009).ff g g j 3. Garcia, P. et al. Acknowledgements g We would like to thank the Women and Children’s Health Research Institute, Canadian Breast Cancer Foundation and Alberta Cancer Foundation for funding this research. Competing interestsh p g The authors declare no competing interests. Author contributions J.M. and I.S.G. conceived and planned the experiments. J.M. performed all experiments and wrote the manuscript with edits by I.S.G. R.M. and R.K. helped perform the mouse experiments. NY helped perform the respirometry experiment with interpretation and analysis from H.L. References 8. Wang, Q. et al. 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Scientific Reports | (2020) 10:355 | https://doi.org/10.1038/s41598-019-57282-1 10 www.nature.com/scientificreports/ Acknowledgements Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-019-57282-1. Correspondence and requests for materials should be addressed to I.S.G. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020 Scientific Reports | (2020) 10:355 | https://doi.org/10.1038/s41598-019-57282-1
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Determination of Optimal Line-Heating Conditions for Flatness Control of Wind Tower Blocks Using Strain as Direct Boundary Method
Materials
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Citation: Yoon, H.-C.; Lim, H.-B.; Noh, H.-J.; Han, Y.-H.; Lee, J.-C.; Yang, H.-I. Determination of Optimal Line-Heating Conditions for Flatness Control of Wind Tower Blocks Using Strain as Direct Boundary Method. Materials 2022, 15, 7962. https:// doi.org/10.3390/ma15227962 Keywords: flatness; tilt angle; line heating; strain as direct boundary Academic Editors: Rafał Goł˛ebski and Antun Stoi´c Academic Editors: Rafał Goł˛ebski and Antun Stoi´c Article Determination of Optimal Line-Heating Conditions for Flatness Control of Wind Tower Blocks Using Strain as Direct Boundary Method Hee-Chan Yoon 1, Hun-Bong Lim 2 , Hong-Jun Noh 1, Young-Hwan Han 1, Jae-Chul Lee 3,* and Hyun-Ik Yang 1,* 1, Hun-Bong Lim 2 , Hong-Jun Noh 1, Young-Hwan Han 1, Jae-Chul Lee 3,* ng 1,* 1 Department of Mechanical Design Engineering, Hanyang University, 222, Wangsimni-ro, Seongdong-gu, Seoul 04763, Korea 2 Department of Mechanical Design Engineering, Myongji College, 134, Gajwa-ro, Seodaemun-gu Seoul 03656, Korea * Correspondence: jc2@kitech.re.kr (J.-C.L.); skynet@hanyang.ac.kr (H.-I.Y.); Tel.: +82-31-8040-6244 (J.-C.L.); +82-31-400-5285 (H.-I.Y.) Abstract: The wind tower block is welded with the flange to assemble the wind tower. The inherent strain due to local heating and cooling of the weld affects the flatness of the flange. Therefore, line heating is performed to satisfy the design criteria of the flange flatness, but the work variables depend on the operator’s empirical judgment. This study proposed a method to determine the optimum linear heating conditions to control the welded flatness of wind tower blocks and flanges. A proposed method uses the inherent strain method, a simple analysis method, and the optimization is performed based on the deformation superposition method. The changes in flange flatness due to welding and single-point heating were calculated. Then, the flatness change due to single-point heating is superimposed with a scale factor, which represents the magnitude of line heating, and is added to the flatness change due to welding. Using the optimization procedure, the line heating conditions used to derive the flatness that satisfies the design criteria were derived and applied to the analytical model for verification. materials materials 1. Introduction [6] proposed a method for predicting plate deformation by organizing the variables for line heating using multivariate analysis. Nomoto et al. [7] developed a deformation analysis method by converting the angular deformation and shrinkage due to line heating into equivalent loads and applying them to a finite-element model. Ko et al. [8] derived the deformation due to line heating by defining the inherent strain for the HAZ to calculate the equivalent load. Ha [9–11] developed a strain as direct boundary (SDB) method that applies inherent strain as an equivalent temperature boundary condition and predicts the thermal deformation of unit specimens and large structures. Noh et al. [12] proposed a method of installing permanent stiffeners such that the effect of line heating does not disappear during the lifting process of the ship. The properties affected by the phase transformation were calculated considering the properties and fractions of each phase for the heating and cooling of line heating. By applying this, the efficiency of line heating as analyzed according to the shape and distance of the stiffeners. p Wind tower blocks are welded with flanges for assembly with other blocks. Resid- ual deformation due to the welding affects the flatness of the flange. Zhang et al. [13] investigated residual stress distribution and evolution in single-pass bead-on-plate TIG welding of S355J2 steels using a thermo-metallurgical–mechanical coupling model. The prediction result of the welding residual stress confirmed that the solid-state phase trans- formation incorporating undercooling austenite and transformation strain is an important factor influencing the welding residual stress. Hensel et al. [14] compared fatigue tests for welded joints, including the microstructure, hardness, phase transformation temperature, and mechanical properties of base materials S355J2 and S960QL using low transformation temperature (LTT) filler and conventional filler. As a result, it was confirmed that the Fe-Cr-Ni-based LTT-metal cored filler material can be reliably welded without cracking or pore formation. Heinze et al. [15] revised the numerical model considering residual stress evolution in the heat-affected zone to welding of low-alloy structural steel S355J2 and heat-resistant steel P91. For structural steel S355J2, no significant influence on the residual stress state due to the usage of the enhanced model was found. For P91, which transforms to become fully martensitic, the residual stress calculation appears to be a difference of up to 200 MPa. 1. Introduction Received: 10 October 2022 Accepted: 9 November 2022 Published: 10 November 2022 The rapid heating and cooling process of welding causes internal expansion and shrinkage of the weld, and residual deformation is generated after welding is completed. Residual deformation reduces the strength of the structure and affects the assembly process according to the tolerance; therefore, a post-treatment process is required to remove it. Line heating is a procedure to remove out-of-plane deformation by generating a symmetrical residual deformation based on a neutral plane through a heating and cooling process using a heat input source such as a torch and induction. In addition, line heating is used to remove the deformation and process it into the desired shape. Therefore, experiments and numerical analysis are being conducted to investigate the relationship between the factors affecting line heating and deformation. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). g g Fujishiro et al. [1] performed a sensitivity analysis on the variables affecting line heating using an experimental method. The analysis was performed on seven variables using an orthogonal array table, and the plate thickness showed the most significant sensitivity. Satoh et al. [2] studied the angular deformation during heating and cooling and found that most of the deformation occurred during cooling. Lim et al. [3] investigated the changes in the physical properties due to the line heating and cooling process. They confirmed that the grains in the heat-affected zone (HAZ) were refined as ferrite and https://www.mdpi.com/journal/materials Materials 2022, 15, 7962. https://doi.org/10.3390/ma15227962 Materials 2022, 15, 7962 2 of 13 pearlite. Nomoto et al. [4] analyzed the deformation caused by line heating by dividing the cooling region of the water-cooled process, considering the actual cooling rate and volume percentage of each phase in the inherent strain region after line heating. Hwang et al. [5] analyzed the thermal deformation of a curved plate due to line heating using the shape of the HAZ derived from a flame-bending experiment. Since the development of numerical analysis, many studies have been conducted. Lee et al. 2. Procedures to Determine Optimal Line Heating Condition 2. Procedures to Determine Optimal Line Heating Condition The process of determining the optimal conditions for lin The process of determining the optimal conditions for line heating to offset the defor- mation due to welding is divided into three procedures, as shown in Figure 1. This section briefly describes each of these procedures. The first procedure calculates the flatness and tilt angle of the flange due to welding the wind tower block and flange. To perform the calculation, the temperature boundary condition of x-groove multilayer butt welding was obtained using the SDB method. Subsequently, the flatness and tilt angle were calculated from the difference in the axial direction displacement of the inner and outer edges of the flange. The second procedure involves calculating the flatness and tilt angle of the flange due to line heating. When line heating is performed on a constant area, the heat-affected zone (HAZ) shape is an equilateral trapezoid. Therefore, the temperature boundary condi- tion can be derived using the SDB method. Line heating was performed at a single point between the inner and outer edges of the flange, and at all single points between the edges to derive the heating position. The flatness and tilt angle due to line heating at a single point were calculated in the same manner as in the first procedure. The third procedure formulates an optimization function based on the deformation superposition theory to cal- culate the scale factor and line-heating position where the flatness and tilt angle of the weld are offset. The input variables for this calculation are the flatness and tilt angle of the flange due to welding and single-point heating. The scale factor is calculated by superposing with the flatness and tilt angle of the line heating and summing the weld deformation in the direction of the line heating. For each procedure, the single point that satisfies the design criteria and where the sum of the scale factors is the minimum is determined as the line heating position. The process of determining the optimal conditions for line heating to offset the de- formation due to welding is divided into three procedures, as shown in Figure 1. This section briefly describes each of these procedures. The first procedure calculates the flat- ness and tilt angle of the flange due to welding the wind tower block and flange. To per- form the calculation, the temperature boundary condition of x-groove multilayer butt welding was obtained using the SDB method. 2. Procedures to Determine Optimal Line Heating Condition 2. Procedures to Determine Optimal Line Heating Condition The process of determining the optimal conditions for lin Subsequently, the flatness and tilt angle were calculated from the difference in the axial direction displacement of the inner and outer edges of the flange. The second procedure involves calculating the flatness and tilt angle of the flange due to line heating. When line heating is performed on a constant area, the heat-affected zone (HAZ) shape is an equilateral trapezoid. Therefore, the temperature boundary condition can be derived using the SDB method. Line heating was performed at a single point between the inner and outer edges of the flange, and at all single points between the edges to derive the heating position. The flatness and tilt angle due to line heating at a single point were calculated in the same manner as in the first procedure. The third procedure formulates an optimization function based on the deformation superpo- sition theory to calculate the scale factor and line-heating position where the flatness and tilt angle of the weld are offset. The input variables for this calculation are the flatness and tilt angle of the flange due to welding and single-point heating. The scale factor is calcu- lated by superposing with the flatness and tilt angle of the line heating and summing the weld deformation in the direction of the line heating. For each procedure, the single point that satisfies the design criteria and where the sum of the scale factors is the minimum is determined as the line heating position. Figure 1. Procedures for determining the line-heating condition. Figure 1. Procedures for determining the line-heating condition. Figure 1. Procedures for determining the line-heating condition. Figure 1. Procedures for determining the line-heating condition. 1. Introduction It was confirmed that the simulation quality of the material exhibiting transforms fully martensitic can be improved by considering the Ms-temperature as a function of pAGS. p In the wind tower assembly process, the flatness of the flange must satisfy the design criteria. Line heating is performed on-site to satisfy the design criteria for flatness; however, the operator’s empirical judgment determines the work variable. Therefore, this study proposes a method of determining optimal line heating conditions to satisfy the design criteria for the flatness change generated by welding the wind tower block and flange. The temperature boundary conditions for the multi-layer butt welding of the wind tower blocks and flanges were obtained using the SDB method proposed by Ha [9]. Subsequently, the temperature boundary conditions were applied to the three-dimensional shell model to obtain the change in the flatness and tilt angle of the flange by welding. In addition, the change in the flatness and tilt angle of the flange was obtained when line heating was performed at a single point. The flatness change for a single point was superimposed in the line-heating direction to offset the flatness change due to welding. At this time, the flatness change due to single-point heating is superimposed with a scale factor, which means the magnitude of line heating, and is added to the flatness change due to welding. The optimal scale factor satisfying the design criteria for the flatness and tilt angle of the flange was obtained using optimization. Materials 2022, 15, 7962 3 of 13 tilt an- 3. Flange Tolerance Requirements 3. Flange Tolerance Requirements Since 2000, there has been a problem with the connecting bolts of the tower block and the flange breaking during the wind tower operation. The leading cause of this problem was identified as the manufacturing and installation tolerance of the flanges. Manufactur- ing and installation tolerances are divided by the flatness and tilt angle of the flange top, which have a decisive effect on the structural integrity after bolting [16,17]. Since 2000, there has been a problem with the connecting bolts of the tower block and the flange breaking during the wind tower operation. The leading cause of this problem was identified as the manufacturing and installation tolerance of the flanges. Manufacturing and installation tolerances are divided by the flatness and tilt angle of the flange top, which have a decisive effect on the structural integrity after bolting [16,17]. which have a decisive effect on the structural integrity after bolting [16,17]. Deutsches Institut für Bautechnik (DIBt) 2004 provided only qualitative requirements for wind towers [18]. In 2008, flatness tolerance was proposed to complement the qualita- tive requirements of DIBt owing to the enlargement of wind towers [19]. A tolerance of Deutsches Institut für Bautechnik (DIBt) 2004 provided only qualitative requirements for wind towers [18]. In 2008, flatness tolerance was proposed to complement the qualitative requirements of DIBt owing to the enlargement of wind towers [19]. A tolerance of local flatness sectors (within 30◦) of 0.5 mm and global flatness (360◦) of 2.0 mm must be satisfied. However, historically, there was no problem when a tolerance of 1.0 mm local flatness was applied. It was determined through experimental research and experience that satisfying Materials 2022, 15, 7962 4 of 13 m local for the 4 of 13 m local for the the tolerance of local flatness is difficult. In DIBt 2012, the tolerance for the local flatness sector (within 30◦) was relaxed to 1.0 mm [20]. An axial direction deformation represents the tilt angle as the slope of the existing and final flange shape. Therefore, the tilt angle can be expressed as the angular displacement. The angular displacement equally changes with the change in flatness, as shown in Figure 2, and a tolerance of 2.0 mm must be satisfied. local flatness sector (within 30°) was relaxed to 1.0 mm [20]. An axial direction defor- mation represents the tilt angle as the slope of the existing and final flange shape. 3. Flange Tolerance Requirements 3. Flange Tolerance Requirements There- fore, the tilt angle can be expressed as the angular displacement. The angular displace- ment equally changes with the change in flatness, as shown in Figure 2, and a tolerance of 2.0 mm must be satisfied. fore, the tilt angle can be expressed as the angular displacement. The angular displace- ment equally changes with the change in flatness, as shown in Figure 2, and a tolerance of 2.0 mm must be satisfied. Figure 2. Design requirements for flanges’ flatness and tilt angle. Figure 2. Design requirements for flanges’ flatness and tilt angle. Figure 2. Design requirements for flanges’ flatness and tilt angle. 4. Calculation of Flatness and Tilt Angle of the Flange by Welding Figure 2. Design requirements for flanges’ flatness and tilt angle. Figure 2. Design requirements for flanges’ flatness and tilt angle. 4. Calculation of Flatness and Tilt Angle of the Flange by Figure 2. Design requirements for flanges’ flatness and tilt angle. Figure 2. Design requirements for flanges’ flatness and tilt angle. 4. Calculation of Flatness and Tilt Angle of the Flange by Figure 2. Design requirements for flanges’ flatness and tilt angle. Figure 2. Design requirements for flanges’ flatness and tilt angle. 4. Calculation of Flatness and Tilt Angle of the Flange by 4. Calculation of Flatness and Tilt Angle of the Flange by Welding 4. Calculation of Flatness and Tilt Angle of the Flange by Welding The research object of this study was a cylindrical block of a 3 with a flange attached for connection between blocks [21] The flang 4. Calculation of Flatness and Tilt Angle of the Flange by Welding The research object of this study was a cylindrical block of a 3.6 MW wind tower, with a flange attached for connection between blocks [21]. The flange was connected to the block via butt welding. The block shape and weld zone are shown in Figure 3. The shape of the welded and welding sequence varies depending on the working environment and among them, the representative shape and welding sequence were selected and re- flected in the study. A three-dimensional model composed of shell elements was con- structed to obtain the deformation due to butt welding of the block and flange; the mate- rial used was S355j2 structure steel, and the material properties are shown in Table 1 The research object of this study was a cylindrical block of a 3.6 MW wind tower, with a flange attached for connection between blocks [21]. The flange was connected to the block via butt welding. The block shape and weld zone are shown in Figure 3. The shape of the welded and welding sequence varies depending on the working environment and among them, the representative shape and welding sequence were selected and reflected in the study. A three-dimensional model composed of shell elements was constructed to obtain the deformation due to butt welding of the block and flange; the material used was S355j2 structure steel, and the material properties are shown in Table 1 [17,22,23] with a flange attached for connection between blocks [21]. The flange was connected to the block via butt welding. The block shape and weld zone are shown in Figure 3. The shape of the welded and welding sequence varies depending on the working environment and among them, the representative shape and welding sequence were selected and re- flected in the study. A three-dimensional model composed of shell elements was con- structed to obtain the deformation due to butt welding of the block and flange; the mate- rial used was S355j2 structure steel, and the material properties are shown in Table 1 [17,22,23] j , p p [17,22,23] Figure 3. The shape of the wind-tower block, flange, and weld zone. Figure 3. The shape of the wind-tower block, flange, and weld zone. Figure 3. The shape of the wind-tower block, flange, and weld zone. Figure 3. The shape of the wind-tower block, flange, and weld zone. 4. Calculation of Flatness and Tilt Angle of the Flange by Welding 4. Calculation of Flatness and Tilt Angle of the Flange by Welding The research object of this study was a cylindrical block of a 3 with a flange attached for connection between blocks [21] The flang Materials 2022, 15, 7962 5 of 13 Table 1. Material properties of S355J2 structure steel. Table 1. Material properties of S355J2 structure steel. Table 1. Material properties of S355J2 structure steel. Property S355J2 Young’s Modulus, Mpa 210 Density, kg/m3 7850 Poisson’s ratio 0.3 Tensile strength, Mpa 630 Yield strength, Mpa 355 Melting point, ◦C 1530 The SDB method was used to obtain the deformation due to butt welding. This method simulates the inherent strain in the member during heating and cooling as internal shrinkage due to welding. Inherent strain refers to strain that is not recovered even when structural restraints or loads are removed. The inherent strain (ϵ∗) is obtained by subtracting the elastic strain (ϵe) from the total strain  ϵtotal , as shown in Equation (1), and this strain is equal to the sum of the thermal strain  ϵth , plastic strain (ϵp), and phase- transformation strain ϵtr [4]. The inherent strain can be expressed as a virtual thermal expansion coefficient and temperature boundary conditions. The virtual thermal expansion coefficient is calculated from the material thermal expansion coefficient and melting point, and the temperature boundary condition is calculated considering the shape of the heat-affected zone. The SDB method was used to obtain the deformation due to butt welding. This method simulates the inherent strain in the member during heating and cooling as internal shrinkage due to welding. Inherent strain refers to strain that is not recovered even when structural restraints or loads are removed. The inherent strain (ϵ∗) is obtained by subtracting the elastic strain (ϵe) from the total strain  ϵtotal , as shown in Equation (1),  th ( ) The SDB method was used to obtain the deformation due to butt welding. This method simulates the inherent strain in the member during heating and cooling as internal shrinkage due to welding. Inherent strain refers to strain that is not recovered even when structural restraints or loads are removed. The inherent strain (ϵ∗) is obtained by subtracting the elastic strain (ϵe) from the total strain  ϵtotal , as shown in Equation (1), and this strain is equal to the sum of the thermal strain  ϵth , plastic strain (ϵp), and phase- transformation strain ϵtr [4]. The inherent strain can be expressed as a virtual thermal expansion coefficient and temperature boundary conditions. 4. Calculation of Flatness and Tilt Angle of the Flange by Welding 4. Calculation of Flatness and Tilt Angle of the Flange by Welding The research object of this study was a cylindrical block of a 3 with a flange attached for connection between blocks [21] The flang The bloc welding sequence is A > C > B > D, and each welding procedure was performe completion of the stacking procedure. The measurement positions of the flat angle after the welding procedure were completed, shown in Figure 5. The calculated as the difference between displacements in the x-direction for th outer edges. The tilt angle was calculated using Equation (4) by measuring th outer points’ x- and y-axis displacements. The flatness and tilt angle were calc and 45 ° intervals, respectively, as shown in Figure 6, and compared with th teria. ௜ ௬𝑦ቁ ௜ ∙ ℎ ℎ௜ ሺ௎ሻ+ ℎ௬ሺ𝑦ሻ ൱𝑑𝑧ൡ𝑑𝑦 ௎ሻ+ ℎ௬ሺ𝑦ሻ 2 , ℎଶ= −ℎ௜ ሺ௎ሻ+ ℎ௬ሺ𝑦ሻ 2 + ൫ℎ௜ ሺ௎ሻ+ ℎ by pass ℎ௜ሺ𝑢ሻ: Weld height by pass ℎ௬ሺ𝑦ሻ 𝑇= ෍𝑇௜ ௠ ௜ୀଵ + ෍𝑇௝ ௡ ௝ୀଵ temperature boundary conditions m,n: N Figure 4. The schematic of the x-groove butt weld modeling. Figure 4. The schematic of the x-groove butt weld modeling. p p bottom passes. The calculated temperature boundary The calculated temperature boundary condition was set at the node where the block and flange were joined according to the welding sequence, as shown in Figure 5. It was assumed that all weld shapes and stacking procedures were the same at this time. The block and flange were joined 1000 mm below the top of the flange. The block and flange welding sequence is A > C > B > D, and each welding procedure was performed following completion of the stacking procedure. The measurement positions of the flatness and tilt angle after the welding procedure were completed, shown in Figure 5. The flatness was calculated as the difference between displacements in the x-direction for the inner and outer edges. The tilt angle was calculated using Equation (4) by measuring the inner and outer points’ x- and y-axis displacements. The flatness and tilt angle were calculated at 5/3 and 45◦intervals, respectively, as shown in Figure 6, and compared with the design criteria. p y and flange were joined according to the welding sequence, as shown in Fig assumed that all weld shapes and stacking procedures were the same at th block and flange were joined 1000 mm below the top of the flange. The bloc welding sequence is A > C > B > D, and each welding procedure was perform completion of the stacking procedure. 4. Calculation of Flatness and Tilt Angle of the Flange by Welding 4. Calculation of Flatness and Tilt Angle of the Flange by Welding The research object of this study was a cylindrical block of a 3 with a flange attached for connection between blocks [21] The flang The virtual thermal expansion coefficient is calculated from the material thermal expansion coefficient and melting point, and the temperature boundary condition is calculated considering the shape of the heat-affected zone. q   , p ( ), phase- transformation strain ϵtr [4]. The inherent strain can be expressed as a virtual thermal expansion coefficient and temperature boundary conditions. The virtual thermal expansion coefficient is calculated from the material thermal expansion coefficient and melting point, and the temperature boundary condition is calculated considering the shape of the heat-affected zone. ϵ∗= ϵth + ϵp + ϵtr = ϵtotal −ϵe (1) (1) The butt welding of thick plates is performed by stacking several passes, and the SDB method considering this stacking was proposed by Ha [24,25]. In the proposed method, assuming that each pass has the same cross-sectional area and is stacked in the layer direction, a temperature boundary condition corresponding to the inherent strain was calculated. Butt welding for the X-groove was performed on the upper and lower surfaces of the plate, and the parameters used to obtain the temperature load for each case were defined, as shown in Figure 4. The temperature boundary condition can be calculated using Equation (2) by considering the weld height and width changes. After all the welding passes are performed, the temperature boundary condition corresponding to the inherent strain of the weld can be obtained by superposing all the temperature boundary conditions for each welding pass. This superpose is expressed in Equation (3), and the temperature boundary conditions for the upper and lower surfaces are reversed and superposed. Ti = R b(U) i 0 1  h(U) i +hy(y)  ·b R h2 h1  h(U) i +hy(y) h ∓ 4z h(U) i +hy(y)· h h(U) i +hy(y)  dz  dy h1 = −h(U) i +hy(y) 2 , h2 = −h(U) i +hy(y) 2 +  h(U) i + h(U) i−1  (2) (2) where bi(u) : Weld width by pass hi(u) : Weld height by pass hy(y) : Lower thickness changes by pass. T = m ∑ i=1 Ti + n ∑ j=1 Tj (3) (3) where Ti,j: Top and bottom temperature boundary conditions m,n: Numer of top and bottom passes. Materials 2022, 15, 7962 6 of 13 ondi mper- Figure 4. The schematic of the x-groove butt weld modeling. Figure 4. The schematic of the x-groove butt weld modeling. 4. Calculation of Flatness and Tilt Angle of the Flange by Welding 4. Calculation of Flatness and Tilt Angle of the Flange by Welding The research object of this study was a cylindrical block of a 3 with a flange attached for connection between blocks [21] The flang 𝑇௜= න 1 ቀℎ௜ ሺ௎ሻ+ ℎ௬ሺ𝑦ሻቁ∙𝑏 ൝න ൭ℎ௜ + ℎ௬ሺ𝑦ሻ ℎ ∓ 4𝑧 ℎ௜ ሺ௎ሻ+ ℎ௬ሺ𝑦ሻ ௛భ ೔ ଴ ∙ ℎ ℎ௜ ሺ௎ሻ+ ℎ௬ሺ𝑦ሻ ൱𝑑𝑧ൡ𝑑𝑦 ℎଵ= −ℎ௜ ሺ௎ሻ+ ℎ௬ሺ𝑦ሻ 2 , ℎଶ= −ℎ௜ ሺ௎ሻ+ ℎ௬ሺ𝑦ሻ 2 + ൫ℎ௜ ሺ௎ሻ+ ℎ௜ିଵ ሺ௎ሻ൯ where 𝑏௜ሺ𝑢ሻ: Weld width by pass ℎ௜ሺ𝑢ሻ: Weld height by pass ℎ௬ሺ𝑦ሻ: Low changes by pass. 𝑇= ෍𝑇௜ ௠ ௜ୀଵ + ෍𝑇௝ ௡ ௝ୀଵ where Ti,j: Top and bottom temperature boundary conditions m,n: Nume bottom passes. The calculated temperature boundary condition was set at the node wh Figure 4. The schematic of the x-groove butt weld modeling. Figure 4. The schematic of the x-groove butt weld modeling. The calculated temperature boundary condition was set at the node where the block and flange were joined according to the welding sequence, as shown in Figure 5. It was assumed that all weld shapes and stacking procedures were the same at this time. The block and flange were joined 1000 mm below the top of the flange. The block and flange welding sequence is A > C > B > D, and each welding procedure was performed following completion of the stacking procedure. The measurement positions of the flatness and tilt angle after the welding procedure were completed, shown in Figure 5. The flatness was calculated as the difference between displacements in the x-direction for the inner and outer edges. The tilt angle was calculated using Equation (4) by measuring the inner and outer points’ x- and y-axis displacements. The flatness and tilt angle were calculated at 5/3 and 45◦intervals, respectively, as shown in Figure 6, and compared with the design criteria. 𝑇௜= න ቀℎ௜ ሺ௎ሻ+ ℎ௬ሺ𝑦ሻቁ∙𝑏 ൝න ൭௜ ௬ሺ𝑦ሻ ℎ ∓ ℎ௜ ሺ௎ሻ+ ℎ௬ሺ𝑦ሻ ௛భ ଴ ∙ ℎ ℎ௜ ሺ௎ሻ+ ℎ௬ሺ𝑦ሻ ൱𝑑𝑧ൡ𝑑𝑦 ℎଵ= −ℎ௜ ሺ௎ሻ+ ℎ௬ሺ𝑦ሻ 2 , ℎଶ= −ℎ௜ ሺ௎ሻ+ ℎ௬ሺ𝑦ሻ 2 + ൫ℎ௜ ሺ௎ሻ+ ℎ௜ିଵ ሺ௎ሻ൯ where 𝑏௜ሺ𝑢ሻ: Weld width by pass ℎ௜ሺ𝑢ሻ: Weld height by pass ℎ௬ሺ𝑦ሻ: Low changes by pass. 𝑇= ෍𝑇௜ ௠ ௜ୀଵ + ෍𝑇௝ ௡ ௝ୀଵ where Ti,j: Top and bottom temperature boundary conditions m,n: Numer bottom passes. The calculated temperature boundary condition was set at the node whe and flange were joined according to the welding sequence, as shown in Fig assumed that all weld shapes and stacking procedures were the same at th block and flange were joined 1000 mm below the top of the flange. ation of Flange Flatness by Line Heating 5. Calculation of Flange Flatness by Line Heating heating is a process that causes deformation by generating inherent strain owing at, which offsets the previously generated deformation. Line heating moves the at source to the desired area with a uniform rule. In addition, knuckles can be d at the end of the heating unit by minimizing the length of the remaining portion heating. If line heating is performed over a constant area, the shape of the HAZ to that of an equilateral trapezoid [5]. The temperature boundary condition can ed using the SDB method for a given HAZ and can be obtained using Equations 6). Line heating is a process that causes deformation by generating inherent strain owing to the heat, which offsets the previously generated deformation. Line heating moves the input heat source to the desired area with a uniform rule. In addition, knuckles can be prevented at the end of the heating unit by minimizing the length of the remaining portion without heating. If line heating is performed over a constant area, the shape of the HAZ is similar to that of an equilateral trapezoid [5]. The temperature boundary condition can be derived using the SDB method for a given HAZ and can be obtained using Equations (5) and (6). 1 𝐵∙ℎන 𝑏ሺ𝑧ሻ∙൬1 −4 ℎ∙𝑧൰ 𝑑𝑧 ௛ଶ ⁄ ି௛ଶ ⁄ (5) = 1 න 𝑏ሺ𝑧ሻ∙൬1 + 4 ∙𝑧൰𝑑𝑧 ௛ଶ ⁄ (6) Ttop = 1 B·h Z h/2 −h/2 b(z)·  1 −4 h·z  dz (5) Tbottom = 1 B·h Z h/2 −h/2 b(z)·  1 + 4 h·z  dz (6) (5) (6) 𝑇௕௢௧௧௢௠ 𝐵∙ℎන 𝑏ሺ𝑧ሻ൬1 + ℎ𝑧൰ 𝑑𝑧 ି௛ଶ ⁄ (6) Width of the HAZ; h: Thickness of the plate; b(z): Width of HAZ with change in where B: Width of the HAZ; h: Thickness of the plate; b(z): Width of HAZ with change in depth z. Width of the HAZ; h: Thickness of the plate; b(z): Width of HAZ with change in e heating with a depth of 5 mm and width of 100 mm was performed on the he flange-flatness results according to the line-heating range are shown in Figure the line heating region was applied in the range of 0–30°, 0–60°, and 0–90° in the rection, it was confirmed that the maximum flange flatness increased to 0.38858, and 0.97117 mm, respectively. The range of flatness change gradually increased rtion to the line-heating range, and flatness changes occurred near the line-heat- e. 4. Calculation of Flatness and Tilt Angle of the Flange by Welding 4. Calculation of Flatness and Tilt Angle of the Flange by Welding The research object of this study was a cylindrical block of a 3 with a flange attached for connection between blocks [21] The flang The measurement positions of the flat angle after the welding procedure were completed, shown in Figure 5. The calculated as the difference between displacements in the x-direction for th outer edges. The tilt angle was calculated using Equation (4) by measuring t outer points’ x- and y-axis displacements. The flatness and tilt angle were calc and 45 ° intervals, respectively, as shown in Figure 6, and compared with th teria. Tilt Angle = tan−1 |xi −xo| |yi −yo|  · 180 π  , (4) 𝑇𝑖𝑙𝑡 𝐴𝑛𝑔𝑙𝑒= tanିଵቆ|𝑥௜−𝑥௢| |𝑦௜−𝑦௢|ቇ∙൬180 𝜋൰, y : Displacement of inner point for each axis x y : Displacement of ou (4) where xi,yi: Displacement of inner point for each axis xo,yo: Displacement of outer point for each axis. where xi,yi: Displacement of inner point for each axis xo,yo: Displacement of ou each axis. Figure 5. The welding position and sequence, and the measurement position of the Figure 5. The welding position and sequence, and the measurement position of the block. Figure 5. The welding position and sequence, and the measurement positi Figure 5. The welding position and sequence, and the measurement position of the block. 7 of 13 Materials 2022, 15, 7962 022, 15, x FOR PEER REVIE (a) (a) (b) Figure 6. The result of welding procedure: (a) flatness changes; (b) tilt angle and angular displace- ment. Figure 6. The result of welding procedure: (a) flatness changes; (b) tilt angle and angular displacement. (b) (b) (a) The result of welding procedure: (a) flatness changes; (b) tilt angle and angular displace- Figure 6. The result of welding procedure: (a) flatness changes; (b) tilt angle and angular displacement. flatness was distributed in the range of 1.72096–1.73046 mm in the negative di- he tilt angle was distributed at 0.129758–0.130618°, with an angular displacement m. The flatness after welding satisfies the design tolerance of 2.0 mm in the global ector (360°) but not 1.0 mm in the local flatness sector (within 30°). The tilt angle the design tolerance of 2.0 mm based on the angular displacement. After the process, the flange is part of the fastening element for connection with the upper d smooth fastening with other blocks is only achieved when the flatness and tilt ow a certain level are satisfied. Therefore, it is necessary to alleviate the flatness different processes, such as line heating. 4. Calculation of Flatness and Tilt Angle of the Flange by Welding 4. Calculation of Flatness and Tilt Angle of the Flange by Welding The research object of this study was a cylindrical block of a 3 with a flange attached for connection between blocks [21] The flang The flatness was distributed in the range of 1.72096–1.73046 mm in the negative direc- tion. The tilt angle was distributed at 0.129758–0.130618◦, with an angular displacement of 1.52 mm. The flatness after welding satisfies the design tolerance of 2.0 mm in the global flatness sector (360◦) but not 1.0 mm in the local flatness sector (within 30◦). The tilt angle satisfies the design tolerance of 2.0 mm based on the angular displacement. After the welding process, the flange is part of the fastening element for connection with the upper block, and smooth fastening with other blocks is only achieved when the flatness and tilt angle below a certain level are satisfied. Therefore, it is necessary to alleviate the flatness through different processes, such as line heating. 6. Determination of Line Heating Position and Scale Factor Using Optimization 6. Determination of Line Heating Position and Scale Factor Using Optimization 6. Determination of Line Heating Position and Scale Factor Using Optimization The process of deriving the optimal line heating conditions is illustrated in Figure 8. The flatness and tilt angle due to the welding blocks and flanges were organized as angle data. The flatness change and tilt angle due to line heating are organized into angle data for every point between the inner and outer points. The flatness result of line heating can be obtained by superimposing the change due to single-point line heating in the line heat- ing direction, as presented in Section 5. At this time, the flatness change due to each single- point line heating was superimposed in the line heating direction with a scale factor. The process of deriving the optimal line heating conditions is illustrated in Figure 8. The flatness and tilt angle due to the welding blocks and flanges were organized as angle data. The flatness change and tilt angle due to line heating are organized into angle data for every point between the inner and outer points. The flatness result of line heating can be obtained by superimposing the change due to single-point line heating in the line heating direction, as presented in Section 5. At this time, the flatness change due to each single-point line heating was superimposed in the line heating direction with a scale factor. The process of deriving the optimal line heating conditions is illustrated in Figure 8. The flatness and tilt angle due to the welding blocks and flanges were organized as angle data. The flatness change and tilt angle due to line heating are organized into angle data for every point between the inner and outer points. The flatness result of line heating can be obtained by superimposing the change due to single-point line heating in the line heat- ing direction, as presented in Section 5. At this time, the flatness change due to each single- point line heating was superimposed in the line heating direction with a scale factor. Figure 8. Optimization procedure for determining the optimal line-heating conditions. Figure 8. Optimization procedure for determining the optimal line-heating conditions. Figure 8. Optimization procedure for determining the optimal line-heating conditions. Figure 8. Optimization procedure for determining the optimal line-heating conditions. Figure 8. Optimization procedure for determining the optimal line-heating conditions. Figure 8. Optimization procedure for determining the optimal line-heating conditions. ation of Flange Flatness by Line Heating 5. Calculation of Flange Flatness by Line Heating Determination of Line Heating Position and Scale Factor Using Optimization 6. Determination of Line Heating Position and Scale Factor Using Optimization ation of Flange Flatness by Line Heating 5. Calculation of Flange Flatness by Line Heating Next, the line heating of 0–60° and 60–90° was superimposed and compared se of 0–90°. The flatness changes were similar. When the difference in each direc- Line heating with a depth of 5 mm and width of 100 mm was performed on the flange. The flange-flatness results according to the line-heating range are shown in Figure 7. When the line heating region was applied in the range of 0–30◦, 0–60◦, and 0–90◦in the radial direction, it was confirmed that the maximum flange flatness increased to 0.38858, 0.72473, and 0.97117 mm, respectively. The range of flatness change gradually increased in proportion to the line-heating range, and flatness changes occurred near the line-heating range. Next, the line heating of 0–60◦and 60–90◦was superimposed and compared with those of 0–90◦. The flatness changes were similar. When the difference in each direction of flatness is expressed as a percentage, the maximum difference is 4.92%, which indicates a considerably similar distribution. Therefore, when line heating is performed at several locations, the flatness result can be obtained by summing those for each line heating. Materials 2022, 15, 7962 8 of 13 ich in- med at ich in- med at y g (b) result can be obtained by summing those for each lin (b) y g g (a) (b) Figure 7. The flatness change by the line heating. (a) Flatness changes according to each line heating range; (b) Flatness changes according to superposing. Figure 7. The flatness change by the line heating. (a) Flatness changes according to each line heating range; (b) Flatness changes according to superposing. , y g g (a) (b) Figure 7. The flatness change by the line heating. (a) Flatness changes according to each line heating range; (b) Flatness changes according to superposing. (a) , (a) (b) (b) (a) (a) Figure 7. The flatness change by the line heating. (a) Flatness changes according to each line heating range; (b) Flatness changes according to superposing. Figure 7. The flatness change by the line heating. (a) Flatness changes according to each line heating range; (b) Flatness changes according to superposing. Figure 7. The flatness change by the line heating. (a) Flatness changes according to each line heating range; (b) Flatness changes according to superposing. 6. Determination of Line Heating Position and Scale Factor Using Optimization h f d h l l h d ll d 6. 6. Determination of Line Heating Position and Scale Factor Using Optimization 6. Determination of Line Heating Position and Scale Factor Using Optimization 6. Determination of Line Heating Position and Scale Factor Using Optimization The optimal line heating conditions to satisfy the design criteria were obtained using the optimization conditions. ฬሼ𝑓௪௘௟ௗሽ + ෍ 𝑚௜∙൛𝑓௜ ௟௜௡௘ ௛௘௔௧௜௡௚ൟ ௡ ௜ୀଵ ฬ≤𝑇𝑜𝑙𝑒𝑟𝑎𝑛𝑐𝑒 ฬሼ𝛿௪௘௟ௗሽ+ ෍ 𝑚௜∙൛𝛿௜ ௟௜௡௘ ௛௘௔௧௜௡௚ൟ ௡ ฬ≤𝑇𝑜𝑙𝑒𝑟𝑎𝑛𝑐𝑒 (8) position of line heating. Equation (8) is a constraint indicating that the superposition results of the flatness and tilt angle of welding and line heating must satisfy the design criteria. The design tolerance of flatness was set to 1.0 mm for the local flatness sector (within 30◦), and the tilt angle was set to 2.0 mm based on angular displacement. The optimal line heating conditions to satisfy the design criteria were obtained using the optimization conditions. ฬሼ𝑓௪௘௟ௗሽ + ෍ 𝑚௜∙൛𝑓௜ ௟௜௡௘ ௛௘௔௧௜௡௚ൟ ௡ ௜ୀଵ ฬ≤𝑇𝑜𝑙𝑒𝑟𝑎𝑛𝑐𝑒 ฬሼ𝛿௪௘௟ௗሽ+ ෍ 𝑚௜∙൛𝛿௜ ௟௜௡௘ ௛௘௔௧௜௡௚ൟ ௡ ฬ≤𝑇𝑜𝑙𝑒𝑟𝑎𝑛𝑐𝑒 (8) min.  ∑ n i=1 mi  (7) y welding; δweld: Tilt angle of flange by weld- (7) ld n f weldo + ∑n i=1 mi· n f line heating i o ≤Tolerance n δweldo + ∑n i=1 mi· n δline heating i o ≤Tolerance (8) g y g; g g y e by line heating at a single point; 𝛿௜ ௟௜௡௘ ௛௘௔௧௜௡௚: Tilt angle e point he scale factor was derived in the range of 0 270677– (8) ngle where mi: scale factor; fweld: Flatness of flange by welding; δweld: Tilt angle of flange by welding; f line heating i : Flatness of flange by line heating at a single point; δline heating i : Tilt angle of flange by line heating at a single point zation result, the scale factor was derived in the range of 0.270677 point. The line-heating position was determined to be the h6 position the scale factors was minimum. The line-heating direction proceeds in d h h f h b d d As an optimization result, the scale factor was derived in the range of 0.270677–0.30085 for every point. The line-heating position was determined to be the h6 position, where the sum of the scale factors was minimum. The line-heating direction proceeds in the circumferential direction at the h6 position of 0◦. The temperature boundary condition of the line heating was multiplied by the optimization result of the scale factor and applied to the analysis model. When optimal line-heating conditions were applied to the welding analysis results, the maximum flatness decreased from 1.73046 mm to 0.99170 mm. 6. Determination of Line Heating Position and Scale Factor Using Optimization 6. Determination of Line Heating Position and Scale Factor Using Optimization 6. Determination of Line Heating Position and Scale Factor Using Optimization The scale factor is a variable that adjusts the magnitude of the line heating, and this adjustment implies that this parameter is related to the magnitude of the flatness. The size of the line heating may vary depending on the line-heating method. However, if constant heating is assumed, the magnitude of the input heat quantity and scale factor are directly proportional. Therefore, the total sum of the scale factors is proportional to the size of the total input heat. By minimizing the scale factor, flatness correction can be performed with The scale factor is a variable that adjusts the magnitude of the line heating, and this adjustment implies that this parameter is related to the magnitude of the flatness. The size of the line heating may vary depending on the line-heating method. However, if constant heating is assumed, the magnitude of the input heat quantity and scale factor are directly proportional. Therefore, the total sum of the scale factors is proportional to the size of the total input heat. By minimizing the scale factor, flatness correction can be performed with The scale factor is a variable that adjusts the magnitude of the line heating, and this adjustment implies that this parameter is related to the magnitude of the flatness. The size of the line heating may vary depending on the line-heating method. However, if constant heating is assumed, the magnitude of the input heat quantity and scale factor are directly proportional. Therefore, the total sum of the scale factors is proportional to the size of the total input heat. By minimizing the scale factor, flatness correction can be performed with minimum input heat. The aforementioned conditions are expressed by Equations (7) and (8). Equation (7) indicates that the sum of the scale factors must have a minimum value as an objective function for optimization. Here, for all single points, the point at which the sum of the scale factors derives the minimum value is selected as the Materials 2022, 15, 7962 9 of 13 (7) position of line heating. Equation (8) is a constraint indicating that the superposition results of the flatness and tilt angle of welding and line heating must satisfy the design criteria. The design tolerance of flatness was set to 1.0 mm for the local flatness sector (within 30◦), and the tilt angle was set to 2.0 mm based on angular displacement. 7. Verification of Methods for Determining Line Heating Conditions 7. Verification of Methods for Determining Line Heating Conditions The size and welding conditions of the wind tower block were varied to verify the proposed method. The size of the wind tower block was divided into 1.5 MW, 3.6 MW, and 5.0 MW. The welding conditions were assumed to be identical for each pass. The flange flatness and tilt angle due to welding and line heating are listed in Table 2. The flatness resulting from welding wind tower blocks and flanges satisfies the design toler- ance of global flatness (360°) for all sizes, but not local flatness (within 30°). The optimal scale factor and line heating position were selected by applying the proposed method. Single-point heating locations were divided into h1-h5 (1.5 MW), h1-h7 (3.6 MW), and h1- h7 (5.0 MW) according to the block size order. Line heating was then performed by ap- plying the selected line heating position and scale factor. The flange flatness after line heating satisfied the tolerance of the local flatness (within 30°), and the decrease in the angular displacement was also confirmed according to the decrease in the tilt angle. The size and welding conditions of the wind tower block were varied to verify the proposed method. The size of the wind tower block was divided into 1.5 MW, 3.6 MW, and 5.0 MW. The welding conditions were assumed to be identical for each pass. The flange flatness and tilt angle due to welding and line heating are listed in Table 2. The flatness resulting from welding wind tower blocks and flanges satisfies the design tolerance of global flatness (360◦) for all sizes, but not local flatness (within 30◦). The optimal scale factor and line heating position were selected by applying the proposed method. Single-point heating locations were divided into h1–h5 (1.5 MW), h1–h7 (3.6 MW), and h1–h7 (5.0 MW) according to the block size order. Line heating was then performed by applying the selected line heating position and scale factor. The flange flatness after line heating satisfied the tolerance of the local flatness (within 30◦), and the decrease in the angular displacement was also confirmed according to the decrease in the tilt angle. Table 2. The verification result for the block-size and welding condition changes. 7. Verification of Methods for Determining Line Heating Conditions 7. Verification of Methods for Determining Line Heating Conditions Design Criteria Procedure Block Size Flatness (mm) Tilt Angle (°) Angular Displacement (mm) Heating Location Welding 1.5_MW 1.20992–1.21582 0.117766–0.117861 1.41 3.6_MW 1.72096–1.73046 0.129758–0.130618 1.51–1.52 5.0_MW 1.51567–1.51682 0.109238–0.109239 1.51 Line heating 1.5_MW 0.99446–0.99899 0.098562–0.099393 1.18–1.19 h4 3.6_MW 0.98234–0.99170 0.084246–0.085105 0.98–0.99 h6 5.0_MW 0.94640–0.94805 0.068737–0.068738 0.95 h7 The welding conditions of the wind tower block and flange depend on the working environment and operator. Changes in welding conditions affect the flange flatness. Therefore, welding was performed by changing the conditions for each pass of the 3.6 MW wind tower block and flange. The optimal conditions for line heating were deter- mined using the proposed method. Thus, it was confirmed that if the difference in flatness was large, the scale factor at the location with high flatness increased. Additionally, it was confirmed that the scale factor of the position that satisfied the flatness tolerance was zero. The position at which the scale factor is zero represents where line heating is unnecessary. Therefore, the proposed method can perform line heating by dividing the section for the Table 2. The verification result for the block-size and welding condition changes. Design Criteria Procedure Block Size Flatness (mm) Tilt Angle (◦) Angular Displacement (mm) Heating Location Welding 1.5_MW 1.20992–1.21582 0.117766–0.117861 1.41 3.6_MW 1.72096–1.73046 0.129758–0.130618 1.51–1.52 5.0_MW 1.51567–1.51682 0.109238–0.109239 1.51 Line heating 1.5_MW 0.99446–0.99899 0.098562–0.099393 1.18–1.19 h4 3.6_MW 0.98234–0.99170 0.084246–0.085105 0.98–0.99 h6 5.0_MW 0.94640–0.94805 0.068737–0.068738 0.95 h7 The welding conditions of the wind tower block and flange depend on the working environment and operator. Changes in welding conditions affect the flange flatness. There- fore, welding was performed by changing the conditions for each pass of the 3.6 MW wind tower block and flange. The optimal conditions for line heating were determined using the proposed method. Thus, it was confirmed that if the difference in flatness was large, the scale factor at the location with high flatness increased. Additionally, it was confirmed that the scale factor of the position that satisfied the flatness tolerance was zero. The position at which the scale factor is zero represents where line heating is unnecessary. Therefore, the proposed method can perform line heating by dividing the section for the position at which Table 2. The verification result for the block-size and welding condition changes. Table 2. The verification result for the block-size and welding condition changes. The welding conditions of the wind tower block and flange depend on the working environment and operator. 6. Determination of Line Heating Position and Scale Factor Using Optimization 6. Determination of Line Heating Position and Scale Factor Using Optimization 6. Determination of Line Heating Position and Scale Factor Using Optimization The maximum tilt angle decreased from 0.130618◦to 0.085105◦, and the maximum angular displacement decreased from 1.52 mm to 0.99 mm. The results of the x-direction deformation of the flange are shown in Figure 9. The flatness and tilt-angle results are shown in Figure 10. It is confirmed that the flatness and tilt angle of the flange satisfy the design tolerance under optimal line heating conditions. l direction at the h6 position of 0°. The temperature boundary condition was multiplied by the optimization result of the scale factor and applied del. When optimal line-heating conditions were applied to the welding e maximum flatness decreased from 1.73046 mm to 0.99170 mm. The le decreased from 0.130618° to 0.085105°, and the maximum angular eased from 1.52 mm to 0.99 mm. The results of the x-direction defor- ge are shown in Figure 9. The flatness and tilt-angle results are shown onfirmed that the flatness and tilt angle of the flange satisfy the design ptimal line heating conditions. (a) (b) Figure 9. The result of the x-direction deformation: (a) after welding; (b) after line heating. Figure 9. The result of the x-direction deformation: (a) after welding; (b) after line heating. (b) (a) (b) (a) of the x-direction deformation: (a) after welding; (b) after line heating. Figure 9. The result of the x-direction deformation: (a) after welding; (b) after line heating. 10 of 13 10 of 13 10 of 13 10 of 13 Materials 2022, 15, 7962 Materials 2022, 15, x FOR (a) (b) Figure 10. The result of line heating procedure: (a) flatness changes; (b) tilt angle and angular dis- placement. Figure 10. The result of line heating procedure: (a) flatness changes; (b) tilt angle and angular displacement. (b) (a) (b) (a) Figure 10. The result of line heating procedure: (a) flatness changes; (b) tilt angle and angular dis- placement. Figure 10. The result of line heating procedure: (a) flatness changes; (b) tilt angle and angular displacement. 7. Verification of Methods for Determining Line Heating Conditions 7. Verification of Methods for Determining Line Heating Conditions deformation of the flange are shown in Figure 11. The flatness and tilt-angle results are shown in Figure 12. (a) (a) (b) (b) (a) (b) Figure 11. The verification result of the x-direction deformation after the line heating between 120° and 240°: (a) after welding; (b) after line heating. Figure 11. The verification result of the x-direction deformation after the line heating between 120◦ and 240◦: (a) after welding; (b) after line heating. (a) (b) Figure 11. The verification result of the x-direction deformation after the line heating between 120° and 240°: (a) after welding; (b) after line heating. (b) (b) (a) (a) Figure 11. The verification result of the x-direction deformation after the line heating between 120° and 240°: (a) after welding; (b) after line heating. Figure 11. The verification result of the x-direction deformation after the line heating between 120◦ and 240◦: (a) after welding; (b) after line heating. Figure 11. The verification result of the x-direction deformation after the line heating between 120° and 240°: (a) after welding; (b) after line heating. (b) t of the line heating procedure: (a) flatness changes; (b) tilt (b) (a) (b) Figure 12. The verification result of the line heating procedure: (a) flatness changes; (b) tilt angle and angular displacement. The welding flatness change occurred at a maximum of 1 21252 mm between 120° (a) (b) Figure 12. The verification result of the line heating procedure: (a) flatness changes; (b) tilt angle and angular displacement. Figure 12. The verification result of the line heating procedure: (a) flatness changes; (b) tilt angle and angular displacement. (a) Fi 12 Th ifi ti (a) (a) i (a) (b) edure (b) igure 12. The verification result of the line heating procedure: (a) flatness changes; (b) tilt angle and angular displacement. h ld fl h d f 1 1 b 1 Figure 12. The verification result of the line heating procedure: (a) flatness changes; (b) tilt angle and angular displacement. Figure 12. The verification result of the line heating procedure: (a) flatness changes; (b) tilt angle and angular displacement. The welding flatness change occurred at a maximum of 1.21252 mm between 120 and 240°, and line heating was performed as described in this section. In the proposed method, line heating was performed at position h7, and the maximum flatness was re- duced to 0.99697 mm. The tilt angle of the flange decreased from 0.090981 to 0.084967°. 7. Verification of Methods for Determining Line Heating Conditions 7. Verification of Methods for Determining Line Heating Conditions Simultaneously with the tilt-angle change, the angular displacement decreased from 1.21 mm to 1.13 mm. It was confirmed that the deformation decreased in the section where the line heating was performed. However, the tilt angle and angular displacement increased and then decreased in the place where the line heating was not performed because the deformation was superposed. The welding flatness change occurred at a maximum of 1.21252 mm between 120° and 240°, and line heating was performed as described in this section. In the proposed method, line heating was performed at position h7, and the maximum flatness was re- duced to 0.99697 mm. The tilt angle of the flange decreased from 0.090981 to 0.084967°. Simultaneously with the tilt-angle change, the angular displacement decreased from 1.21 mm to 1.13 mm. It was confirmed that the deformation decreased in the section where the line heating was performed. However, the tilt angle and angular displacement increased and then decreased in the place where the line heating was not performed because the deformation was superposed. The welding flatness change occurred at a maximum of 1.21252 mm between 120◦ and 240◦, and line heating was performed as described in this section. In the proposed method, line heating was performed at position h7, and the maximum flatness was reduced to 0.99697 mm. The tilt angle of the flange decreased from 0.090981 to 0.084967◦. Simul- taneously with the tilt-angle change, the angular displacement decreased from 1.21 mm to 1.13 mm. It was confirmed that the deformation decreased in the section where the line heating was performed. However, the tilt angle and angular displacement increased and then decreased in the place where the line heating was not performed because the deformation was superposed. 7. Verification of Methods for Determining Line Heating Conditions 7. Verification of Methods for Determining Line Heating Conditions Changes in welding conditions affect the flange flatness. Therefore, welding was performed by changing the conditions for each pass of the 3.6 MW wind tower block and flange. The optimal conditions for line heating were deter- mined using the proposed method. Thus, it was confirmed that if the difference in flatness was large, the scale factor at the location with high flatness increased. Additionally, it was confirmed that the scale factor of the position that satisfied the flatness tolerance was zero. The position at which the scale factor is zero represents where line heating is unnecessary. Therefore, the proposed method can perform line heating by dividing the section for the The welding conditions of the wind tower block and flange depend on the working environment and operator. Changes in welding conditions affect the flange flatness. There- fore, welding was performed by changing the conditions for each pass of the 3.6 MW wind tower block and flange. The optimal conditions for line heating were determined using the proposed method. Thus, it was confirmed that if the difference in flatness was large, the scale factor at the location with high flatness increased. Additionally, it was confirmed that the scale factor of the position that satisfied the flatness tolerance was zero. The position at which the scale factor is zero represents where line heating is unnecessary. Therefore, the proposed method can perform line heating by dividing the section for the position at which Materials 2022, 15, 7962 Materials 2022, 15, x FOR Materials 2022, 15, x FOR 11 of 13 11 of 13 11 of 13 11 of 13 11 of 13 11 of 13 the deviation of the line heating is large. The results of the x-direction deformation of the flange are shown in Figure 11. The flatness and tilt-angle results are shown in Figure 12. deformation of the flange are shown in Figure 11. The flatness and tilt-angle results are shown in Figure 12. deformation of the flange are shown in Figure 11. The flatness and tilt-angle results are shown in Figure 12. the deviation of the line heating is large. The results of the x-direction deformation of the flange are shown in Figure 11. The flatness and tilt-angle results are shown in Figure 12. deformation of the flange are shown in Figure 11. The flatness and tilt-angle results are shown in Figure 12. 8. Conclusions The asse b 8. Conclusions 8. Conclusions The assembly of the wind tower block is bolted, and the flatness affects the installa- tion tolerance in the bolting process. In order to control flatness in the manufacturing stage, milling and line heating are performed in the field. Among these methods, line heating is performed by the operator’s empirical judgment without clear standards. There The assembly of the wind tower block is bolted, and the flatness affects the installa- tion tolerance in the bolting process. In order to control flatness in the manufacturing stage, milling and line heating are performed in the field. Among these methods, line heating is performed by the operator’s empirical judgment without clear standards. There The assembly of the wind tower block is bolted, and the flatness affects the installation tolerance in the bolting process. In order to control flatness in the manufacturing stage, milling and line heating are performed in the field. Among these methods, line heating is performed by the operator’s empirical judgment without clear standards. There are many Materials 2022, 15, 7962 12 of 13 12 of 13 studies on line heating, but the studies need to suggest clear standards. Therefore, this study proposed a method to determine the optimal line-heating conditions to control the flatness generated by welding a wind-tower block and flange. The scale factor for line heating that offsets welding deformation was determined using the deformation superposition method and optimization. The proposed method was verified by changing the size and welding conditions of the wind tower. The size of the wind tower is 1.5 MW, 3.6 MW, and 5.0 MW; the welding conditions are the same. Scale factors for size were derived in the range 0.022865–0.032911, 0.270677–0.30085, and 0.116164–0.18979 at positions h4, h6, and h7, respectively. Welding conditions were changed for each pass at 3.6 MW. The scale factor for the change in welding conditions was in the range of 0.159787–0.771813 at the h7 position and was derived from the position that did not satisfy the flatness tolerance. It was confirmed that the flatness satisfies all the design tolerances when the scale factor for size and welding condition change is superimposed with the temperature boundary condition of line heating. The scale factor is a variable that adjusts the size of the line heating. Adjusting the line heating size means that the flatness level can be changed. 8. Conclusions The asse b 8. Conclusions 8. Conclusions Using these scale factors, the size and location of the line heating are determined; based on this, the width and height of the line heating and the starting position can be suggested. In addition, line heating may be performed by dividing the position where the scale factor is zero according to the deviation of the flatness. Therefore, this can be used for cross-, lattice-, and point-heating. Author Contributions: Conceptualization, H.-C.Y.; methodology, H.-C.Y.; software, H.-C.Y. and H.-J.N. and Y.-H.H.; validation, H.-J.N.; formal analysis, H.-B.L.; writing—review and editing, H.-I.Y. and J.-C.L.; visualization, H.-C.Y. and Y.-H.H. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 1. Hashimoto, T.; Fujishiro, Y. 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Reproductive Strategies of the Seagrass Zostera japonica Under Different Geographic Conditions in Northern China
Frontiers in marine science
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ORIGINAL RESEARCH published: 07 December 2020 doi: 10.3389/fmars.2020.574790 ORIGINAL RESEARCH published: 07 December 2020 doi: 10.3389/fmars.2020.574790 Reproductive Strategies of the Seagrass Zostera japonica Under Different Geographic Conditions in Northern China Shidong Yue1,2,3,4,5†, Xiaomei Zhang1,2,3,4†, Shaochun Xu1,2,3,4,5, Yu Zhang1,2,3,4,5, Peng Zhao6, Xiaodong Wang7 and Yi Zhou1,2,3,4* 1 CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China, 2 Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China, 3 Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, China, 4 CAS Engineering Laboratory for Marine Ranching, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China, 5 University of Chinese Academy of Sciences, Beijing, China, 6 State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, China, 7 Mashan Group Co., Ltd., Weihai, China Edited by: Dongyan Liu, East China Normal University, China Edited by: Dongyan Liu, East China Normal University, China Seagrasses form a unique group of submerged marine angiosperms capable of both sexual and asexual reproduction. The amounts of sexual and asexual reproduction differ within some species relying on geographic location and environmental factors. Here, we studied the reproductive strategies of different geographic Zostera japonica populations, S1 and S2 at Swan Lake lagoon (SLL), and H1 and H2 at Huiquan Bay (HQB), in northern China. The duration of flowering at SLL was longer than at HQB, whereas flowering initiation at HQB occurred earlier than at SLL. In addition, the timing of seed maturation at HQB occurred earlier than at SLL. The allocation to sexual reproduction at SLL was greater than at HQB. The maximum potential seed production was greatest at S1 (22228.52 ± 8832.46 seeds · m−2), followed by S2 (21630.34 ± 9378.67 seeds · m−2), H2 (7459.60 ± 1779.33 seeds · m−2), and H1 (2821.05 ± 1280.57 seeds · m−2). The seasonal changes in total shoot density and biomass were small at HQB. There was a relatively large number of overwintering shoots at HQB because of the higher average temperature during winter. The allocation to sexual reproduction was lower than at SLL, and no seedlings were observed at HQB during our study. Thus, the population of Z. japonica at HQB was maintained by asexual reproduction. Compared with HQB, the biomass of overwintering shoots at SLL was less than 30 g dry weight · m−2. The Z. japonica at SLL relied on asexual and sexual reproduction to maintain the population. The results show the necessity of understanding local reproductive strategies before starting restoration and management projects. The study provides fundamental information and guidance for the conservation and restoration of seagrass beds. Reviewed by: Qiuying Han, Hainan Tropical Ocean University, China Jennifer Li Ruesink, University of Washington, United States *Correspondence: Yi Zhou yizhou@qdio.ac.cn †These authors have contributed equally to this work Specialty section: This article was submitted to Marine Ecosystem Ecology, a section of the journal Frontiers in Marine Science Received: 21 June 2020 Accepted: 27 October 2020 Published: 07 December 2020 Keywords: seagrass, asexual reproduction, sexual reproduction, seed bank, seedling INTRODUCTION 1972; Tomlinson, 1974), while in other populations, annual seagrass is established each year from seeds (Felger and McRoy, 1975; Keddy and Patriquin, 1978; Bayer, 1979; Harrison, 1979). Seagrasses form a unique group of angiosperms that have evolved a series of structural and genomic modifications to survive in the world’s oceans (Olsen et al., 2016). As important habitat-formers, seagrasses are the basis of one of the most productive and widespread coastal ecosystems worldwide (Costanza et al., 1997; Duffy, 2006; York et al., 2015). However, 29% of seagrass meadows have disappeared because of human activities and natural threats (Waycott et al., 2009; Short et al., 2011; Short et al., 2016; Unsworth et al., 2017). Therefore, effective management programs and active restoration work are becoming increasingly important (Zhou et al., 2014; Unsworth et al., 2015; Cullen- Unsworth and Unsworth, 2016; van Katwijk et al., 2016; Lefcheck et al., 2017). Understanding their reproductive strategies is essential for the conservation, management, and restoration of seagrasses. Z. japonica is an intertidal seagrass species that is native to the Western Pacific Ocean from Russia to Vietnam (Miki, 1933), and has been successfully introduced to the coastlines of British Columbia (Canada), as well as Washington, Oregon, and northern California (United States) (Shafer et al., 2014). However, it is widely threatened by human disturbances in its native range of China, Korea, and Japan (Lee et al., 2004; Abe et al., 2009; Hodoki et al., 2013; Zhang et al., 2019, 2020a). Thus, conservation and restoration efforts are required urgently for Z. japonica in its native habitat. Limited studies have been conducted on the reproductive strategy of Z. japonica. Henderson and Hacker (2015) investigated the reproductive allocation of Z. japonica under different sediment disturbance regimes. Flowering biomass was negatively correlated with vegetative biomass as the sediment disturbance increased. Suonan et al. (2017) showed that sexual reproductive effort in Z. japonica tends to be enhanced under disturbed (due to calm harvesting) and inundated environmental conditions for population persistence. Zhang et al. (2019) reported on the temporal and spatial changes in population recruitment in Z. japonica meadows located in the Yellow River Delta, China. The positive effects of Z. japonica’s sexual reproduction on the population’s genetic diversity was confirmed by a microsatellite analysis (Zhang et al., 2020b). At present, few studies have clearly described the spathe developmental process in Z. japonica. Additionally, understanding the differences in Z. INTRODUCTION japonica reproductive strategies at large spatial scales is essential for the conservation and management of this species. Seagrasses are submerged marine angiosperms capable of both sexual and asexual reproduction. It is usually assumed that the recruitment of seagrass populations occurs mainly through asexual reproduction (Williams, 1990; Procaccini and Mazzella, 1998; Rasheed, 2004). The recovery of gaps within seagrass meadows solely by asexual reproduction may be common. In a multi-species Caribbean seagrass meadow, all the recolonization occurred through vegetative propagation (Williams, 1990). Rasheed (1999) found that the recolonization of artificially cleared plots in a seagrass meadow dominated by Zostera capricorni occurred principally by asexual growth from surrounding rhizomes. A microsatellite marker- based study reported that clonal growth is important in the maintenance of Posidonia oceanica populations (Procaccini and Mazzella, 1998). Successful sexual recruitment is limited owing to low pollination rates, restricted pollen and seed dispersal, and low seed and seedling survival rates (Les, 1988; Laushman, 1993; Reusch, 2003). However, sexual reproduction is the only way to maintain the genetic diversity of a seagrass population (Ackerman, 2006; Reynolds et al., 2012, 2013). Sexual recruitment can provide a means for seagrasses to colonize new areas or to establish new patches of seagrasses (Rasheed, 2004; Kendrick et al., 2012). Under highly disturbed conditions, in which there is few remnant seagrass available, initial recolonization may occur by seeding (Marba and Walker, 1999; Greve et al., 2005; Lee et al., 2007). Here, we studied the reproductive strategies of two different geographic populations of Z. japonica in northern China, Swan Lake (SLL) and Huiquan Bay (HQB). The winter at SLL is relatively cold, and on the coldest days the lagoon is covered with ice. In contrast, the winter at HQB is warmer, with a very low likelihood of snow and ice. Thus, at SLL, the Z. japonica meadow is almost bare in winter owing to the lower temperature, while Z. japonica survives the winter well at HQB. The aims of this study were as follows: (1) to compare the differences in reproductive strategies and determine the causes; and (2) to describe the spathe developmental process in Z. japonica. Our study will provide basic information to clarify the roles of sexual and asexual reproduction in population recruitment and to establish restoration and conservation strategies. INTRODUCTION The amounts of sexual and asexual reproduction differ within some species relying on geographic location and environmental conditions (Rasheed, 2004; Xu S.et al., 2018; Zhang et al., 2020b). Z. capricorni flowering occurs from September to April in Botany Bay, New South Wales (Larkum et al., 1984). However, there is only a low incidence of Z. capricorni flowering at Ellie Point (September and October), northern Queensland (McKenzie, 1994). The flowering duration is longer in the midshore zone (7 months), compared with the offshore (4 months), and inshore (3 months) zones (Conacher et al., 1994). Many local populations of Z. marina are maintained mainly by clonal growth from perennial rhizomes (Phillips, Citation: Yue SD, Zhang XM, Xu SC, Zhang Y, Zhao P, Wang XD and Zhou Y (2020) Reproductive Strategies of the Seagrass Zostera japonica Under Different Geographic Conditions in Northern China. Front. Mar. Sci. 7:574790. doi: 10.3389/fmars.2020.574790 December 2020 | Volume 7 | Article 574790 1 Frontiers in Marine Science | www.frontiersin.org Reproductive Strategy of Zostera japonica Yue et al. Frontiers in Marine Science | www.frontiersin.org Study Sites The SSL (Figure 1; 122◦34′E, 37◦21′N) is located east of Weihai City, northern China. It is ∼2.1 km long and ∼1.8 km wide, covering an area of ∼4.8 km2 (Xu S.et al., 2018). The lagoon has irregular semidiurnal mixed tides (tidal December 2020 | Volume 7 | Article 574790 Frontiers in Marine Science | www.frontiersin.org 2 Reproductive Strategy of Zostera japonica Yue et al. FIGURE 1 | Zostera japonica study sites in Swan Lake (SLL) and Huiquan Bay (HQB). FIGURE 1 | Zostera japonica study sites in Swan Lake (SLL) and Huiquan Bay (HQB). range = 1.65 m) (Zhang et al., 2020b). The lagoon is shallow (average depth < 1.5 m) and is colonized by Z. marina and Z. japonica (Zhang et al., 2014, 2015; Zhou et al., 2015; Xu Q.et al., 2018; Xu et al., 2019). Z. japonica principally occurs in the narrow mid-upper intertidal zone, which is divided into two parts (Zhang et al., 2015). Thus, two study sites were designed based on Z. japonica’s spatial distribution. The sediment of S1 is mainly sandy, whereas the sediment of S2 is mainly mud. from June 2018 to June 2019. Instantaneous photosynthetic photon flux densities (PPFDs; mol photons · m−2 · s−1) were measured every 15 min and daily PPFDs (mol photons · m−2 · d−1) were calculated as the sum of the quantum flux within a 24 h period. Three sediment cores (diameter = 10.5 cm and depth = 12 cm) were collected at each site for the determination of the grain size distribution using a laser diffraction analysis (Short and Coles, 2001). The HQB (Figure 1; 120◦34′E, 36◦05′N) is an open bay in Qingdao, northern China (Zhou et al., 2014). It has regular semidiurnal tides (tidal range = 4.8 m). The sediment of the bay is mainly sandy and two seagrass species (Z. marina and Z. japonica) colonize the southeastern corner of this bay (Xu S.et al., 2018). We surveyed two study sites based on the spatial distribution of Z. japonica (Figure 1). H1 was located in a non- continuous Z. japonica patch area, while H2 was located in the species’ continuous area. Biological Measurements During the flowering periods in 2018, seven replicate sediment cores (diameter = 10.5 cm and depth = 12 cm) were collected semimonthly at the four sites. To investigate seedling recruitment, seven replicate sediment cores (diameter = 10.5 cm and depth = 12 cm) were collected semimonthly from March to May in 2019 at all the sites. From June to December 2019, seven replicate sediment cores (diameter = 10.5 cm and depth = 12 cm) were collected monthly at the four sites. Environmental Parameters For each core, the reproductive shoot density (shoots · m−2) and the height of reproductive shoot (cm) were determined. Flowering frequency (%) was determined as the percentage of reproductive shoot density to total shoot density. The reproductive effort (%) was calculated as the percentage of reproductive shoot biomass to total shoot biomass. For each reproductive shoot, the number of seed spathes per shoot and the number of spathes per shoot were counted. Spathes in which seed development had initiated (“seed spathes”) were distinguished from those that contained flowers. For each spathe, the number of female flowers per flowering spathe or the number of seeds per seed spathe were counted. The potential seed production per unit area (PSP, seeds · m−2) in each core was calculated using the following equation: ¯Nija: the average number of spathes per shoot in sampling time j at site i, ¯Nija: the average number of spathes per shoot in sampling time j at site i, above. For each core, the reproductive shoot density (shoots · m−2) and the height of reproductive shoot (cm) were determined. Flowering frequency (%) was determined as the percentage of reproductive shoot density to total shoot density. The reproductive effort (%) was calculated as the percentage of reproductive shoot biomass to total shoot biomass. For each reproductive shoot, the number of seed spathes per shoot and the number of spathes per shoot were counted. Spathes in which seed development had initiated (“seed spathes”) were distinguished from those that contained flowers. For each spathe, the number of female flowers per flowering spathe or the number of seeds per seed spathe were counted. The potential seed production per unit area (PSP, seeds · m−2) in each core was calculated using the following equation: ¯Ni: the average number of seeds per seed spathe at site i, RSDijk: the reproductive shoot density of core k in sampling time j at site i. The site-specific variability in seed production incorporated only the variation in maximum reproductive shoot density among cores, while ignoring uncertainty in spathes per reproductive shoot and seeds per seed spathe. Seedling recruitment was investigated using the same cores described above. For each core, the seedling density (shoots · m−2) was determined, and seedling frequency was calculated as the ratio of seedlings to total shoots (%). Environmental Parameters All the samples were sieved (2 mm) with seawater in situ to remove most of the sediment, and the seagrass materials were taken to the laboratory and cleaned using tap water. For each sample, the total number of shoots (including reproductive and vegetative shoots) was recorded to provide shoot density (shoots · m−2). Next, they were dried to a constant weight at 60◦C to estimate total shoot biomass [g dry weight (DW) · m−2]. A HOBO Pendant light/temp MX 2202 (ONSET, United States) was used to record water temperatures (◦C) at SLL (S1 and S2) and HQB at 15-min interval from June 2018 to June 2019. The average daily values of the water temperatures were calculated, and the daily temperatures were averaged monthly. An ECO- PARSB sensor (Sea-Bird Scientific, United States) deployed in the center of the Z. japonica meadows was used to record light intensity at the canopy in SLL (S1 and S2) and HQB During the flowering periods, reproductive shoots and their seed production were investigated using the same cores described December 2020 | Volume 7 | Article 574790 Frontiers in Marine Science | www.frontiersin.org 3 Reproductive Strategy of Zostera japonica Yue et al. Yue et al. FIGURE 2 | Seasonal changes in temperature (A) and light intensity (B) in the study sites from June 2018 to June 2019 (Huiquan Bay is represented by H1 and H2). Values are mean ± SD. FIGURE 2 | Seasonal changes in temperature (A) and light intensity (B) in the study sites from June 2018 to June 2019 (Huiquan Bay is represented by H1 and H2). Values are mean ± SD. FIGURE 2 | Seasonal changes in temperature (A) and light intensity (B) in the study sites from June 2018 to June 2019 (Huiquan Bay is represented by H1 and H2). Values are mean ± SD FIGURE 3 | Sediment grain sizes at the four study sites Values are mean ± SD (N = 3 cores per site) FIGURE 3 | Sediment grain sizes at the four study sites. Values are mean ± SD (N = 3 cores per site). FIGURE 3 | Sediment grain sizes at the four study sites. Values are mean ± SD (N = 3 cores per site). December 2020 | Volume 7 | Article 574790 Frontiers in Marine Science | www.frontiersin.org 4 Yue et al. Reproductive Strategy of Zostera japonica above. December 2020 | Volume 7 | Article 574790 Data Analysis The significance levels of the differences in total shoot density and biomass among the sampling times and the four sites were tested using a two-way ANOVA. A one-way ANOVA was used to test the significance of differences in the maximum height of reproductive shoots, maximum reproductive shoot density, maximum flowering frequency, maximum reproductive FIGURE 5 | Seasonal changes in the reproductive shoot densities (A), reproductive shoot height (B), flowering frequency (C), and reproductive effort (D) of Zostera japonica at the four study sites from June 2018 to December 2019. Values are mean ± SD (N = 7 cores per site). FIGURE 5 | Seasonal changes in the reproductive shoot densities (A), reproductive shoot height (B), flowering frequency (C), and reproductive effort (D) of Zostera japonica at the four study sites from June 2018 to December 2019. Values are mean ± SD (N = 7 cores per site). FIGURE 5 | Seasonal changes in the reproductive shoot densities (A), reproductive shoot height (B), flowering frequency (C), an japonica at the four study sites from June 2018 to December 2019. Values are mean ± SD (N = 7 cores per site). n the reproductive shoot densities (A), reproductive shoot height (B), flowering frequency (C), and reproductive effort (D) of Zostera rom June 2018 to December 2019. Values are mean ± SD (N = 7 cores per site). TABLE 2 | Flowering traits of Zostera japonica at the four study sites from June 2018 to December 2019. Environmental Parameters To estimate the seed density in the sediment seed bank, seven random sediment cores (diameter = 10.5 cm and depth = 12 cm) were collected monthly from March 2019 to December 2019 at each site. The sediment cores were sieved twice using 2 and 0.7 mm and mesh. Larger plant matter and other detritus were retained in the 2 mm mesh, while seeds mixed with small detritus were retained PSPijk = ¯Nija × ¯Ni × RSDijk i: study site (S1 or S2 or H1 or H2), j: sampling time, i: study site (S1 or S2 or H1 or H2), y j: sampling time, k: core serial number (1–7), FIGURE 4 | Seasonal changes in total shoot densities (A) and biomasses (B) of Zostera japonica at the four study sites from June 2018 to December 2019. Values are mean ± SD (N = 7 cores per site). GURE 4 | Seasonal changes in total shoot densities (A) and biomasses (B) of Zostera japonica at the four study sites from June 2018 to December 2019 Values FIGURE 4 | Seasonal changes in total shoot densities (A) and biomasses (B) of Zostera japonica at the four study sites from June 2018 to December 2019. Values are mean ± SD (N = 7 cores per site). TABLE 1 | The total shoot density and biomass of Zostera japonica among the four study sites at the beginning of spring (2019.3.15). Variable S1 S2 H1 H2 Total shoot density (shoots · m−2) 2162.35 ± 717.60ab 165.06 ± 186.99c 1733.18 ± 411.23b 2674.05 ± 773.46a Total shoot biomass (g DW · m−2) 27.57 ± 8.48c 2.64 ± 3.70c 114.39 ± 42.82a 78.41 ± 29.69b Different letters indicate significant differences at p < 0.05. Values are mean ± SD (N = 7 cores per site). he total shoot density and biomass of Zostera japonica among the four study sites at the beginning of spring (2019.3.15). December 2020 | Volume 7 | Article 574790 5 Reproductive Strategy of Zostera japonica Yue et al. in the 0.7 mm mesh. The number of viable seeds with hard pericarps was recorded to calculate seed bank density (seeds · m−2). Environmental Parameters effort, maximum number of spathes per reproductive shoot, maximum number of seed spathes per reproductive shoot, maximum number of female flowers per flowering spathe, maximum number of seeds per seed spathe, maximum potential seed production, maximum seed density in sediment, maximum seedling density and maximum seedling frequency among the four sites. When these data did not satisfy the homogeneity of variance, a Kruskal–Wallis Test was used to test the significances of differences. Multiple comparisons were performed using the Duncan method or Independent-Samples T-tests, and the level of significance was set at p < 0.05. Statistical analyses Data Analysis Variable S1 S2 H1 H2 Maximum reproductive shoot density (shoots · m−2) 4357.70 ± 1731.53a(2018.8.30) 4060.59 ± 1299.44a(2018.8.30) 1568.11 ± 711.82b(2018.7.30) 2063.31 ± 492.16b(2018.7.30) Maximum flowering frequency (%) 49.32 ± 11.02ab(2018.8.30) 59.03 ± 16.58a(2018.8.30) 37.29 ± 13.58b(2018.7.30) 57.45 ± 10.55a(2018.7.30) Maximum reproductive effort (%) 55.31 ± 10.26a(2018.9.15) 61.15 ± 14.57a(2018.8.30) 31.96 ± 11.97b(2018.7.30) 51.90 ± 13.24a(2018.7.30) Maximum height of reproductive shoots (cm) 9.85 ± 1.83b(2019.9.15) 23.49 ± 8.37a(2019.9.15) 15.09 ± 2.59b(2018.7.30) 22.46 ± 4.98a(2018.7.30) Maximum number of spathes per reproductive shoot 2.66 ± 0.40b(2018.9.30) 3.41 ± 0.48a(2019.10.15) 2.15 ± 0.28b(2019.8.15) 2.45 ± 0.66b(2018.7.30) Maximum number of seed spathes per reproductive shoot 2.24 ± 0.40b(2019.10.15) 2.75 ± 0.50a(2019.10.15) 1.25 ± 0.34d(2019.8.15) 1.75 ± 0.39c(2018.7.30) Maximum number of female flowers per flowering spathe 5.85 ± 0.40(2019.7.15) 5.95 ± 0.49(2018.8.30) 3.81 ± 2.76(2018.6.30) 5.99 ± 1.07(2018.7.15) Maximum number of seeds per seed spathe 3.73 ± 1.03ab(2018.8.15) 4.55 ± 0.48a(2019.9.15) 2.70 ± 1.62c(2019.7.15) 3.88 ± 0.67ab(2018.7.30) Different letters indicate significant differences at p < 0.05. Values are mean ± SD (N = 7 cores per site). TABLE 2 | Flowering traits of Zostera japonica at the four study sites from June 2018 to December 2019. Variable S2 H1 S1 December 2020 | Volume 7 | Article 574790 Frontiers in Marine Science | www.frontiersin.org 6 Reproductive Strategy of Zostera japonica Yue et al. were conducted using SPSS 17.0. All values are reported as mean ± SD. warmer at HQB, with extremely low probabilities of snow and ice (Figure 2A). The water temperature averaged 3.37 ± 2.81◦C at S1, 2.34 ± 3.24◦C at S2, and 6.53 ± 2.42◦C at HQB in winter. The annual maximum temperature was highest at HQB (28.23◦C), and the annual minimum temperature was lowest at S2 (−4.56◦C). The negative accumulated temperature was −14.60◦C at S1, −37.46◦C at S2, and 0◦C at HQB. The light increased in spring and summer and decreased in fall and winter at all the study sites (Figure 2B). The sediment particulate size composition at each site is shown in Figure 3. The sediments at all the sites were mainly composed of sand (0.063–2.0 mm), whereas the proportion of silt (0.039–0.063 mm) at S2 (21.55 ± 1.60%) was greater than at the other sites. Any data collected at the shoot level (i.e., reproductive shoot height and number of spathes per reproductive shoot), as well as data collected at the spathe level (i.e., seeds per seed spathe) were first averaged to generate core-specific numbers. Thus, a core was the sample in all the statistical tests and in calculations of mean and SD. For maximum- value trait comparisons, data were used from different dates across sites, if maximum values were reached on different dates. Environmental Parameters Environmental Parameters Winter was relatively colder at SLL, and ice formed on the water surface on the coldest days. By comparison, the winter was There were significant site × time interactions for total shoot density and biomass [F(66,555) = 4.981, p < 0.05; F(66,555) = 5.170, FIGURE 6 | Seasonal changes in the number of Zostera japonica spathes per reproductive shoot (A), and the number of seed spathes per reproductive shoot (B) at the four study sites from June 2018 to December 2019. Values are mean ± SD (N = 7 cores per site). Frontiers in Marine Science | www.frontiersin.org 7 December 2020 | Volume 7 | Article 574790 FIGURE 6 | Seasonal changes in the number of Zostera japonica spathes per reproductive shoot (A), and the number of seed spathes per reproductive shoot (B) at the four study sites from June 2018 to December 2019. Values are mean ± SD (N = 7 cores per site). December 2020 | Volume 7 | Article 574790 7 Frontiers in Marine Science | www.frontiersin.org Reproductive Strategy of Zostera japonica Yue et al. during 2018 (49.32% ± 11.02% at S1 and 59.03% ± 16.58% at S2) and in September during 2019 (35.49% ± 6.62% at S1 and 55.77% ± 20.33% at S2, Figure 5C). At HQB, the flowering frequencies were greatest in July during 2018 (37.29% ± 13.58% at H1 and 57.45% ± 10.55% at H2) and 2019 (29.62% ± 19.47% at H1 and 24.26% ± 9.14% at H2). Therefore, the maximum flowering frequency at S2 was greater than those at other sites [F(3, 24) = 4.003, p < 0.05; Table 2]. during 2018 (49.32% ± 11.02% at S1 and 59.03% ± 16.58% at S2) and in September during 2019 (35.49% ± 6.62% at S1 and 55.77% ± 20.33% at S2, Figure 5C). At HQB, the flowering frequencies were greatest in July during 2018 (37.29% ± 13.58% at H1 and 57.45% ± 10.55% at H2) and 2019 (29.62% ± 19.47% at H1 and 24.26% ± 9.14% at H2). Therefore, the maximum flowering frequency at S2 was greater than those at other sites [F(3, 24) = 4.003, p < 0.05; Table 2]. p < 0.05, respectively]. Environmental Parameters At the beginning of spring (early March), the total shoot density was greatest at H2, followed by S1, H1, and S2 [F(3,24) = 24.930, p < 0.05; Figure 4 and Table 1]; and the total shoot biomass was greatest at H1 (114.39 ± 42.82 g DW · m−2), followed by H2 (78.41 ± 29.69 g DW · m−2), S1 (27.57 ± 8.48 g DW · m−2), and S2 [2.64 ± 3.70 g DW · m−2; Chi-square(3) = 23.499, p < 0.05; Figure 4 and Table 1]. DISCUSSION In this study, we report the differences in reproductive strategies of Z. japonica under different geographic and environmental conditions in northern China. The seasonal changes in total shoot density and biomass were small in HQB. There was a relatively large number of overwintering shoots at HQB because of the higher average temperature in winter. The allocation to sexual reproduction was lower than at SLL, and no seedlings were observed at HQB during our study. Thus, the population of Z. japonica at HQB was maintained by asexual reproduction. Compared with HQB, the biomass of overwintering shoots at SLL was less than 30 g DW · m−2. The Z. japonica In this study, we report the differences in reproductive strategies of Z. japonica under different geographic and environmental conditions in northern China. The seasonal changes in total shoot density and biomass were small in HQB. There was a relatively large number of overwintering shoots at HQB because of the higher average temperature in winter. The allocation to sexual reproduction was lower than at SLL, and no seedlings were observed at HQB during our study. Thus, the population of Z. japonica at HQB was maintained by asexual reproduction. Compared with HQB, the biomass of overwintering shoots at SLL was less than 30 g DW · m−2. The Z. japonica Flowering Characteristics The reproductive effort at S1 was greatest in September during 2018 (55.31% ± 10.26%) and 2019 (38.08% ± 8.99%). At S2, the reproductive effort was greatest in August during 2018 (61.15% ± 14.57%) and October during 2019 (59.07% ± 9.32%). At HQB, the reproductive effort was greatest in July during 2018 (31.96% ± 11.97% at H1 and 26.46% ± 25.10 at H2) and 2019 (51.90% ± 13.24% at H1 and 17.98% ± 5.77% at H2). Therefore, the maximum reproductive effort at S2 was greater than those at other sites [F(3, 24) = 7.065, p < 0.05; Figure 5D and Table 2]. At SLL, reproductive shoots were observed first in June, and they lasted until November in 2018, while the reproductive shoots at HQB were observed from June to October in 2018. The flowering duration at SLL was longer than at HQB. g The maximum reproductive shoot density at SLL was greater than at HQB [F(3, 24) = 10.123, p < 0.05; Figure 5A and Table 2]. The maximum height of reproductive shoots varied significantly [Chi-square(3) = 19.087, p < 0.05] among the four study sites (Figure 5B). The maximum height of reproductive shoots at S1 (9.85 ± 1.83 cm) was lower than at other sites (Table 2). The flowering frequency at SLL was greatest in August The maximum number of spathes per reproductive shoot and the maximum number of seed spathes per FIGURE 7 | Seasonal changes in the number of Zostera japonica female flowers per flowering spathe (A) and the number of seeds per seed spathe (B) at the four study sites from June 2018 to December 2019. Values are mean ± SD (N = 7 cores per site). FIGURE 7 | Seasonal changes in the number of Zostera japonica female flowers per flowering spathe (A) and the number of seeds per seed spathe (B) at the four study sites from June 2018 to December 2019. Values are mean ± SD (N = 7 cores per site). December 2020 | Volume 7 | Article 574790 8 Frontiers in Marine Science | www.frontiersin.org Reproductive Strategy of Zostera japonica Yue et al. other sites [Chi-square(3) = 18.049, p < 0.05; Figure 9B and Table 3]. reproductive shoot were significantly different among the four study sites [F(3,24) = 8.931, p < 0.05; F(3, 24) = 16.994, p < 0.05, respectively; Figure 6]. Flowering Characteristics The values at S2 (3.41 ± 0.48; 2.75 ± 0.50, respectively) were higher than at S1 (2.66 ± 0.40; 2.24 ± 0.40, respectively), H2 (2.45 ± 0.66; 1.75 ± 0.39, respectively), and H1 (2.15 ± 0.28; 1.25 ± 0.34, respectively) (Table 2). The maximum seedling density and frequency varied significantly among the four study sites [Chi-square(3) = 17.709, p < 0.05; Chi-square(3) = 13.478, p < 0.05, respectively; Figure 10].The maximum seedling density and frequency at S2 were 1,254.49 ± 1,490.83 shoots · m−2 and 30.90% ± 30.15%, respectively. The maximum seedling density and frequency at S1 were 82.53 ± 114.26 shoots · m−2 and 3.83% ± 5.79%, respectively. At SLL, seedlings were observed first in early April, were most prevalent in early May and lasted until June (Figure 10). No seedlings were found in situ at HQB. There were no significant differences in the maximum number of female flowers per flowering spathe among the four study sites [Chi-square(3) = 6.354, p > 0.05; Figure 7A]. The maximum number of seeds per seed spathe varied significantly among the four study sites [Chi-square(3) = 7.984, p < 0.05; Figure 7B]. The value was highest at S2 (4.55 ± 0.48), followed by H2 (3.88 ± 0.67), S1 (3.73 ± 1.03), and H1 (2.70 ± 1.62) (Table 2). Based on a large number of observations, we recorded the spathe developmental process and the morphology of female and male flowers (Figure 8). Frontiers in Marine Science | www.frontiersin.org Potential Seed Production, Seed Density in Sediment and Seedling Data The maximum potential seed production varied significantly among the four study sites [Chi-square(3) = 20.390, p < 0.05; Figure 9A]. The value was highest at S1 (22228.52 ± 8832.46 seeds · m−2), followed by S2 (21630.34 ± 9378.67 seeds · m−2), H2 (7459.60 ± 1779.33 seeds · m−2), and H1 (2821.05 ± 1280.57 seeds · m−2) (Table 3). The maximum seed density in sediment at S2 (2954.65 ± 2549.72 seeds · m−2) was higher than those at FIGURE 8 | The Zostera japonica spathe developmental process and the morphology of male (A) and female (B) flowers. FIGURE 8 | The Zostera japonica spathe developmental process and the morphology of male (A) and female (B) flowers. December 2020 | Volume 7 | Article 574790 Frontiers in Marine Science | www.frontiersin.org 9 Yue et al. Reproductive Strategy of Zostera japonica FIGURE 9 | Seasonal changes in potential Zostera japonica seed production (A) and seed density in sediments (B) at the four study sites from June 2018 to December 2019. Values are mean ± SD (N = 7 cores per site). TABLE 3 | Maximum potential seed production and maximum seed density in sediment of Zostera japonica at the four study sites from March 2019 to December 2019 FIGURE 9 | Seasonal changes in potential Zostera japonica seed production (A) and seed density in sediments (B) at the four study sites from June 2018 to December 2019. Values are mean ± SD (N = 7 cores per site). December 2019. Values are mean ± SD (N = 7 cores per site). TABLE 3 | Maximum potential seed production and maximum seed density in sediment of Zostera japonica at the four study sites from March 2019 to December 2019. Variable S1 S2 H1 H2 Maximum potential seed production (seeds · m−2) 22228.52 ± 8832.46a(2018.8.30) 21630.34 ± 9378.67a(2019.9.15) 2821.05 ± 1280.57b(2018.7.30) 7459.60 ± 1779.33b(2018.7.30) Maximum seed density in sediment (seeds · m−2) 561.22 ± 367.99b(2019.9.15) 2954.65 ± 2549.72a(2019.11.15) 33.01 ± 56.38b(2019.11.15) 379.65 ± 1004.46b(2019.12.15) Different letters indicate significant differences at p < 0.05. Values are mean ± SD (N = 7 cores per site). December 2019. Values are mean ± SD (N = 7 cores per site). TABLE 3 | Maximum potential seed production and maximum seed density in sediment of Zostera japonica at the four study sites from March 2019 to December 2019. Potential Seed Production, Seed Density in Sediment and Seedling Data Because the photoperiod and precipitation at the four sites in the present study were similar, higher temperature is likely the main reason for the difference in flowering phenology. Temperature is a main influencing factor for many plant growth processes, and in many cases, higher temperatures accelerate plant growth, leading to an earlier progression to the next stage (Saxe et al., 2001; Badeck et al., 2004). Qin et al. (2020) observed that the flowering duration of Z. marina in the colder region (5–7.5 months) was longer than in the warmer region (3.5 months). Blok et al. (2018) reported that Z. marina reproductive shoots appeared later in the colder regions. number seeds per seed spathe if pollination was 100% successful. However, the number of female flowers per flowering spathe was greater than the number of seeds per seed spathe at the four sites. Pollen limitation is a common reason for this phenomenon (van Tussenbroek et al., 2016). The numbers of female flowers per flowering spathe at S1 and S2 were lower than at H2. However, the potential seed production levels at these sites were greater than at HQB. van Tussenbroek et al. (2016) reported that the pollination success rate of Z. noltei was significant positively correlated with reproductive shoot density (<600 shoots · m−2). The higher reproductive output at SLL might result from the greater reproductive shoot density compared with at HQB. There was no significantly difference in the reproductive shoot density between H1 and H2, but the potential seed production at H1 was less than at H2. Therefore, isolated patches (H1) have a much lower seed-set than continuous populations (H2). During the flowering periods, there were differences in the allocation to sexual reproduction at the four study sites. At SLL, the flowering frequency, reproductive effort and the potential seed production were greater than at HQB. In addition, the maximum height of reproductive shoots at S1 was lower than at HQB, while the maximum number of spathes per reproductive shoot at S1 was higher than at HQB. These data indicated that the allocation to sexual reproduction was greater at SLL. During the spathe developmental process (Figure 8), the number of female flowers per flowering spathe should be equal to the Although the potential seed production at SLL was relatively high, the seed density in sediment was still at a low level. Potential Seed Production, Seed Density in Sediment and Seedling Data Variable S1 S2 H1 H2 Maximum potential seed production (seeds · m−2) 22228.52 ± 8832.46a(2018.8.30) 21630.34 ± 9378.67a(2019.9.15) 2821.05 ± 1280.57b(2018.7.30) 7459.60 ± 1779.33b(2018.7.30) Maximum seed density in sediment (seeds · m−2) 561.22 ± 367.99b(2019.9.15) 2954.65 ± 2549.72a(2019.11.15) 33.01 ± 56.38b(2019.11.15) 379.65 ± 1004.46b(2019.12.15) Different letters indicate significant differences at p < 0.05. Values are mean ± SD (N = 7 cores per site). Different letters indicate significant differences at p < 0.05. Values are mean ± SD (N = 7 cores per site). The flowering frequency at H2 was nearly 20%, while the flowering frequencies at S1 and S2 were both less than 6% in the mid June 2018. Thus, the flowering initiation at HQB might have occurred before mid June 2018. In 2019, reproductive shoots were recorded in the early June at HQB (Figure 5B). Thus, the flowering initiation may occur earlier at HQB than at SLL, while the flowering duration at SLL was longer than at HQB (Figure 5A). In addition, the timing of seed maturation at HQB was earlier than at SLL. of SLL relied on asexual and sexual reproduction to maintain its population. Zhang et al. (2019) reported that Z. japonica located in the Yellow River Delta mainly relied on seedlings for population maintenance owing to the low temperature in winter. In spring, there were more germinated seeds of Z. marina in the area where ice had removed the aboveground tissue of Z. marina on its meadows (Robertson and Mann, 1984). This phenomenon is similar to the seedling recruitment of Z. japonica at SLL. December 2020 | Volume 7 | Article 574790 Frontiers in Marine Science | www.frontiersin.org 10 Yue et al. Reproductive Strategy of Zostera japonica FIGURE 10 | Changes in Zostera japonica seedling densities (A) and seedling frequency (B) at the four study sites from March 2019 to December 2019. Values are mean ± SD (N = 7 cores per site). FIGURE 10 | Changes in Zostera japonica seedling densities (A) and seedling frequency (B) at the four study sites from March 2019 to December 2019. Values are mean ± SD (N = 7 cores per site). Thus, the SLL and HQB populations differed in flowering phenology. Factors that affect the phenology of plants are genes, photoperiod, temperature and precipitation (Forrest and Miller- Rushing, 2010). Frontiers in Marine Science | www.frontiersin.org DATA AVAILABILITY STATEMENT The datasets presented in this article are not readily available because the follow-up research is still in progress. Requests to access the datasets should be directed to yueshidong17@mails.ucas.ac.cn. Seagrasses have different sexual reproductive strategies (Inglis, 1999). Some species such as Halodule spp. and Cymodocea spp. form seed banks of small seeds with hard shells that can remain dormant for long periods; whereas other species, such as Posidonia spp. and Thalassia spp., form buoyant fruits with large inner non-dormant seeds (Kuo et al., 1990; Kuo and Den Hartog, 2006; Guerrero-Meseguer et al., 2018). Harrison (1991) showed that recently shed Z. marina seeds may undergo physiological and physical dormancy. We found that Z. japonica seeds also experienced dormancy (unpublished data). Thus, seeds do not germinate immediately after maturity. Therefore, seed banks in sediments are important for the successful sexual recruitment of Z. japonica. Seed germination of Z. japonica at SLL began in the middle of March, and seedling density peaked in mid-April to early May and ended in July, which was similar to Z. japonica in the Yellow River Delta (Zhang et al., 2019). No seedlings were found in situ at HQB in the present study; and no seedlings have been observed in monospecific meadows of Z. japonica on the southern coast of Korea (Suonan et al., 2017). Potential Seed Production, Seed Density in Sediment and Seedling Data The ratio of sediment seed banks to potential seed production was less than 10% at all four sites, and no stable sediment seed banks were found at HQB. Similar phenomena have been reported previously. Harrison (1993) reported that a large amount of Z. marina seeds in a seed bank was lost owing to death and that only a limited number of the seeds remained in the persistent December 2020 | Volume 7 | Article 574790 11 Reproductive Strategy of Zostera japonica Yue et al. seed bank in the sediment. We also observed a large amount of seed coats in SLL. However, we found hardly any seed coat in HQB, which indicates that seeds had disappeared. Their fate is still unknown. We compared the dry weights, wet weights, and moisture contents of Z. japonica seeds at H2 and S1, and there were no significant differences (unpublished data). Additionally, the seeds were similar in size. Fishman and Orth (1996) found that seed predation can decrease Z. marina seeds by 65% in a seed bank. Dispersal is an important process in the life history of nearly all plant species and can be facilitated by both abiotic and biotic mechanisms (Sumoski and Orth, 2012). Therefore, the disappearance of seeds in HQB may be to the result of seed predation and dispersal. This will help us to choose effective methods for seagrass bed restoration. Owing to the disappearance of seeds in HQB, they should not be used to restore this seagrass meadow. Our research provides fundamental information and guidance for the conservation and restoration of seagrass beds. ACKNOWLEDGMENTS We would like to thank Kai Xiao and Gang Li for the help in the field survey, and Mingjie Liu, Yongliang Qiao, and Jingchun Sun for the assistance during the laboratory experiments. FUNDING This research was supported by the National Science & Technology Basic Work Program (2015FY110600), the National Key R&D Program of China (2019YFD0901301), the National Natural Science Foundation of China (No. 41606192/41176140), the Key Research Project of Frontier Sciences of CAS (QYZDB- SSW-DQC041-1), the International Partners Program of the Chinese Academy of Sciences (133137KYSB20180069), and the Taishan Scholars Program (Distinguished Taishan Scholars). In conclusion, we showed that SLL and HQB populations adapted to their habitats and maintained different reproductive strategies. The population of Z. japonica in HQB is maintained by asexual reproduction, whereas the Z. japonica of SLL relies on asexual and sexual reproduction to maintain its population. The results indicate the necessity of understanding local reproductive strategies before starting restoration and management projects. AUTHOR CONTRIBUTIONS SY and YiZ carried out the experiments and data analysis, and prepared the figures. XZ, SX, YuZ, PZ, and XW prepared materials and carried out the experiments. YiZ and SY participated in the design of the work. SY revised the manuscript. All authors read and approved the final manuscript. Blok, S. E., Olesen, B., and Krause-Jensen, D. (2018). Life history events of eelgrass Zostera marina L. populations across gradients of latitude and temperature. Mar. Ecol. Prog. Ser. 590, 79–93. doi: 10.3354/meps12479 Blok, S. E., Olesen, B., and Krause-Jensen, D. (2018). Life history events of eelgrass Zostera marina L. populations across gradients of latitude and temperature. Mar. Ecol. Prog. Ser. 590, 79–93. doi: 10.3354/meps12479 Conacher, C. A., Poiner, I. R., and Odonohue, M. (1994). Morphology, flowering and seed production of Zostera capricorni Aschers in subtropical Australia. Aquat. 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Y., Song, X. Y., Xu, S. C., Yue, S. D., Gu, R. T., et al. (2019). A unique meadow of the marine angiosperm Zostera japonica, covering a large area in the turbid intertidal Yellow River Delta, China. Sci. Total Environ. 686, 118–130. doi: 10.1016/j.scitotenv.2019. 05.320 Copyright © 2020 Yue, Zhang, Xu, Zhang, Zhao, Wang and Zhou. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Frontiers in Marine Science | www.frontiersin.org December 2020 | Volume 7 | Article 574790 REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2020 Yue, Zhang, Xu, Zhang, Zhao, Wang and Zhou. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zhang, X. M., Zhou, Y., Adams, M. P., Wang, F., Xu, S. C., Wang, P. M., et al. (2020a). Plant morphology and seed germination responses of seagrass (Zostera japonica) to water depth and light availability in Ailian Bay, December 2020 | Volume 7 | Article 574790 Frontiers in Marine Science | www.frontiersin.org 14
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Wogonin Has Multiple Anti-Cancer Effects by Regulating c-Myc/SKP2/Fbw7α and HDAC1/HDAC2 Pathways and Inducing Apoptosis in Human Lung Adenocarcinoma Cell Line A549
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Abstract Wogonin is a plant monoflavonoid which has been reported to inhibit cell growth and/or induce apoptosis in various tumors. The present study examined the apoptosis-inducing activity and underlying mechanism of action of wogonin in A549 cells. The results showed that wogonin was a potent inhibitor of the viability of A549 cells. Apoptotic protein changes detected after exposure to wogonin included decreased XIAP and Mcl-1 expression, increased cleaved-PARP expression and increased release of AIF and cytotchrome C. Western blot analysis showed that the activity of c-Myc/Skp2 and HDAC1/ HDAC2 pathways, which play important roles in tumor progress, was decreased. Quantitative PCR identified increased levels of c-Myc mRNA and decreased levels of its protein. Protein levels of Fbw7a, GSK3b and Thr58-Myc, which are involved in c- Myc ubiquitin-dependent degradation, were also analyzed. After exposure to wogonin, Fbw7a and GSK3b expression decreased and Thr58-Myc expression increased. However, MG132 was unable to prevent c-Myc degradation. The present results suggest that wogonin has multiple anti-cancer effects associated with degradation of c-Myc, SKP2, HDAC1 and HDAC2. Its ability to induce apoptosis independently of Fbw7a suggests a possible use in drug-resistance cancer related to Fbw7 deficiency. Further studies are needed to determine which pathways are related to c-Myc and Fbw7a reversal and whether Thr58 phosphorylation of c-Myc is dependent on GSK3b. Citation: Chen X-m, Bai Y, Zhong Y-j, Xie X-l, Long H-w, et al. (2013) Wogonin Has Multiple Anti-Cancer Effects by Regulating c-Myc/SKP2/Fbw7a and HDAC1/ HDAC2 Pathways and Inducing Apoptosis in Human Lung Adenocarcinoma Cell Line A549. PLoS ONE 8(11): e79201. doi:10.1371/journal.pone.0079201 Editor: Jian-Xin Gao, Shanghai Jiao Tong University School of Medicine, China Received January 23, 2013; Accepted September 20, 2013; Published November 12, 2013 en et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. pyright:  2013 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution L restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright:  2013 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attr unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: The study was supported by grants from the Ministry of Science and Technology of Guizhou Province, China ([2012] 7006 and NY [2011]3072); Guangzhou Medical College (2012C16); the Foundation for Young Scientists of Guangzhou Educational Committee (2012C118). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ytjq0629_cn@sina.com (QJ); xiaoh_wang@126.com (XHW) * E-mail: ytjq0629_cn@sina.com (QJ); xiaoh_wang@126.com (XHW) . These authors contributed equally to this work. Wogonin Has Multiple Anti-Cancer Effects by Regulating c-Myc/SKP2/Fbw7a and HDAC1/HDAC2 Pathways and Inducing Apoptosis in Human Lung Adenocarcinoma Cell Line A549 Xin-mei Chen1., Yang Bai2., Yu-jian Zhong1., Xiao-lin Xie3, Han-wu Long3, Yu-yin Yang3, Shi-gen Wu3, Qiang Jia3,4*, Xiao-hua Wang1* 1 Guangzhou Medical University, Guangzhou, Guangdong, PR China, 2 Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, PR China, 3 Institute of Biology, Guizhou Academy of Sciences, Guiyang, Guizhou, PR China, 4 Fanjingshan Forest Ecosystem Research Station, Guizhou Academy of Sciences, Jiangkou, Guizhou, PR China November 2013 | Volume 8 | Issue 11 | e79201 Introduction different recognition subunits of the SCF-type E3 ligase (SCF, Skp1/Cullin/F-box protein complexes) that recognize specific substrates for proteasomal degradation. Regulation of c-Myc involves phosphorylation of c-Myc at Thr58, resulting in Fbw7- mediated proteasomal degradation. Glycogen synthase kinase 3b(GSK3b) is the only kinase known to phosphorylate c-Myc at Thr58 [24]. different recognition subunits of the SCF-type E3 ligase (SCF, Skp1/Cullin/F-box protein complexes) that recognize specific substrates for proteasomal degradation. Regulation of c-Myc involves phosphorylation of c-Myc at Thr58, resulting in Fbw7- mediated proteasomal degradation. Glycogen synthase kinase 3b(GSK3b) is the only kinase known to phosphorylate c-Myc at Thr58 [24]. Methylthiazolyldiphenyl-tetrazolium Bromide (MTT) Cell Vaibility Assay Cells harvested with trypsin were seeded into 96-well plates at a density of 16104 per well. After overnight incubation, the culture medium was removed and the cells were incubated with different concentrations of wogonin. After exposure to wogonin for 24, 48 or 72 h the cells were incubated with MTT at 37uC for an additional 4 h. This allowed mitochondrial dehydrogenase to convert MTT into insoluble formazan crystals. The culture medium was then discarded, and 100 mL of dimethylsulfoxide (DMSO) was added to each well to dissolve the formazan crystals. The absorption of solubilized formazan was measured at 490 nm using a EL340 microplate reader (Bio-Tek. Instruments, Wi- nooske, VT). Skp2 is a promising target for restricting cancer stem cell and cancer progression [12], it’s overexpression is frequently observed in human cancer and it may, therefore, act as an oncogene. In support of this hypothesis, Skp2 has been shown to recognize Cyclin-dependent kinase (Cdk) inhibitors and tumor suppressive proteins such as p27 Kip1 (Cyclin-dependent kinase inhibitor 1B), p57 Kip2 (Cyclin-dependent kinase inhibitor 1C), p130 (130 kDa retinoblastoma-associated protein) and Tob1 (transducer of ERBB-2 1), but not c-Myc [13–16]. Skp2-mediated formation of ubiquitin from c-Myc occurs independently of phosphorylation [25]. Fbw7 deficiency is thought to be involved in drug resistance in human cancers [22,23]. It has been shown to be inactivated by mutation, deletion, or promoter hypermethylation in breast cancer [17,18], colon cancer [19,20], and leukemia [21]. Fbw7 is expressed as three different isoforms (designated a, b, and c) respectively located in the a-nucleus, b-cytoplasm, and c-nucleolus [26,27]. As there are no antibodies for these three isoforms we used the best described and longest of the three isoforms Fbw7a, in our experiments with wogonin. Real-time PCR Analysis Quantitative RT-PCR was undertaken using a SYBR Green reporter. A549 cells exposed to wogonin were washed with PBS and total RNA was purified by using RNAiso Plus (TAKARA, Japan). The resultant RNA was first reverse transcribed into cDNA using a PrimeScriptH RT Master Mix kit (TAKARA, Japan). Gene-specific primers were combined with SYBRH Premix Ex TaqTM (TAKARA, Japan) and amplified using an ABI 7500 real-time PCR machine (Applied Biosystems, USA). All qPCR reactions were performed independently on five samples. The relative mRNA expression was calculated using the 22DDCt method. The primer sequences used are listed in Table 1. Apoptosis Assay In this study, we evaluated the effects of wogonin on cell viability and apoptosis in the human lung adenocarcinoma epithelial cell line A549. We also assessed the regulation and function of c-Myc/Skp2/Fbw7a and HDAC1/HDAC2 pathways involved in its apoptotic effect. Cells were labeled with FITC-labeled Annexin V and propidium iodide (PI) using an Annexin V-FITC apoptosis detection kit (KeyGEN BioTECH, China), according to the manufacturer’s instructions. Briefly, after 48 h exposure to different concentrations of wogonin, the cells were washed with cold PBS and resuspended in 1X binding buffer. Aliquots of 105 cells were mixed with 5 mL of Annexin V-FITC and 5 mL of PI for 15 min at room temperature in the dark. Fluorescence (530 nm) was detected by flow cytometry (FACS Aria, BD Biosciences, USA) within 1 h. Mitochondrial Membrane Potential The mitochondrial membrane potential (Dym) of A549 cells was measured usimg a fluorescent, lipophilic, cationic probe, JC-1 (Beyotime, China), according to the manufacturer’s directions. Briefly, cells exposed to wogonin were incubated with 1X JC-1 staining solution for 20 min at 37uC. They were then rinsed twice with JC-1 staining buffer and images were taken with an Eclipse Ti Nikon microscope (Nikon, Japan). Introduction angiogenesis [4]. Data from preclinical studies have demonstrated that naturally occurring and synthetic histone deacetylase inhib- itors have potent anticancer activity. Despite of the large number of clinical trials aimed at improving patient survival, lung cancer remains a leading cause of cancer- related mortality worldwide in both men and women. Approxi- mately 85% of all lung cancer cases are categorized as non-small cell lung cancer (NSCLC), which is typically diagnosed at advanced stages [1]. Lung adenocarcinoma, the predominant histological subtype of NSCLC, accounts for 20 to 30% of primary lung cancer cases among subjects under 45 years of age, regardless of smoking history [2]. Most cases of NSCLC are unsuitable for surgery and chemotherapy remains the cornerstone of treatment for advanced disease. HDAC1 and HDAC2 belong to the Class I histone deacetylase (HDAC) family. In vivo, these enzymes form complexes with Sin3, NuRD and Co-REST [5]. HDAC1 and HDAC2 also bind directly to DNA binding proteins such as YY1, Rb binding protein-1 and Sp1 [5]. HDAC1 and HDAC2 belong to the Class I histone deacetylase (HDAC) family. In vivo, these enzymes form complexes with Sin3, NuRD and Co-REST [5]. HDAC1 and HDAC2 also bind directly to DNA binding proteins such as YY1, Rb binding protein-1 and Sp1 [5]. c-Myc is a transcription factor that is responsible for regulating an array of genes involved in cellular proliferation, growth, apoptosis and differentiation [6]. Deregulated c-Myc expression is observed in roughly 70% of all human tumors [10]. c-Myc expression is regulated by gene transcription, and is dependent on mRNA stability and posttranslational control of protein stability [7–9]. Histone deacetylases (HDACs) are enzymes that remove histone acetylation products. This process compacts the structure of chromatin and represses transcription [3]. HDACs act on various nonhistone protein substrates which play a role in the regulation of gene expression, cell proliferation, cell migration, cell death and Posttranslational regulation of c-Myc can be mediated by Skp2 (S-phase kinase-associated protein 2) and Fbw7(F-box and WD repeat domain-containing 7) [11]. Skp2 and Fbw7 are two PLOS ONE | www.plosone.org November 2013 | Volume 8 | Issue 11 | e79201 November 2013 | Volume 8 | Issue 11 | e79201 1 Wogonin Inducing Apoptosis in A549 medium with 10% fetal bovine serum (Sijiqing, China) at 37uC and 5% CO2. medium with 10% fetal bovine serum (Sijiqing, China) at 37uC and 5% CO2. Nuclear Staining A549 cells were stained with a DAPI (49, 6-diamidino-2- phenylindole) staining kit (KeyGEN, China). After exposure to graded concentrations of wogonin for 48 h, the cells were washed and incubated with a DAPI working solution (1–2 mg/mL) for 15 min at 37uC. The cells were then rinsed with methanol and Buffer A (60% glycerol in 10 mM phosphate-buffered saline (PBS, pH7.6)) was added to the suspension. A549 cells were viewed using an Eclipse Ti Nikon microscope (Nikon, Japan). Wogonin (5, 7-dihydroxy-8-methoxyflavanon) is a naturally monoflavonoid extracted from Scutellaria baicalensis radix [28] that has been recognized as an anticancer drug candidate with potentially low toxicity [29]. The anticancer activity of wogonin has been reported various human cell lines including myeloma cell RPMI 8226 [30], hepatocellular carcinoma SK-HEP-1 [31] and SMMC-7721 [32], glioma cancer cells [33], nasopharyngeal carcinoma cells [34], human breast cancer cells [35,36] and human cervical carcinoma HeLa cells [37]. Its activity is mediated by the induction of apoptosis and cell differentiation, and is regulated by various genes and proteins [38–41]. Reagents and Antibodies Wogonin was purchased from Guangzhou IDC (China) and MG132 (carbobenzoxy-Leu-Leu-leucinal) was obtained from (Beyotime, China). The following antibodies were used: Fbw7(Cdc4, H-300) and p-c-Myc (Thr 58) (Santa Cruz, USA); c-Myc, GSK3b, AIF (apoptosis inducing factor, Proteintech Group, Inc., USA); HDAC1, HDAC2, Skp2, Survivin, Bcl-2 (B- cell lymphoma 2), b-Actin (Boster, China); Mcl-1 (myeloid cell leukemia sequence 1), XIAP(X-linked inhibitor of apoptosis protein, Bioss, China); Cytochome c (KeyGEN, China); PARP (poly ADP-ribose polymerase, Sino Biological Inc., China). Wogonin Down-regulates c-Myc and Skp2 at the Protein Level, and Increases the mRNA Level of c-Myc Both c-Myc and Skp2 were down-regulated at the protein level following exposure to wogonin (0, 15, 25, 35 mg/mL) for 48 h, (Fig. 4B). As shown in Fig. 4A, the mRNA level of Skp2 decreased 0.81-fold, whereas the mRNA level of c-Myc increased approx- imately 1.6-fold (uncertainties related to fold-changes both ,2). These findings indicate that a proteasomal degradation pathway may be involved in the regulation of c-Myc and Skp2. However, as wogonin resulted in decreased protein expression in Skp2, further experiments focused on the proteasome recognition subunit, Fbw7a, which targets c-Myc. The soluble protein concentration was determined with a BCA protein assay kit (Beyotime, China). The cell lysates were boiled for 5 min in loading buffer and separated on an SDS-PAGE gel. After electrophoresis, the proteins were transferred to a PVDF membrane (Millipore, USA). The membranes were blocked with nonfat milk, probed with various primary antibodies and HRP- conjugated secondary antibodies, and visualized with enhanced chemiluminescence (ECL) detection reagents (Beyotime, China). Wogonin Inducing Apoptosis in A549 Wogonin Inducing Apoptosis in A549 DAPI staining (Fig. 1D) identified condensed and cleaved nuclei in cells exposed to 35 mg/mL wogonin, while only clear nuclei with pale blue staining were observed in the control group. Table 1. Primer sequences for real-time PCRs. Gene Primer sequences (59R39) GAPDH Forward GAAATCCCATCACCATCTTCCAGG Reverse GAGCCCCAGCCTTCTCCATG HDAC1 Forward TAAATTCTTGCGCTCCATCC Reverse AACAGGCCATCGAATACTGG HDAC2 Forward CGTGTAATGACGGTATCATTCC Reverse ACCAGATAATGAGTCTGCACC c-Myc Forward AGCGACTCTGAGGAGGAACAAG Reverse GTGGCACCTCTTGAGGACCA Skp2 Forward TGGGAATCTTTTCCTGTCTG Reverse GAACACTGAGACAGTATGCC Fbw7a Forward AGTAGTATTGTGGACCTGCCCGTT Reverse GACCTCAGAACCATGGTCCAACTT GSK3b Forward GGCAGCATGAAAGTTAGCAGA Reverse GGCGACCAGTTCTCCTGAATC doi:10.1371/journal.pone.0079201.t001 Table 1. Primer sequences for real-time PCRs. As shown in Fig. 2A, Wogonin was associated with a dose- dependent decrease in mitochondria potential (Dym) which resulted in decreased red fluorescence (JC-1 polymer) and increased of green fluorescence (JC-1 monomer). This may have been related to down-regulation of Mcl-1 as there was no obvious decrease in Bcl-2 levels. Wogonin also promoted the release of AIF and cytochrome C into the cytoplasm providing further evidence of mitochondrial damage (Fig. 2C). Down-regulation of XIAP, survivin, and of cleaved fragments from PARP indicated that the process of apoptosis continued after mitochondria damage (Fig. 2B). Western Blot Analysis Cells washed with PBS were lysed with RIPA (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA and leupeptin, (Beyotime, China) supplemented with protease inhibitor PMSF (Beyotime, China). Cytoplasmic proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit (Key- GEN BioTECH, China). Wogonin Inhibits Cell Viability and Induces Cell Apoptosis Experiments were conducted with the proteasome inhibitor MG132 to further understand the proteasomal degradation pathway involved in the regulation of c-Myc. The results in Fig. 5C show that MG132 was unable to reverse c-Myc degradation induced by 25 mg/mL wogonin. Further research is therefore needed to define the exact pathway involved in c-Myc degradation. Cell viability was assessed using an MTT assay in association with DAPI staining and flow cytometric analysis after exposure to different concentrations of wogonin. MTT analysis indicated that wogonin inhibited cell viability in a dose-dependent and time- dependent manner (Fig. 1B). This was confirmed by results of flow cytometric analysis using AnnexinV-PI (Fig. 1C). Compared with the control group, the rate of both early and late stage apoptosis increased after exposure to all concentrations of wogonin. The total apoptosis rate exceeded 50% in the 35 mg/ mL group. Statistical Analysis Statistical analyses was undertaken using SPSS version 17.0 software. Results are expressed as means and standard errors (Mean 6 SEM) from at least three independent experiments. One- way analysis of variance (ANOVA) was used to determine statistical significance between groups. Values of P,0.05 were considered statistically significant. Protein levels of Fbw7a decreased following exposure to wogonin, (Fig. 5B), but there was no corresponding decrease in mRNA expression (Fig. 5A). Thr58 phophorylation of c-Myc increased (Fig. 5B). Thr58 phophorylation of c-Myc is a required component of its degradation and is mediated by GSK3b. However, GSK3b expression decreased at both the mRNA (0.78-fold at 35 mg/mL) (Fig. 5A) and protein level (Fig. 5B). These findings suggest that phophorylation of c-Myc at Thr58 may occur independently of GSK3b. However, this requires further study. Wogonin Down-regulates HDAC1 and HDAC2 at Both mRNA and Protein Levels Protein levels of HDAC1 and HDAC2 were down-regulated in a dose-dependent manner after exposure to different concentra- tions of wogonin for 48 h (Fig. 3B). This result is consistent with the down-regulation of mRNA detected by qPCR showing that HDAC1 was decreased by 0.69-fold and HDAC2 by 0.73-fold, (uncertainties related to fold-changes were ,2) (Fig. 3A). These changes may result in a proportional increase in histone acetylation and promote the expression of tumor suppressive proteins. doi:10.1371/journal.pone.0079201.t001 Cell Culture The human pulmonary adenocarcinoma cell line A549 was obtained from the Cell Bank of the Animal Experiment Center, North School Region, Sun Yat-Sen University. The cells used in the experiments were maintained in our laboratory in RPMI 1640 November 2013 | Volume 8 | Issue 11 | e79201 PLOS ONE | www.plosone.org 2 Discussion Wogonin is a naturally occurring monoflavonoid extracted from Scutellaria baicalensis radix [28]. It has been reported to have PLOS ONE | www.plosone.org November 2013 | Volume 8 | Issue 11 | e79201 3 Wogonin Inducing Apoptosis in A549 Figure 1. Chemical structure of wogonin and wogonin exerted potent anti- A549 activity in vitro. (A) Chemical structure of wogonin (C16H12O5, MW: 284.27). (B) Cell viability was analyzed using the MTT assay. Cells were incubated with wogonin for 24 h, 48 h or 72 h. The bars represent the mean values 6 SEM (n = 3). (C) Apoptosis assessed by Annexin V/PI staining in A549 cells. Cells were incubated with wogonin at concentrations of 0, 15, 25, 35 mg/mL, for 24 h, 48 h and 72 h. (D) Nuclei stained by DAPI and observed by fluorescence microscope. Apoptotic cells are indicated by arrows. *P,0.05 vs control group, **P,0.01 vs control group. doi:10.1371/journal.pone.0079201.g001 Figure 1. Chemical structure of wogonin and wogonin exerted potent anti- A549 activity in vitro. (A) Chemical structure of wogonin (C16H12O5, MW: 284.27). (B) Cell viability was analyzed using the MTT assay. Cells were incubated with wogonin for 24 h, 48 h or 72 h. The bars represent the mean values 6 SEM (n = 3). (C) Apoptosis assessed by Annexin V/PI staining in A549 cells. Cells were incubated with wogonin at concentrations of 0, 15, 25, 35 mg/mL, for 24 h, 48 h and 72 h. (D) Nuclei stained by DAPI and observed by fluorescence microscope. Apoptotic cells are indicated by arrows. *P,0.05 vs control group, **P,0.01 vs control group. doi:10.1371/journal.pone.0079201.g001 progression [4]. Our results show that the mRNA and protein levels of HDAC1 and HDAC2 were both decreased in the presence of wogonin, indicating that acetylated histone protein may promote expression of tumor suppressive proteins and thereby inhibit tumor progression. antineoplastic activity in various types of cancer by the induction of apoptosis and cell differentiation [30–37], and to be regulated by various genes and proteins [38–41]. It is known that the c- Myc/Skp2/Fbw7a and HDAC1/HDAC2 pathways, are associ- ated with tumor progression. Here we investigate their role in the anticancer effects of wogonin in NSCLC A549 cells. An inter-relationship exists between c-Myc and Skp2 such that c-Myc promotes Skp2 expression, and Skp2 targets c-Myc for ubiquitin-dependent degradation [11,12,24,25]. November 2013 | Volume 8 | Issue 11 | e79201 Discussion They suppress gene expression, and modify tumor specific proteins involved in November 2013 | Volume 8 | Issue 11 | e79201 PLOS ONE | www.plosone.org November 2013 | Volume 8 | Issue 11 | e79201 4 Wogonin Inducing Apoptosis in A549 Figure 2. Effects of wogonin on mitochondrial membrane potential and apoptotic proteins in A549. (A) Analysis of the mitochondrial membrane potential (DYm) using JC-1 staining after exposure to wogonin for 48 h. A fluorescence microscope was used to visualize the results. Mitochondrial depolarization was indicated by an increase in green fluorescence and a decrease in red fluorescence intensity. (B) Protein levels of Bcl- 2, Mcl-1, XIAP, survivin and PARP assayed by western blot. (C) Cytoplasmic proteins were extracted for western blot analysis of released Cytochrome c and AIF. In these experiments, cells were exposed to wogonin 0, 15, 25, 35 mg/mL for 48 h. *P,0.05 vs control group, **P,0.01 vs control group. doi:10.1371/journal.pone.0079201.g002 Figure 2. Effects of wogonin on mitochondrial membrane potential and apoptotic proteins in A549. (A) Analysis of the mitochondrial membrane potential (DYm) using JC-1 staining after exposure to wogonin for 48 h. A fluorescence microscope was used to visualize the results. Mitochondrial depolarization was indicated by an increase in green fluorescence and a decrease in red fluorescence intensity. (B) Protein levels of Bcl- 2, Mcl-1, XIAP, survivin and PARP assayed by western blot. (C) Cytoplasmic proteins were extracted for western blot analysis of released Cytochrome c and AIF. In these experiments, cells were exposed to wogonin 0, 15, 25, 35 mg/mL for 48 h. *P,0.05 vs control group, **P,0.01 vs control group. doi:10.1371/journal.pone.0079201.g002 Figure 3. Effects of wogonin on HDAC1 and HDAC2 in A549. (A) Relative mRNA levels of HDAC1 and HDAC2 were detected using real-time PCR with GAPDH as an internal control. Results are expressed as the mean 6 SEM of five independent experiments. #P,0.01 vs control group. (B) Protein levels of HDAC1 and HDAC2 assayed by western blot. In these experiments, cells were exposed to wogonin 0, 15, 25 and 35 mg/mL for 48 h. *P,0.05 vs control group, **P,0.01 vs control group. doi:10.1371/journal.pone.0079201.g003 Figure 3. Effects of wogonin on HDAC1 and HDAC2 in A549. (A) Relative mRNA levels of HDAC1 and HDAC2 were detected using real-time PCR with GAPDH as an internal control. Results are expressed as the mean 6 SEM of five independent experiments. #P,0.01 vs control group. Discussion In our experi- ments, wogonin down-regulated c-Myc and Skp2 at the protein level, but the mRNA expression of c-Myc increased 1.6-fold. These findings suggest that the proteasomal degradation pathway may be related to the reversal of c-Myc. However, since Skp2 expression decreased, we focused our further experiments on Fbw7a, another proteasome recognition subunit that targets c- Myc. We first evaluated the anti-viability and apoptotic effects of wogonin using MTT and Annexin V-PI double staining assays. Our results indicated that wogonin caused dose-dependent and time-dependent inhibition of cell viability (IC50,35 mg/mL at 48 h). The early increase in apoptosis rate in response to wogonin occurred in parallel, with Annexin V-positive cells gradually becoming Annexin-V negative. At the IC50 (35 mg/mL) cells showed evidence of marked apoptosis, consistent with the nuclear morphology changes seen with DAPI staining. Thr58 phophorylation of c-Myc is required for Fbw7a mediated c-Myc degradation [24]. As Thr58 is phophorylated by GSK3b, we evaluated expression levels of Fbw7a, Thr58 c-Myc and GSK3b. In these experiments Fbw7a expression decreased at the protein level but not at the mRNA level. Thr58 phophorylation of c-Myc increased to some degree and GSK3b expression at both the mRNA and protein level decreased. These results highlight lack of conformity between Fbw7a mRNA and protein levels, the decreased expression of GSK3b, and the increased phophoryla- tion of c-Myc at Thr58. The expression of apoptosis related proteins, such as Bcl-2, Mcl- 1, PARP, XIAP, Survivin, cytochrome c and AIF was evaluated to further identify the apoptotic effects of wogonin at the protein level. The results indicate that wogonin is able to influence mitochondrial membrane stability and decrease mitochondria membrane potential (Dym). This was evidenced by JC-1 staining, decreased mcl-1 expression and the release of cytochrome C and AIF into the cytoplasm. Cleaved PARP and decreased XIAP and survivin expression may also have contributed to the progression of apoptosis. A previous study demonstrated that phosphorylation of Thr58 in A549 cells occurred independently of GSK3b [42]. However, GSK3b is the only known kinase that phosphorylates c-Myc at Thr58. In our studies the proteasome inhibitor MG132 was HDAC1 and HDAC2 are Class I HDACs that deacetylate histone and non-histone proteins [5]. Discussion (B) Protein levels of HDAC1 and HDAC2 assayed by western blot. In these experiments, cells were exposed to wogonin 0, 15, 25 and 35 mg/mL for 48 h. *P,0.05 vs control group, **P,0.01 vs control group. doi:10.1371/journal.pone.0079201.g003 November 2013 | Volume 8 | Issue 11 | e79201 PLOS ONE | www.plosone.org 5 Wogonin Inducing Apoptosis in A549 Figure 4. Effects of wogonin on c-Myc and Skp2 in A549. (A) Relative mRNA levels of c-Myc and Skp2 were detected using real-time PCR with GAPDH as an internal control. Results are expressed as the mean 6 SEM of five independent experiments. #P,0.01 vs control group. (B) Protein levels of c-Myc and Skp2 assayed by western blot. In these experiments, cells were exposed to wogonin 0, 15, 25 and 35 mg/mL for 48 h. *P,0.05 vs control group, **P,0.01 vs control group. doi:10.1371/journal.pone.0079201.g004 Figure 4. Effects of wogonin on c-Myc and Skp2 in A549. (A) Relative mRNA levels of c-Myc and Skp2 were detected using real-time PCR with GAPDH as an internal control. Results are expressed as the mean 6 SEM of five independent experiments. #P,0.01 vs control group. (B) Protein levels of c-Myc and Skp2 assayed by western blot. In these experiments, cells were exposed to wogonin 0, 15, 25 and 35 mg/mL for 48 h. *P,0.05 vs control group, **P,0.01 vs control group. doi:10.1371/journal.pone.0079201.g004 Figure 5. Effects of wogonin on Fbw7a and GSK3b in A549. (A) Relative mRNA levels of Fbw7a and GSK3b were detected using real-time PCR with GAPDH as an internal control. Results are expressed as the mean 6 SEM of five independent experiments. #P,0.01 vs control group. (B) Protein levels of Fbw7a, Thr58-Myc and GSK3b assayed by western blot. (C) Protein levels of c-Myc assayed by western blot. In these experiments, 1 mM MG132 was added incubated with or without 25 mg/mL wogonin for 48 h. *P,0.05 vs control group, **P,0.01 vs control group. doi:10.1371/journal.pone.0079201.g005 Figure 5. Effects of wogonin on Fbw7a and GSK3b in A549. (A) Relative mRNA levels of Fbw7a and GSK3b were detected using real-time PCR with GAPDH as an internal control. Results are expressed as the mean 6 SEM of five independent experiments. #P,0.01 vs control group. (B) Protein levels of Fbw7a, Thr58-Myc and GSK3b assayed by western blot. (C) Protein levels of c-Myc assayed by western blot. 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Crocetti E, Paci E (2002) Trends in lung adenocarcinoma incidence and survival, Lung Cancer 2: 2152216. 25. Kim SY, Herbst A, Tworkowski KA, Salghetti SE, Tansey WP (2003) Skp2 regulates Myc protein stability and activity. Mol Cell 11: 117721188. 3. Ruthenburg AJ, Li H, Patel DJ, Allis CD (2007) Multivalent engagement of chromatin modifications by linked binding modules. Nat Rev Mol Cell Biol 8: 983–994. 26. Spruck CH, Strohmaier H, Sangfelt O, Mu¨ller HM, Hubalek M, et al. (2002) hCDC4 gene mutations in endometrial cancer. Cancer Res 62: 453524539. 27. Welcker M, Orian A, Grim JE, Eisenman RN, Clurman BE (2004) A nucleolar isoform of the Fbw7 ubiquitin ligase regulates c-Myc and cell size. Curr Biol 14: 185221857. 4. Marks PA, Xu WS (2009) Histone deacetylase inhibitors: potential in cancer therapy, J Cell Biochem 107: 6002608. 5. de Ruijter AJ, van Gennip AH, Caron HN, Kemp S, van Kuilenburg AB (2003) Histone deacetylases (HDACs): characterization of the classical HDAC family. BioChem J 370: 7392749. 28. Chi YS, Cheon BS, Kim HP (2001) Effect of wogonin, a plant flavone from Scutellaria radix, on the suppression of cyclooxygenase-2 and the induction of inducible nitric oxide synthase in lipopolysaccharide-treated RAW 264.7 cells. Biochem Pharmacol 61: 1195–1203. J 6. Dang CV, Resar LM, Emison E, Kim S, Li Q, et al. (1999) Function of the c- Myc oncogenic transcription factor. Exp Cell Res 253: 63277. 29. Li-Weber M (2009) New therapeutic aspects of flavones: the anticancer properties of Scutellaria and its main active constituents wogonin, baicalein and baicalin. Cancer Treat Rev 35: 57–68. 7. Kelly K, Cochran H, Stiles CD, Leder P (1983) Cell-specific regulation of the c- Myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell 35: 6032610. 30. Zhang M, Liu LP, Chen Y, Tian XY, Qin J, et al. (2013) Wogonin induces apoptosis in RPMI 8226, a human myeloma cell line, by downregulating phospho-Akt and overexpressing Bax. Life Sci 1: 55–62. 8. Discussion In these experiments, 1 mM MG132 was added incubated with or without 25 mg/mL wogonin for 48 h. *P,0.05 vs control group, **P,0.01 vs control group. doi:10.1371/journal.pone.0079201.g005 PLOS ONE | www.plosone.org November 2013 | Volume 8 | Issue 11 | e79201 6 Wogonin Inducing Apoptosis in A549 unable to prevent the degradation of c-Myc suggesting that decreased Fbw7a and Skp2 might be involved in this process. However, the exact mechanism involved requires further research. Author Contributions Conceived and designed the experiments: QJ XHW. Performed the experiments: YJZ YB XMC XLX HWL. Contributed reagents/materials/ analysis tools: XMC XLX HWL. Wrote the paper: XMC YB YJZ. Analyzed the data and interpreted the results: YYY SGW. Read and approved the final manuscript: XMC YB YJZ XLX HWL YYY SGW GJ XHW. Taken together our findings suggest that the ability of wogonin to influence different biochemical pathways may explain its activity against a variety of different cancers. Our data may also in part explain why some gene deficient cells (e.g. those with Fbw7a deficiency) are resistant to wogonin. References Toxicology 282: 122– 128. p 16. Hiramatsu Y, Kitagawa K, Suzuki T, Uchida C, Hattori T, et al. (2006) Degradation of Tob1 mediated by SCFSkp2-dependent ubiquitination. Cancer Res 66: 847728483. 37. Yang L, Zhang HW, Hu R, Yang Y, Qi Q, et al. (2009) Wogonin induces G1 phase arrest through inhibiting Cdk4 and cyclin D1 concomitant with an elevation in p21Cip1 in human cervical carcinoma HeLa cells. Biochem Cell Biol 87: 933–942. 17. Zhao D, Zheng HQ, Zhou Z, Chen C (2010) The Fbw7 tumor suppressor targets KLF5 for ubiquitin-mediated degradation and suppresses breast cell proliferation. Cancer Res 70: 4728–4738. 18. Akhoondi S, Lindstrom L, Widschwendter M, Corcoran M, Bergh J, et al. (2010) Inactivation of FBXW7/hCDC4-beta expression by promoter hyper- methylation is associated with favorable prognosis in primary breast cancer. Breast Cancer Res 12: R105. 38. Lee E, Enomoto R, Koshiba C, Hirano H (2009) Inhibition of P-glycoprotein by wogonin is involved with the potentiation of etoposide-induced apoptosis in cancer cells. Ann NY Acad Sci 1171: 132–136. 39. Ding J, Polier G, Ko¨hler R, Giaisi M, Krammer PH, et al. (2012) Wogonin and related natural flavones overcome tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) protein resistance of tumors by down-regulation of c- FLIP protein and up-regulation of TRAIL receptor 2 expression. J Biol Chem 1: 641–9. 19. Inuzuka H, Shaik S, Onoyama I, Gao D, Tseng A, et al. (2011) SCF(FBW7) regulates cellular apoptosis by targeting MCL1 for ubiquitylation and destruction. Nature 471: 104–109. 20. Sancho R, Jandke A, Davis H, Diefenbacher ME, Tomlinson I, et al. (2010) F- box and WD repeat domain-containing 7 regulates intestinal cell lineage commitment and is a haploinsufficient tumor suppressor. Gastroenterology 139: 929–941. 40. Song X, Yao J, Wang F, Zhou M, Zhou Y, et al. (2013) Wogonin inhibits tumor angiogenesis via degradation of HIF-1a protein. Toxicol Appl Pharmacol 2: 144–55. 21. O’Neil J, Look AT (2007) Mechanisms of transcription factor deregulation in lymphoid cell transformation. Oncogene 26: 6838–6849. 41. Yang L, Wang Q, Li D, Zhou Y, Zheng X, et al. (2013) Wogonin enhances antitumor activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo through ROS-mediated downregulation of cFLIPL and IAP proteins. Apoptosis 5: 618–26. y p g 22. Wang Z, Fukushima H, Gao D, Inuzuka H, Wan L, et al. (2011) The two faces of FBW7 in cancer drug resistance. Bioessays 11: 8512859. of FBW7 in cancer drug resistance. 42. Li Q, Kluz T, Sun H, Costa M (2009) Mechanisms of c-Myc Degradation by Nickel Compounds and Hypoxia. PLoS ONE 4: e8531. References Bioessays 11: 8512859. 23. Wertz IE, Kusam S, Lam C, Okamoto T, Sandoval W, et al. (2011) Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7. Nature 471: 110–114. 42. Li Q, Kluz T, Sun H, Costa M (2009) Mechanisms of c-Myc Degradation by Nickel Compounds and Hypoxia. PLoS ONE 4: e8531. November 2013 | Volume 8 | Issue 11 | e79201 7 PLOS ONE | www.plosone.org
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Phagocytosis-like cell engulfment by a planctomycete bacterium
Nature communications
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1 Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-0053, Japan. 2 Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa 237-0061, Japan. 3 National Institute for Environmental Studies, Tsukuba, Ibaraki 305-0053, Japan. 4These authors contributed equally: Takashi Shiratori, Shigekatsu Suzuki. *email: tshiratori@jamstec.go.jp NATURE COMMUNICATIONS | (2019) 10:5529 | https://doi.org/10.1038/s41467-019-13499-2 | www.nature.com/naturecommunications ARTICLE ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 T here are significant differences between eukaryotes and prokaryotes in terms of cellular and genomic complex- ity1,2. During the emergence of ancestral eukaryotes (eukaryogenesis), cells increased in size and developed an endo- membrane system, a nucleus and other membrane-bounded organelles. They also developed actin- and tubulin-based cytos- keletons, which facilitated uptake of large particles from the environment (phagocytosis)3. Phagocytosis not only supports efficient nutrient acquisition via consumption of other organisms (phagotrophy), but is also considered by some eukaryogenesis theories to be involved in the acquisition of the mitochondrion4–6. However, it remains unclear how the ancestral eukaryote acquired the phagocytosis ability since no known present organism exhibits a primitive phagocytosis. T Our attempts to establish axenic cultures were unsuccessful. We found that ‘Ca. Uab amorphum’ has a flat, round or oval cell shape that is ~4–5 μm in diameter (Fig. 1a–c). However, cells occasionally reached 10 μm in diameter when cultures included many co-cultured bacterial cells (Supplementary Fig. 1a). ‘Ca. Uab amorphum’ moved on solid substrates at approximately 8–16 μm/min while frequently changing cell shape (Supplemen- tary Movie 1). In addition, ‘Ca. Uab amorphum’ engulfed other bacteria in cultures (Fig. 1d, e, Supplementary Movie 2); the species description is included in the ‘Methods’ section. p p Amplification of ribosomal RNA (rRNA) using PCR and fluorescent in situ hybridization (FISH) with specific oligonucleo- tide probes for ‘Ca. Uab amorphum’ revealed that this organism was not a eukaryotic amoeba, but a bacterium whose 16 S rRNA gene sequence similarity with the closest known species was only 79% (Supplementary Fig. 2, Supplementary Table 1). Genome sequencing of the monoxenic culture produced a circular genome that encoded the 16 S rRNA of ‘Ca. Uab amorphum’; the remaining sequences were small portions of the A. macleodii genomes (Supplementary Table 2). The ‘Ca. Uab amorphum’ genome was AT rich, 9.5 Mb in size and encoded 6660 protein- coding genes without plasmids (Supplementary Table 3). Recent metagenomic studies have identified an archaeal group, Asgard, most closely related to Eukarya7,8. Asgard archaea pos- sess genes that are homologous to eukaryotic genes involved in cytoskeleton formation, vesicle/membrane trafficking or remo- delling and phagocytosis7,8. Recent cultivation and physiological studies of an Asgard archaeon, ‘Candidatus Prometheoarchaeum syntrophicum’, have shown that this organism is syntrophic with bacteria and occasionally produces long, branching protrusions9. However, it has not been reported that ‘Ca. Prometheoarchaeum syntrophicum’ displays any phagocytic-like behaviour. ARTICLE g g p ( pp y ) To identify the phylogenetic position of ‘Ca. Uab amorphum’, we performed molecular phylogenetic analyses with two datasets, one using 16S rRNA gene sequences and the other using 171 proteins. Both analyses indicated that ‘Ca. Uab amorphum’ belongs to the bacterial phylum Planctomycetes (Fig. 2, Supple- mentary Fig. 3). ‘Ca. Uab amorphum’ was genetically separated from three subgroups of Planctomycetes: Planctomycetia, Phyci- sphaerae and anammox (anaerobic ammonium oxidation) bacteria10, with anammox bacteria being its closest relative. We found that various environmental rRNA gene sequences formed a clade with ‘Ca. Uab amorphum’ (Supplementary Fig. 4). This “Uab clade” included sequences derived from freshwater, hypersaline and marine environments, as well as the bovine rumen. The similarity between ‘Ca. Uab amorphum’ and these y y y g y Here, we describe a planctomycete bacterium, ‘Candidatus Uab amorphum’ (named after a hungry giant in Palauan mythology, called Uab) that exhibits several intriguing eukaryotic-like fea- tures, including the ability to engulf other microorganisms (bacteria and eukaryotes) through a phagocytosis-like process. Phagocytosis-like cell engulfment by a planctomycete bacterium Takashi Shiratori 1,2,4*, Shigekatsu Suzuki 3,4, Yukako Kakizawa1 & Ken-ichiro Ishida1 Takashi Shiratori 1,2,4*, Shigekatsu Suzuki 3,4, Yukako Kakizawa1 & Ken-ichiro Ishida1 Phagocytosis is a key eukaryotic feature, conserved from unicellular protists to animals, that enabled eukaryotes to feed on other organisms. It could also be a driving force behind endosymbiosis, a process by which α-proteobacteria and cyanobacteria evolved into mito- chondria and plastids, respectively. Here we describe a planctomycete bacterium, ‘Candidatus Uab amorphum’, which is able to engulf other bacteria and small eukaryotic cells through a phagocytosis-like mechanism. Observations via light and electron microscopy suggest that this bacterium digests prey cells in specific compartments. With the possible exception of a gene encoding an actin-like protein, analysis of the ‘Ca. Uab amorphum’ genomic sequence does not reveal any genes homologous to eukaryotic phagocytosis genes, suggesting that cell engulfment in this microorganism is probably not homologous to eukaryotic phagocytosis. The discovery of this “phagotrophic” bacterium expands our understanding of the cellular complexity of prokaryotes, and may be relevant to the origin of eukaryotic cells. 1 Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-0053, Japan. 2 Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa 237-0061, Japan. 3 National Institute for Environmental Studies, Tsukuba, Ibaraki 305-0053, Japan. 4These authors contributed equally: Takashi Shiratori, Shigekatsu Suzuki. *email: tshiratori@jamstec.go.jp 1 NATURE COMMUNICATIONS | (2019) 10:5529 | https://doi.org/10.1038/s41467-019-13499-2 | www.nature.com/naturecommunicatio NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 0.2 Planctomyces maris Isosphaera pallida Chlamydiae Cyanobacteria Betaproteobacteria Chloroflexi ‘Candidatus Scalindua brodae’ ‘Candidatus Scalindua wagneri’ Phycisphaera mikrensis Planctomyces limnophilus ‘Candidatus Brocadia sp.’ Nostocoida limicola Pirellula marina ‘Candidatus Brocadia anammoxidans’ Spirochaetes Singulisphaera acidiphila ‘Candidatus Kuenenia stuttgartiensis’ ‘Candidatus Anammoxoglobus propionicus’ Gammaproteobacteria Firmicutes ‘Candidatus Uab amorphum’ Gemmata obscuriglobus Alphaproteobacteria Pirellula staleyi Verrucomicrobia Actinobacteria Chlorobi Aquificae 83/1.00 74/0.98 92/1 57/– 76/1.00 59/1.00 55/– 97/1.00 97/1.00 77/1.00 74/1.00 59/0.98 83/1.00 92/1.00 94/1.00 64/1.00 92/1.00 82/1.00 70/0.99 97/1.00 Thermotogae Phycisphaerae Planctomycetia Anammox bacteria Planctomycetes Fig. 2 Maximum likelihood tree of ‘Ca. Uab amorphum’ and other bacterial 16S rRNA gene sequences. Left and right values on nodes indicate bootstrap value and Bayesian posterior probability, respectively. Scale bar indicates nucleotide substitution rate per site. Chlamydiae Actinobacteria Chloroflexi 74/1.00 Thermotogae Fig. 2 Maximum likelihood tree of ‘Ca. Uab amorphum’ and other bacterial 16S rRNA gene sequences. Left and value and Bayesian posterior probability, respectively. Scale bar indicates nucleotide substitution rate per site. Fig. 2 Maximum likelihood tree of ‘Ca. Uab amorphum’ and other bacterial 16S rRNA gene sequences. Left and right values on nodes indicate bootstrap value and Bayesian posterior probability, respectively. Scale bar indicates nucleotide substitution rate per site. e of ‘Ca. Uab amorphum’ and other bacterial 16S rRNA gene sequences. Left and right values on nodes indicate bootst robability, respectively. Scale bar indicates nucleotide substitution rate per site. respectively14,15. We found that the genome of ‘Ca. Uab amorphum’ includes genes encoding proteins similar to Gram- negative bacterial type II secretion systems (T2SS) (Supplemen- tary Table 4), which would be consistent with a Gram-negative bacterial cell plan. environmental sequences varied between 79.4% (KC604713) and 98.4% (KU631341). These data suggest that relatives of ‘Ca. Uab amorphum’ live in various environments and have high genetic diversity. We also screened 16S rRNA data from the Tara Oceans expedition11 and found sequences related to the “Uab clade” in datasets derived from stations in the North and South Atlantic Oceans, the Indian Ocean, the North and South Pacific Ocean, the Red Sea and the Southern Ocean (Supplementary Fig. 5). The genome of ‘Ca. Uab amorphum’ possesses most of the genes required for peptidoglycan biosynthesis (Supplementary Table 5), but the presence and properties of a peptidoglycan layer remain untested. Considering its cell flexibility, this organism is unlikely to have a rigid peptidoglycan layer. Results h Characteristics of ‘Ca. Uab amorphum’. ‘Ca. Uab amorphum’ was discovered from a surface seawater sample collected at the Republic of Palau. It was maintained as an enriched or as a monoxenic culture with Alteromonas macleodii (NBRC 102226). a b c 0 s 8 s 26 s 44 s 76 s 128 s d 0 min 3 min 9 min 15 min 36 min 24 min j e f g h i k o m l n Fig. 1 Light and fluorescent micrographs of ‘Candidatus Uab amorphum’. a Cells of ‘Ca. Uab amorphum’ (arrowheads) in the xenic culture. b, c A cell of ‘Ca. Uab amorphum’. Double arrowheads indicate an engulfed bacterium. d–i Selected images of time-lapse video showing prey engulfment process of ‘Ca. Uab amorphum’. j–o Selected images of confocal fluorescent time-lapse video showing prey engulfment and digestion process of ‘Ca. Uab amorphum’. Green fluorescence indicates AcGFP1-labelled Escherichia coli. Scale bars, 10 μm (a) and 5 μm (b–o). 2 15 min m a 0 min j 44 s g 0 s d m d a g 0 min 44 s 24 min n 3 min k h 8 s e 76 s h k n 3 min 8 s 76 s b c c b 36 min o 26 s f i 9 min l l f 128 s i l o o 26 s Fig. 1 Light and fluorescent micrographs of ‘Candidatus Uab amorphum’. a Cells of ‘Ca. Uab amorphum’ (arrowheads) in the xenic culture. b, c A cell of ‘Ca. Uab amorphum’. Double arrowheads indicate an engulfed bacterium. d–i Selected images of time-lapse video showing prey engulfment process of ‘Ca. Uab amorphum’. j–o Selected images of confocal fluorescent time-lapse video showing prey engulfment and digestion process of ‘Ca. Uab amorphum’. Green fluorescence indicates AcGFP1-labelled Escherichia coli. Scale bars, 10 μm (a) and 5 μm (b–o). NATURE COMMUNICATIONS | (2019) 10:5529 | https://doi.org/10.1038/s41467-019-13499-2 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 NATURE COMMUNICATIONS | (2019) 10:5529 | https://doi.org/10.1038/s41467-019-13499-2 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 However, the actin-like protein appears to be more closely related to certain archaeal actins than to eukaryotic actins (see the section ‘Fibrous structures and an actin-like protein’). activity of F-ATPase), cells did not show any locomotion or cell engulfment, which suggest that these may be energy-dependent processes (Supplementary Fig. 1b, c). Feeding experiments by using an AcGFP1-labelled E. coli showed that the rod-shaped signals were separated into several globules (Fig. 1j–o, Supple- mentary Video 4) that disappeared over time and did not diffuse into the cell. We detected few acidic and reactive oxygen species (ROS) signals in cells of ‘Ca. Uab amorphum’ (Supplementary Fig. 1d–i). Electron microscopy analyses showed invagination of the ‘Ca. Uab amorphum’ cell surface around prey cells (Fig. 3d, e). Consistent with the prediction that both outer and cytoplasmic membranes invaginate during prey engulf, the resulting com- partment enclosing the prey cell (phagosome-like vacuole, PV) appeared to be surrounded by two membranes, i.e. the inner membrane of the PV would be the putative Gram-negative outer membrane, and the outer membrane of the PV would be the putative cytoplasmic membrane (Fig. 3f). Most prey cells in PVs were found to be disrupted to some extent (Fig. 3g, h), which suggests that digestion may occur in PVs. Some PVs exhibited activity of F-ATPase), cells did not show any locomotion or cell engulfment, which suggest that these may be energy-dependent processes (Supplementary Fig. 1b, c). Feeding experiments by using an AcGFP1-labelled E. coli showed that the rod-shaped signals were separated into several globules (Fig. 1j–o, Supple- mentary Video 4) that disappeared over time and did not diffuse into the cell. We detected few acidic and reactive oxygen species (ROS) signals in cells of ‘Ca. Uab amorphum’ (Supplementary Fig. 1d–i). Electron microscopy analyses showed invagination of the ‘Ca. Uab amorphum’ cell surface around prey cells (Fig. 3d, e). Consistent with the prediction that both outer and cytoplasmic membranes invaginate during prey engulf, the resulting com- partment enclosing the prey cell (phagosome-like vacuole, PV) appeared to be surrounded by two membranes, i.e. the inner membrane of the PV would be the putative Gram-negative outer membrane, and the outer membrane of the PV would be the putative cytoplasmic membrane (Fig. 3f). Most prey cells in PVs were found to be disrupted to some extent (Fig. 3g, h), which suggests that digestion may occur in PVs. Some PVs exhibited Phagocytosis-like cell engulfment. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 g d PE PV OM CM f NB NB NB NB C PE C C PV PV PE a b OM CM PE OM NB NB NB CM c h e Fig. 3 Ultrastructure of ‘Candidatus Uab amorphum’. a Transmission electron micrograph of ‘Ca. Uab amorphum’ showing cytoplasm (C), cytopla membrane (CM), nuclear body (NB), outer membrane (OM), periplasm (PE) and phagosome-like vacuole (PV). Arrows indicate dense granules tha probably glycogen granules. b, c High-magnification transmission electron micrograph showing the membrane structures. Cytoplasmic membrane ( nuclear body (NB), outer membrane (OM) and periplasm (PE). d Scanning electron micrograph of a ‘Ca. Uab amorphum’ that is about to engulf a bacterium (double arrowhead). e Transmission electron micrograph of a ‘Ca. Uab amorphum’ that is about to engulf a bacterium (double arrowhe f Ultrastructure of PV that is surrounded by outer membrane (OM), periplasm (PE) and cytoplasmic membrane (CM). Double arrowhead indicate engulfed prey bacterium g, h that disrupted prey bacteria in PVs. Scale bars, 1 μm (a, d, e), 500 nm (b, h) and 200 nm (c, f, g). PE b OM CM PE b OM CM PE OM NB NB NB CM c NB NB NB NB C PE C C PV PV a b a c PE OM NB NB NB CM c OM d e f PE PV OM CM f g h g f g h h Fig. 3 Ultrastructure of ‘Candidatus Uab amorphum’. a Transmission electron micrograph of ‘Ca. Uab amorphum’ showing cytoplasm (C), cytoplasmic membrane (CM), nuclear body (NB), outer membrane (OM), periplasm (PE) and phagosome-like vacuole (PV). Arrows indicate dense granules that are probably glycogen granules. b, c High-magnification transmission electron micrograph showing the membrane structures. Cytoplasmic membrane (CM), nuclear body (NB), outer membrane (OM) and periplasm (PE). d Scanning electron micrograph of a ‘Ca. Uab amorphum’ that is about to engulf a prey bacterium (double arrowhead). e Transmission electron micrograph of a ‘Ca. Uab amorphum’ that is about to engulf a bacterium (double arrowhead). f Ultrastructure of PV that is surrounded by outer membrane (OM), periplasm (PE) and cytoplasmic membrane (CM). Double arrowhead indicates engulfed prey bacterium g, h that disrupted prey bacteria in PVs. Scale bars, 1 μm (a, d, e), 500 nm (b, h) and 200 nm (c, f, g). proteins, with the exception of an actin-like protein and four WD40 repeat-containing proteins (Supplementary Table 7). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 Synthesis and degradation of the peptidoglycan layer are known to be required for engulfment of the daughter cell during spore formation in other bacterial species16,17. pp y g Transmission electron microscopy (TEM) demonstrated that ‘Ca. Uab amorphum’ has two membrane-bounded regions: an electron-dense ribosome-free region and a ribosome-containing region (Fig. 3a–c). The ribosome-free region located immediately beneath the outermost membrane and was highly invaginated into the ribosome-containing region, which showed a reticulate pattern in thin-section micrographs. The ribosome-containing region had multiple nuclear bodies that appeared to be separated by the invaginated ribosome-free region. This cellular structure consisting of ribosome-free and ribosome-containing regions with complex invaginations is typical of some planctomycete bacteria such as Gemmata obscuriglobus12. On the basis of electron microscopic observation and distribution of RNA13, it has been previously proposed that the outermost membrane surrounding the ribosome-free region is the cytoplasmic membrane, and the membrane surrounding the ribosome- containing region is an intracytoplasmic membrane. However, recent genomic and cell biological studies indicate that the cellular structure of planctomycetes resembles that of typical Gram-negative bacteria, with the outermost and inner mem- branes being equivalent to the outer membrane and cytoplasmic membrane, and the ribosome-free and ribosome-containing regions corresponding to the periplasm and cytoplasm, In comparison with other planctomycetes, ‘Ca. Uab amor- phum’ exhibited a relatively large and gene-rich genome (Supplementary Table 3), with many genes involved in signal transduction (Supplementary Fig. 6). In addition, we identified 1515 genes of unknown function, and 1823 genes (27.6% of all genes) potentially derived from horizontal gene transfer (HGT) (Supplementary Table 6). Most of these HGT candidates (1778 genes) were apparently derived from bacteria. We detected only two genes potentially derived from eukaryotes, encoding proteins similar to actin (UABAM_01738) and acyloxyacyl hydrolase (AOAH; UABAM_05422), respectively. ‘Ca. Uab amorphum’ apparently lacks other eukaryotic-derived genes previously reported in Planctomycetes, such as genes associated with sterol biosynthesis, membrane coat protein, integrin and the inter- alpha-trypsin inhibitor18–20. We searched for homologues of eukaryote signature proteins (ESPs)21 and found 27 ESP candidates (Supplementary Table 7). Most of these candidates were more similar to other bacterial proteins than to eukaryotic 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 Purple enzymes are predicted to be secreted in PV or localized at the outer membrane and involve to degradation of prey. Gree line indicates the cytoplasmic membrane. Grey line indicates the outer membrane. Brown layers indicate periplasm. Blue circles indicate nucleoside an nucleotide transporters. Green circles indicate sugar transporters. Orange circles indicate peptide and amino acid transporters. DA R H+ TR Uridine Cytidine Uracil UMP UDP UTP RNA (pyrimidine) CDP CTP dCDP dCTP DNA (pyrimidine) Deoxyuridine dUMP dTMP dTDP dTTP Guanosine Adenosine Guanine Adenine GMP/AMP GDP/ADP GTP/ATP Deoxyguannosine/ deoxyadenosine dGMP/dAMP dGDP/dADP dGTP/dATP RNA (purine) DNA (purine) DNA/RNA interconversitons Fig. 4 Putative transporters, enzymes and pathways of ‘Candidatus Uab amorphum’. Arrows indicate pathways that are present. Pathways that are absent are crossed out. Purple enzymes are predicted to be secreted in PV or localized at the outer membrane and involve to degradation of prey. Green line indicates the cytoplasmic membrane. Grey line indicates the outer membrane. Brown layers indicate periplasm. Blue circles indicate nucleoside and nucleotide transporters. Green circles indicate sugar transporters. Orange circles indicate peptide and amino acid transporters. pathway and T2SS in the ‘Ca. Uab amorphum’ genome (Supplementary Table 4). We speculate that these secretion systems may be used to secrete digestive enzymes into the PV. Other genes, encoding putative transporters of amino acids, nucleosides and sugars (Fig. 4), might be involved in the utilization of prey components. narrow ducts that appeared to be connected to the outside of the cell, or to another PV (Supplementary Fig. 8a, b). However, serial sectional observation showed that some PVs apparently lacked such ducts. It is therefore possible that ducts may disappear over time, and that PVs may eventually close (Supplementary Fig. 8c–k). De novo phospholipid synthesis may be important for formation and repair of PV membranes in ‘Ca. Uab amorphum’. Phosphatidylethanolamine (PE) is one of the main phospholipids in bacterial membranes and is synthesized from phosphatidyl- serine (PS) by phosphatidylserine decarboxylases (PSD) in bacteria26. In addition to a PSD gene candidate, we found that ‘Ca. Uab amorphum’ has two genes potentially encoding ethanolamine phosphotransferase (EPT1; UABAM_02218 and UABAM_03998), which is also found in a few myxobacteria and eukaryotes (Supplementary Fig. 10a). EPT1 synthesizes PE from 1,2-diacyl-sn-glycerol (DAG), which is the degradation product of PE and phosphatidylcholine27. The ‘Ca. Uab amorphum’ genome includes genes encoding putative digestive enzymes, which might degrade prey compo- nents. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 Glycogen Protein DNA Peptidoglycan Phospholipid AMY Malltose CPA DPP Peptid,amino acids 1,2-Diacyl-sn-glycerol PLC LYTC Stem peptides Amino sugars Nucleotide Prey ENDA T2SS Sec/tat Enzymes ATP Phagosome-like vacuole OM PG IM OM IM Nucleoside 5Nt? Flipping Fumarate/malate/ succinate/citric acid RNA RNase (prey) Pi antimicrobial Ala/gly Na+ AGCS Solute H+ APC Gly/Betaine BCCT Organic acid Na+ BASS Na+ CaCA Ca2+ H+ Metal CDF Nucleoside H+ or Na+ CNT CPA1 Na+ H + Amino acid H+ or Na+ DAACS Fumarate/malate/ succinate Na+ DAAS Dcu Asp/fumarate/malate/succinate Asp/fumarate/ malate/succinate DcuC Glu Na+ ESS Gluconate H+ GntP Sugar H+ or Na+ GPH ? MFS Na+ Antimicrobial MATE Nucleobase H+ NCS2 NhaC Na+ H+ Solute Na+ NSS Oligopeptide H+ OPT Pi Na+ PNaS RhtB Amino acid H+ H+ Antimicrobial RND Solute Na+ SSS SO4 2– H+ SulP Glu H+ TRAP-T K+ H+ Trk FeoB ATP Fe2+ MgtE Mg2+/Co2+ ATP Maltose Maltose transporter Na+ K+ ATP Sodium pomp Nucleobase Nucleoside H+ or Na+ EPT1 T2SS Sec/tat Enzymes ATP 1,2-Diacyl-sn-glycerol Triacylglycerol Fatty acid Phosphatidyl -ethanolamine Asp Asn Glu Gln L-Glutamyl-P L-Glutamate 5-semialdehyde Pro Pyruvate Ala Sugar Glycolysis TCA cycle Oxaloacetate 2-Oxoglutarate Uridine Cytidine Uracil UMP UDP UTP RNA (pyrimidine) CDP CTP dCDP dCTP DNA (pyrimidine) Deoxyuridine dUMP dTMP dTDP dTTP Guanosine Adenosine Guanine Adenine GMP/AMP GDP/ADP GTP/ATP Deoxyguannosine/ deoxyadenosine dGMP/dAMP dGDP/dADP dGTP/dATP RNA (purine) DNA (purine) DNA/RNA biosynthesis DNA/RNA interconversitons Arg His Ile Leu Lys Phe Trp Tyr Val Essential amino acids Amino acid biosynthesis Amino acid Proteins Met Ser Gly Thr Cys ala/gly Na+ AGCS Solute H+ APC ATP Gly/betaine BCCT Organic acid Na+ BASS Na+ CaCA Ca2+ H+ Metal CDF Nucleoside H+ or Na+ CNT CPA1 Na+ H+ Amino acid H+ or Na+ DAACS Fumarate/malate/ succinate Na+ DAAS Dcu Asp/fumarate/ malate/succinate Asp/fumarate/ malate/succinate DcuC Glu Na+ ESS Gluconate H+ GntP Sugar H+ or Na+ GPH ? MFS Na+ Antimicrobial MATE Nucleobase H+ NCS2 NhaC Na+ H+ NSS Oligopeptide H+ OPT Pi Na+ PNaS RhtB Amino acid H+ H+ Antimicrobial RND Solute Na+ SSS SO4 2– H+ SulP Glu H+ TRAP-T K+ H+ Trk FeoB ATP Fe2+ MgtE Mg2+/Co2+ ATP Maltose Maltose transporter Na+ K+ ATP Sodium pomp Solute Na+ Triacylglycerol biosynthesis Fig. 4 Putative transporters, enzymes and pathways of ‘Candidatus Uab amorphum’. Arrows indicate pathways that are present. Pathways that are absent are crossed out. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 The most notable feature of ‘Ca. Uab amorphum’ is its ability to engulf other organisms (prey), including Gram-negative bacteria (Alteromonas macleodii and Escherichia coli), Gram-positive bacteria (Lactobacillus far- ciminis and Staphylococcus condimenti) and a picoeukaryotic alga (Bathycoccus prasinos) (Supplementary Table 8, Supplementary Fig. 7). However, it was not able to engulf a marine yeast Debaryomyces hansenii, possibly due to its relatively large cell size (ca. 5 μm in diameter, Supplementary Movie 3). Light and fluorescent microscopy showed that prey cells were taken up by surface invagination of the ‘Ca. Uab amorphum’ cell. In the presence of sodium azide (which inhibits the ATP hydrolase NATURE COMMUNICATIONS | (2019) 10:5529 | https://doi.org/10.1038/s41467-019-13499-2 | www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS | (2019) 10:5529 | https://doi.org/10.1038/s41467-019-13499-2 | www.nature.com/naturecommunications ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 HGT. We speculate that these genes might confer protection against prey antibiotics that are released into the PV. and cylindrical structures that contain linear fibres (Fig. 5a–d). Such conspicuous and large fibrous structures have not been reported in other bacteria as far as we know; we therefore propose that these structures may be involved in the specific locomotion and engulfment processes displayed by ‘Ca. Uab amorphum’. Dependence on phagotrophy can often result in starvation due to unstable food source. Therefore, certain predatory bacteria have developed specific resting forms to resist starvation23,29. However, we were unable to observe any ‘Ca. Uab amorphum’ resting forms via microscopy. As we previously mentioned, the ‘Ca. Uab amorphum’ genome includes a gene encoding an actin-like protein. Molecular phylogenetic analysis showed that this protein was distant from the bacterial actin homolog MreB and the Crenarchaeal actin, and formed a clade with eukaryotic actins, actin-related proteins (ARPs) 1–3, bacterial actin-related protein (BARP) from Haliangium ochraceum35, ActM from Microcystis aeruginosa36 and Asgard actins (Fig. 6). This analysis suggests that the Uab actin may not have been directly transferred from eukaryotes, but possibly derived from an Asgard or related archaeon. We investigated the presence of genes encoding actin-binding proteins (known to be involved in assembly, stabilisation and cross-link of actin filament37) and found two candidate proteins (UABAM_01722 and UABAM_04996) that are similar to Aip1 and Crn1, respectively (Supplementary Table 11). We used F- actin-binding phalloidin and an antibody specific for eukaryotic actin but failed to observe a signal on ‘Ca. Uab amorphum’ cells when examined by fluorescent microscopy. Therefore, it remains unclear whether the actin-like protein, or the putative actin- binding proteins, form fibrous structures and/or whether they are involved in the organism’s unique locomotion and engulfment abilities. ‘Ca. Uab amorphum’ possesses genes potentially encoding enzymes for the synthesis of triacylglycerol (TAG) from phospholipid via DAG (Supplementary Fig. 10b–d). Given that genes for this pathway were not detected in the genomes of non- phagotrophic relatives, we speculate that ‘Ca. Uab amorphum’ may use this metabolic pathway to store energy as TAG in order to counter starvation. Phagotrophy can drive degenerative evolution in metabolism. Our genomic analysis suggested that ‘Ca. Uab amorphum’ may be able to synthesize only six amino acids de novo (Fig. 4, Supplementary Data 2). ARTICLE The inability to synthesize some amino acids is found in animals, phagotrophic and parasitic protists and predatory bacteria that acquire nutrients from preys or hosts30,31. Interestingly, ‘Ca. Uab amorphum’ also lacked candidate genes for de novo purine and pyrimidine biosynthesis, but had genes for salvage pathways (Fig. 4, Supplementary Figs. 11 and 12). It is therefore possible that ‘Ca. Uab amorphum’ must acquire some amino acids and nucleotides from prey. The absence of de novo purine and pyrimidine biosynthesis pathways in some obligate parasites has previously been reported32,33. Fibrous structures and an actin-like protein. Actin is a eukaryote-specific protein that forms filaments by polymerization and plays crucial roles in cellular motility, cytoskeleton formation and phagocytosis34. TEM observation showed that ‘Ca. Uab amorphum’ seems to have at least four different types of fibrous structures. These include large striated fibres that occasionally reached 2 µm in length, small striated fibres, short bundled fibres NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 We detected 179 putative peptidases (Supplementary Data 1), and some digestive proteins that may be secreted or localized at the outer membrane (Fig. 4, Supplementary Table 9). Genes encoding α-amylase, carboxypeptidase and deoxyribonu- clease I may have been acquired via HGT from Bdellovibrionales or Myxococcales, which are known to digest bacteria outside of the cells22,23 (Supplementary Fig. 9a–c). In mammals, AOAH (one of the proteins that might have been obtained by HGT from eukaryotes, Supplementary Fig. 9d) degrades and inactivates lipopolysaccharides of the bacterial outer membrane24. Therefore, it is possible that the putative AOAH counterpart in ‘Ca. Uab amorphum’ could potentially be also involved in degradation of bacterial lipopolysaccharides. Metabolic adaptation to phagotrophy. The ‘Ca. Uab amorphum’ genome includes many genes similar to antibiotic-resistance genes (Supplementary Table 10). Planctomycetes are known to have β-lactamase;28 however, we found genes encoding putative drug efflux systems, most of which are potentially derived from In Gram-negative bacteria, the Sec/Tat pathway transports proteins from the cytoplasm to the periplasm, and the T2SS transports proteins from the periplasm to the outside of cells25. We found genes encoding most of the components of the Sec/Tat TURE COMMUNICATIONS | (2019) 10:5529 | https://doi.org/10.1038/s41467-019-13499-2 | www.nature.com/naturecommunications 5 Discussion I hi Discussion In this study, we describe the bacterium ‘Candidatus Uab amorphum’, which was discovered from a surface seawater sample collected at the Republic of Palau. Molecular phylogenetic analyses indicate that the organism belongs to the phylum Planctomycetes and is closely related to anammox bacteria. Planctomycetes are known to have unusual eukaryote-like fea- tures such as budding cell division, sterol synthesis and uptake of macromolecules via endocytosis-like behaviour. The endocytosis- like uptake was reported in Gemmata obscuriglobus as an ATP- dependent, membrane-involving process with similarities to eukaryotic endocytosis;38 however, a recent study reported that the macromolecules are accumulated in the periplasm13. Species of anammox bacteria have a unique membrane-bounded orga- nelle called the anammoxosome that is involved in ammonium oxidation39. ‘Ca. Uab amorphum’ presents other eukaryotic-like features, such as a large and flexible cell, amoeba-like locomotion and uptake of other organisms by phagocytosis-like engulfment. In contrast to the endocytosis-like process previously reported for G. obscuriglobus, the phagocytosis-like engulfment observed in ‘Ca. Uab amorphum’ appears to involve invagination of both outer and inner membrane, and the resulting PV seems to be surrounded by a double membrane. In G. obscuriglobus, macro- molecules are accumulated in the cytoplasm or periplasm13,38, while in ‘Ca. Uab amorphum’ the AcGFP1 signals of engulfed E. coli cells appear to be compartmentalized in PVs (Fig. 1j–o, Supplementary Movie 4). b c d a Fig. 5 Fibrous structures in ‘Candidatus Uab amorphum’. a Large striated fibre. b Small striated fibre. c Short bundled fibre. d Cylindrical structures containing a linear fibre. Scale bars, 200 nm. a c c d Our microscopy studies show that ‘Ca. Uab amorphum’ can engulf various bacteria and picoeukaryotes, and appears to digest the engulfed organisms in specific compartments. The genomic analyses suggest that the organism may be able to utilize prey components such as sugar, amino acids and nucleotides from their prey. Therefore, we propose that engulfment by ‘Ca. Uab amorphum’ may serve a phagocytosis-like function. Fig. 5 Fibrous structures in ‘Candidatus Uab amorphum’. a Large striated fibre. b Small striated fibre. c Short bundled fibre. d Cylindrical structures containing a linear fibre. Scale bars, 200 nm. Eukaryotic phagocytosis is regulated by many eukaryote- specific genes that are involved in recognition of prey, signal transduction, rearrangement of cytoskeleton, membrane NATURE COMMUNICATIONS | (2019) 10:5529 | https://doi.org/10.1038/s41467-019-13499-2 | www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 OLS26637.1_HeimC3_07390 Lokiarch_09100 OLS26636.1_HeimC3_07380 Lokiarch_41030 OLS22914.1_HeimC3_28570 OLS12485.1_RBG13Loki_3906 OLS26907.1_HeimC3_06640 OLS21836.1_HeimC3_33580 31/0.98 100/1 25/0.7807 100/1 99/1 81/0.97 97/1 98/1 100/1 72/0.9999 100/1 50/0.9655 99/1 15/0.6279 100/1 13/0.9249 41/0.8769 38/0.742 88/0.9998 76/0.9985 61/0.8167 100/1 29/0.8666 Arp3 Conserved ‘lokiactin’ Arp1 Arp2 Actin Crenarcheal actin MreB MamK OLS15788.1_RBG13Loki_0585 ParM AlfA Diverse ‘lokiactin’ UABAM_01738 ‘Ca. Uab amorphum’ UABAM_01571 ‘Ca. Uab amorphum’ 99/1 59/0.9396 0.5 ActM Microcystis aeruginosa BARP Haliangium ochraceum Actin Fig. 6 Maximum likelihood tree of actin-like proteins. Left and right values on nodes indicate bootstrap value and Bayesian posterior probability, respectively. Bacterial actin-related protein (BARP) of Haliangium ochraceum and ActM of Microcystis aeruginosa form a clade with eukaryotic actin. Scale bar indicates amino acid substitution rate per site. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 ARTICLE Arp1 29/0.8666 Conserved ‘lokiactin’ 31/0.98 AlfA Fig. 6 Maximum likelihood tree of actin-like proteins. Left and right values on nodes indicate bootstrap value and Bayesian posterior probability, respectively. Bacterial actin-related protein (BARP) of Haliangium ochraceum and ActM of Microcystis aeruginosa form a clade with eukaryotic actin. Scale bar indicates amino acid substitution rate per site. the absence of an energy-producing symbiont. Therefore, pri- mitive phagocytosis may have originated in a mitochondrion-less eukaryote ancestor, with mitochondrion acquisition by phago- cytosis being a later event. remodelling and phagosome maturation40. ‘Ca. Uab amorphum’ possesses an actin-like gene that might be derived from an Asgard archaeon or a relative. However, we did not identify any other genes with homology to eukaryotic genes involved in phagocy- tosis. In addition, the structure of the ‘Ca. Uab amorphum’ PV seems to be different from that of the eukaryotic phagosome. Therefore, the phagocytosis-like process observed in ‘Ca. Uab amorphum’ is probably not homologous to eukaryotic phagocy- tosis, despite apparent morphological and functional similarities. The endokaryotic hypothesis proposes that the origin of an ancestral eukaryote involved a Gram-negative bacterial host engulfing an archaeon, accounting for origin of the nucleus as an engulfed cell41. ‘Ca. Uab amorphum’ is unlikely to be a descen- dant of such Gram-negative bacterial host, but it may be a good bacterial model to test this hypothesis. Although it has been suggested that phagocytosis without mitochondria is implausible due to the energetic cost of phagocytic functions42,43, our study indicates that prokaryotes can take up other microorganisms in remodelling and phagosome maturation40. ‘Ca. Uab amorphum’ possesses an actin-like gene that might be derived from an Asgard archaeon or a relative. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 However, we did not identify any other genes with homology to eukaryotic genes involved in phagocy- tosis. In addition, the structure of the ‘Ca. Uab amorphum’ PV seems to be different from that of the eukaryotic phagosome. Therefore, the phagocytosis-like process observed in ‘Ca. Uab amorphum’ is probably not homologous to eukaryotic phagocy- tosis, despite apparent morphological and functional similarities. The endokaryotic hypothesis proposes that the origin of an ancestral eukaryote involved a Gram-negative bacterial host engulfing an archaeon, accounting for origin of the nucleus as an engulfed cell41. ‘Ca. Uab amorphum’ is unlikely to be a descen- dant of such Gram-negative bacterial host, but it may be a good bacterial model to test this hypothesis. Although it has been suggested that phagocytosis without mitochondria is implausible due to the energetic cost of phagocytic functions42,43, our study indicates that prokaryotes can take up other microorganisms in Our analysis of environmental 16S rRNA gene data indicates that many uncultured lineages detected in various environments belong to the ‘Ca. Uab amorphum’ clade and have high genetic diversity. Therefore, it is tempting to speculate that some of these uncultured organisms may display other unusual phenotypes, may be acting as bacterial predators in marine, freshwater and terrestrial microbial ecosystems. Culture establishment and comparative analysis of Uab lineages will help to unveil their ecological importance and the evolution of eukaryotic-like fea- tures, and may provide insights about the origin of eukaryotes. NATURE COMMUNICATIONS | (2019) 10:5529 | https://doi.org/10.1038/s41467-019-13499-2 | www.nature.com/naturecommunicatio ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 containing 0.1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 10 min in the dark. Coverslips were washed with distilled water, air-dried, mounted with Slow- Fade Diamond (Invitrogen, Carlsbad, CA) and sealed with nail polish. Specimens were then observed using a Leica DMRD microscope (Leica, Wetzlar, Germany) equipped with an Olympus DP73 CCD camera (Olympus, Tokyo, Japan). AcGFP1-labelled Escherichia coli was prepared by transformation using TOP10- competent cells (Thermo Fisher Scientific, MA, USA) and pAcGFP1 Vector (Takara, Tokyo, Japan). Cells of AcGFP1-labelled E. coli were added to glass- bottomed dishes together with ‘Ca. Uab amorphum’ and immediately observed under Nikon A1 confocal microscope (Nikon, Tokyo, Japan). For detection of acidic compartments in ‘Ca. Uab amorphum’ cells, ‘Ca. Uab amorphum’ and AcGFP1-labelled E. coli in glass-bottomed dishes were incubated with 1 μM L−1 LysoTracker Red DND-99 (Thermo Fisher Scientific) in the dark for 1 h. Cells were washed three times by ESM medium and observed under Nikon A1 confocal microscope. For detection of reactive oxygen species, ‘Ca. Uab amorphum’ in glass- bottomed dishes was incubated with 1 μM L−1 LysoTracker Red DND-99 and 1 mM L−1 DCFH-DA (OxiSelect Intracellular ROS Assay Kit, Cell Biolabs, CA, USA) in the dark for 1 h. Cells were washed three times by ESM medium and observed under Nikon A1 confocal microscope. Sample collection and culture establishment. Surface seawater was collected in the Republic of Palau (longitude = 7.181386° N, latitude = 134.336947° E) on 27 October 2015. Approximately 500 mL of the seawater sample was filtered through 5 µm Isopore membrane filters (Millipore, Billerica, USA), and was subsequently concentrated to approximately 10 mL using 0.6 µm Isopore membrane filters (Millipore, Billerica, USA). The concentrated sample was serially diluted across 12 wells by using a 96-well culture plate filled with ESM medium44. Samples were incubated at 24 °C in the dark for 10 days. ‘Candidatus Uab amorphum’ and other bacterial cells were observed in several wells under light microscope following the incubation period. Single ‘Ca. Uab amorphum’ cells were isolated from the incu- bated sample with a micropipette, and were transferred into a new 96-well cell culture plate filled with the ESM medium. A small amount of the incubated sample was also added in the new 96-well cell culture plate together with isolated ‘Ca. Uab amorphum’ cell in order to inoculate bacteria as food sources. As a result, a xenic culture that consists of clonal ‘Ca. ARTICLE Resin replacement was performed with a 1:1 mixture of acetone and Agar Low Viscosity Resin R1078 (Agar Scientific Ltd., Stansted, England) for 30 min, and then with Low Viscosity Resin R1078 for 2 h. Resin was polymerized at 60 °C for 12 h. Ultra-thin sections were prepared on a Reichert Ultracut S ultramicrotome (Leica, Vienna, Austria). Cell sections were double-stained with 2% (w/v) uranyl acetate and lead citrate, and were observed using a Hitachi H-7650 electron microscope (Hitachi High-Technologies Corp., Tokyo, Japan) equipped with a Veleta TEM CCD camera (Olympus, Tokyo, Japan). DNA extraction, PCR and sequencing. Cells of ‘Ca. Uab amorphum’ and uni- dentified prey bacteria in the xenic culture were collected by centrifugation. Total DNA was extracted using the DNeasy plant mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. PCR was performed using primer pairs specific for the bacterial 16S rRNA gene (27F and 1492R)45,46. The PCR cycles (30 cycles) consisted of denaturation at 96 °C for 10 s, annealing at 55 °C for 30 min and extension at 68 °C for 2 min. The amplicon was purified with the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany), and was then cloned into the p-GEM T-easy vector (Promega, Tokyo, Japan). Ten clones were completely sequenced with a 3130 Genetic Analyser (Applied Biosystems, Foster City, CA), using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems). Sequenced clones were assembled into six sequences by the CodonCode Aligner (CodonCode Co., Centerville, MA) with a sequence similarity threshold of 99%. The 16-S rRNA gene sequence of ‘Ca. Uab amorphum’ was deposited in the GenBank database with accession code LC496071. Molecular phylogenetic analyses using 16S rRNA gene. We prepared a dataset containing 16S rRNA gene sequences of major bacterial lineages and ‘Ca. Uab amorphum’. The sequences were first automatically aligned in MAFFT v7.27349 with the G-INS-i algorithm at default settings, and were then manually edited with SeaView version 4.650. Ambiguous regions in the alignment were trimmed with SeaView. The final alignment consisted of 71 OTUs and 1307 sites. The best substitution model was searched for using jModelTest 2.1.1051, and the GTR + Γ + I model was selected. A maximum likelihood (ML) tree was heuristically searched for using RAxML version 8.1.1552 under the GTR + Γ + I model. Tree searches began with 20 randomized maximum-parsimony trees, and the highest log likelihood (lnL) was selected as the ML tree. ARTICLE Uab amorphum’ and unidentified bacteria was established. The xenic culture was maintained in a culture flask or a culture plate in ESM medium at 20 °C in the dark, and was subcultured into A new medium every month. containing 0.1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 10 min in the dark. Coverslips were washed with distilled water, air-dried, mounted with Slow- Fade Diamond (Invitrogen, Carlsbad, CA) and sealed with nail polish. Specimens were then observed using a Leica DMRD microscope (Leica, Wetzlar, Germany) equipped with an Olympus DP73 CCD camera (Olympus, Tokyo, Japan). AcGFP1-labelled Escherichia coli was prepared by transformation using TOP10- competent cells (Thermo Fisher Scientific, MA, USA) and pAcGFP1 Vector (Takara, Tokyo, Japan). Cells of AcGFP1-labelled E. coli were added to glass- bottomed dishes together with ‘Ca. Uab amorphum’ and immediately observed under Nikon A1 confocal microscope (Nikon, Tokyo, Japan). For detection of acidic compartments in ‘Ca. Uab amorphum’ cells, ‘Ca. Uab amorphum’ and AcGFP1-labelled E. coli in glass-bottomed dishes were incubated with 1 μM L−1 LysoTracker Red DND-99 (Thermo Fisher Scientific) in the dark for 1 h. Cells were washed three times by ESM medium and observed under Nikon A1 confocal microscope. For detection of reactive oxygen species, ‘Ca. Uab amorphum’ in glass- bottomed dishes was incubated with 1 μM L−1 LysoTracker Red DND-99 and 1 mM L−1 DCFH-DA (OxiSelect Intracellular ROS Assay Kit, Cell Biolabs, CA, USA) in the dark for 1 h. Cells were washed three times by ESM medium and observed under Nikon A1 confocal microscope. y To establish a monoxenic culture of ‘Ca. Uab amorphum’, Alteromonas macleodii (NBRC 102226), which was obtained from NITE Biological Resource Centre (NBRC), was inoculated into a 96-well culture plate filled with the ESM medium. A single cell of ‘Ca. Uab amorphum’ isolated by the micropipette was added into the culture plate. The monoxenic culture of ‘Ca. Uab amorphum’ was maintained in the same condition as the xenic culture. No antibodies were used during the culture establishment. The monoxenic culture was deposited at the Japan Collection of Microorganisms (JCM) as JCM 39082. Electron microscopy. For scanning electron microscopy, cells of ‘Ca. Uab amor- phum’ were cultured on 8.5-mm-diameter glass SEM plates (Okenshoji Co., Tokyo, Japan) treated with 0.1% (w/v) poly L-lysine (Sigma Chemical Co., St. Louis, MO) for one week. ARTICLE A nonparametric bootstrap analysis with 1000 replicates was conducted using RAxML under the GTR + Γ + I model. A Bayesian analysis was run using MrBayes 3.2.253 with the GTR + Γ + I model for each dataset. One cold and three heated Markov chain Monte Carlo chains with default temperatures were run for 5 × 106 generations; lnL values and trees were sampled at 100-generation intervals. The first 2.2 × 106 generations with an average standard deviation of split frequencies (ASDSF) greater than 0.01 were discarded as “burnin”. Bayesian posterior probabilities (BPP) and branch lengths were calcu- lated from the remaining trees. Fluorescent microscopy. Fluorescein isothiocyanate (FITC)-labelled oligonu- cleotide probes EUB338 (ref. 47) and PLA886 (ref. 48) were purchased from FASMAC Co., Ltd. (Kanagawa, Japan). EUB338 completely matched all sequences derived from the xenic culture, while PLA886 matched only to the 16S rRNA gene sequence of ‘Ca. Uab amorphum’, and had at least three mismatches to other sequences (Supplementary Fig. 2) The xenic culture of ‘Ca. Uab amorphum’ was cultured on coverslips that were treated with 0.1% (w/v) poly L-lysine (Sigma Chemical Co., St. Louis, MO) for one week prior to observation. Fixation and hybridization were performed as described in a previous study47. Cells grown on coverslips were fixed with 4% (w/v) paraformaldehyde for 16 h at 4 °C, and were then treated with a series of 50%, 80% and 96% ethanol for 3 min each and air- dried. Hybridization buffer containing 0.9 M NaCl, 20 mM Tris/HCl (pH 8.0), 0.01% (w/v) SDS, 5 ng/μL probe and a specific amount of formamide (Supple- mentary Table 12) were mounted on coverslips and incubated at 46 °C for 1.5 h in humid chambers. In the case of PLA886, an equimolar amount of a competitor oligonucleotide (cPLA886) was added to the hybridization buffer to avoid binding to non-target bacteria. Coverslips were washed in pre-heated (48 °C) washing buffer containing 0.9 M NaCl, 20 mM Tris/HCl (pH 8.0) and 0.01% (w/v) SDS, and were then placed in the washing buffer for 20 min at 48 °C. They were then washed with distilled water and air-dried. Each coverslip was incubated with PBS g In order to conduct molecular phylogenetic analysis of environmental sequences related to ‘Ca. Uab amorphum’, we prepared a 16 S rRNA gene dataset including major subgroups of Planctomycetes, which were selected based on a previous phylogenetic study54. ARTICLE Cells were pre-fixed with vapour of 4% (w/v) osmium tetroxide (OsO4) for 30 min, and were subsequently post-fixed with 1% (w/v) osmium tetroxide in culture medium for 2 h. Fixed cells were dehydrated in a series of 15–100% (v/v) ethanol. After dehydration, specimens were placed once in a 1:1 mixture of 100% ethanol and t-butyl alcohol, and then twice in 100% t-butyl alcohol, and chilled in the freezer. The specimens were freeze-dried using a VFD- 21S (SHINKU-DEVICE, Ibaraki, Japan) freeze drier, and were then mounted on aluminium stubs using carbon paste. Specimens were sputter-coated with platinum–palladium using a Hitachi E-102 sputter-coating unit (Hitachi High- Technologies Corp., Tokyo, Japan), and were observed using a JSM-6360F field emission SEM (JEOL, Tokyo, Japan). Light and time-lapse microscopic observation. Cells of ‘Ca. Uab amorphum’ and unidentified prey bacteria in the xenic culture were inoculated into glass-bottomed dishes filled with the ESM medium. Cells were incubated for 1–5 days prior to observation. Light micrographs and time-lapse videos were taken using an Olympus IX71 inverted microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP73 CCD camera (Olympus, Tokyo, Japan). Sodium azide (76.9 mM) was added to the xenic culture 15 min before observation. For the feeding experiment, cells of Debaryomyces hansenii JCM 1439, Lactobacillus farciminis JCM 1097 and Staphylococcus condimenti JCM 6074 (obtained from JCM) were added to glass-bottomed dishes together with ‘Ca. Uab amorphum’ and observed immediately under light microscope. Cells of Bathycoccus prasinos NIES-2670 (obtained from the National Institute for Environmental Studies, NIES) were added to glass-bottomed dishes together with ‘Ca. Uab amorphum’, and were incubated for one day prior to light microscopic observation and specimen preparation for transmission electron microscopic (TEM) observation. y p For TEM observation, cells were collected by centrifugation, and were pre-fixed with a mixture containing 1% glutaraldehyde, 0.1 M cacodylate buffer and 0.25 M sucrose for 1 h. Pelleted cells were washed twice with 0.2 M sodium cacodylate buffer (pH 7.2), and were post-fixed with a mixture containing 1% osmium tetroxide and 0.2 M cacodylate buffer. Fixed cells were dehydrated in a series of 30–100% (v/v) ethanol. After dehydration, cells were placed in a 1:1 mixture of 100% ethanol and acetone for 10 min, and 100% acetone for 10 min two times each. Methods Data reporting. No statistical calculations were performed to pre-determine the sample size. TURE COMMUNICATIONS | (2019) 10:5529 | https://doi.org/10.1038/s41467-019-13499-2 | www.nature.com/naturecommunications 7 ARTICLE Periplasm (or paryphoplasm) highly invaginates into the cytoplasm (or pirellulosome) and shows a reticulate pattern (Fig. 3a–c). The cytoplasm includes multiple nuclear bodies (Fig. 3a, c). The cell interior includes four types of fibrous structures: large striated fibres, small striated fibres, short bundle fibres and cylindrical structures that contain linear fibres (Fig. 5). Cells can engulf bacteria and picoeukaryotes (Bathycoccus prasinos) (Fig. 1d–o, Fig. 3d–h, Supplementary Fig. 7, Supplementary Movies 2 and 4). Engulfed bacteria and algae can be found in phagosome-like vacuoles (PVs) (Fig. 2f–h, Supplementary Fig. 7s–t). Some PVs connect to other phagosome-like structures or outside of the cell by narrow ducts (Supplementary Fig. 8a, b). Similarity of the 16 S rRNA gene sequence (LC496071) to that of the closest species (the planctomycete ‘Candidatus Kuenenia stuttgartiensis’) is 79% (Supplementary Table 1). The genome of ‘Ca. Uab amorphum’ (AP019860) is circular and 9,503,110 bp in size. The G + C content of the genome is 39.4 mol%. A co-culture of ‘Ca. Uab amorphum’ has been deposited at the JCM as JCM 39082. Genome assembly and annotation. Raw long reads were error-corrected and assembled using Canu v1.4, and 49 contigs were acquired. To remove prey sequences, homology search was performed using BLASTn against the NCBI nr database; only the longest contig was derived from Ca. Uab amorphum (Supple- mentary Table 2), which was manually circularized. Any other sequences, e.g. plasmids, were not found in ‘Ca. Uab amorphum’. The contig was polished using raw long reads by Pbjelly 0.3.055 and Quiver 2.1.0 (Pacific Biosciences). Gene models were constructed using the DFAST web server56 with the Refseq database. Functional annotation was performed using the EggNOG mapper web server57. Molecular phylogenetic analysis using 171 proteins. Molecular phylogenetic analysis was performed using 171 highly conservative orthologues. The dataset was composed of 29 operation taxonomic units (OTU), which contained most available genomes from planctomycetes. Four species in Verrucomycrobia or Chlamydiae were used as an outgroup. The highly conserved orthologues were searched for by reciprocal blast best hit with the cut-off of coverage ≥30% and similarity ≥40%; 171 proteins (52,272 amino acids) were conserved among ≥90% of the OTU, and were used for the phylogenetic analysis. The amino acids were aligned using Mafft v7.221, and highly diversified regions were manually trimmed on MEGA 758. Model test was performed using IQ-TREE 1.4.359, following the Bayesian infor- mation criterion (BIC). ARTICLE The first 7 × 106 generations with an average standard deviation of split frequencies (ASDSF) value greater than 0.03 were discarded as “burnin”. Bayesian posterior probabilities (BPP) and branch lengths were calculated from the remaining trees. carboxypeptidase, 173 amino acid positions and 11 OTUs for DNase I and 154 amino acid positions and 10 OTUs for EPT1. ML trees were constructed using IQ- TREE 1.5.5 following the best-fit model, which was chosen in accordance with BIC (WAG + I + G4 for α-amylase, LG + G4 for actin, DGAT and DNase I, LG + I + G4 for AOAH and PLC, WAG + G4 for carboxypeptidase and mtZOA + I + G4 for EPT1) with 200 replicates of nonparametric bootstrap. A Bayesian analysis was run using MrBayes 3.2.6 with the LG + Γ model for the actin dataset. One cold and three heated Markov chain Monte Carlos with default temperatures were run for 1 × 107 generations; lnL values and trees were sampled at 100-generation intervals. The first 7 × 106 generations with an average standard deviation of split frequencies (ASDSF) value greater than 0.03 were discarded as “burnin”. Bayesian posterior probabilities (BPP) and branch lengths were calculated from the remaining trees. Description of ‘Candidatus Uab amorphum’. ‘Candidatus Uab amorphum’ (U.a.b masc. n. a giant of Palauan mythology. a.mor’phus. L. neut. adj. amorphum amorphous, deformed). Genome sequencing. For genome sequencing, total DNA was extracted from the monoxenic culture of ‘Ca. Uab amorphum’ that was cultivated until most prey bacteria were consumed. Long reads (157,852 reads, 1.4 Gb) were sequenced using PacBio RS II (Pacific Biosciences, Melon Park, CA) with one cell (P6–C3 chem- istry). Mean read length was 9.0 kbp. Sequencing was performed by Macrogen (Seoul, Korea). p ) Marine free-living aerobic Gram-negative bacterium was collected from surface seawater in the Republic of Palau (7.181386° N, 134.336947° E). Cells are flattened and round or oval shape with granular cytoplasm (Fig. 1a–c). Flagellum is absent. Cells were 3.2–7.8 μm (4.5 ± 0.85 μm) in the long axis and 2.8–5.5 μm (4.0 ± 0.57 μm) in the short axis (n = 79). When prey bacteria were abundant in the culture, cells occasionally reached 10 μm in diameter (Supplementary Fig. 1a). Cells attach to substrate and show gliding motility with changing shape (Supplementary Movie 1). Cells reproduce by binary fission. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 identity = 30 and coverage = 40. In this analysis, based on the phylogenetic relation- ships, “selfGroup” and “closeGroup” were set as ‘Candidatus Brocadiaceae’ (taxonomic ID: 1127830) and the PVC (Planctomycetes–Verrucomicrobia–Chlamydiae) group (taxonomic ID: 1783257), respectively. identity = 30 and coverage = 40. In this analysis, based on the phylogenetic relation- ships, “selfGroup” and “closeGroup” were set as ‘Candidatus Brocadiaceae’ (taxonomic ID: 1127830) and the PVC (Planctomycetes–Verrucomicrobia–Chlamydiae) group (taxonomic ID: 1783257), respectively. GTR + Γ model. We removed BLASTn-derived environmental sequences that did not form a clade with ‘Ca. Uab amorphum’ from the final dataset. Sequences in the final dataset were aligned and trimmed as shown above; the final alignment consisted of 100 OTUs and 1,351 sites. The best model was identified using jModelTest, and the GTR + Γ + I model was selected. The ML tree was heuristically searched for using RAxML under the GTR + Γ + I model. Tree searches began with 20 randomized maximum-parsimony trees, and the highest lnL was selected as the ML tree. A nonparametric bootstrap analysis with 1,000 replicates was conducted using RAxML under the GTR + Γ + I model. A Bayesian analysis was conducted using MrBayes with the GTR + Γ + I model for each dataset. One cold and three heated Markov chain Monte Carlo chains with default temperatures were run for 5 × 106 generations; lnL values and trees were sampled at 100-generation intervals. The first 1.0 × 106 generations with ASDSF greater than 0.01 were discarded as “burnin”. BPP and branch lengths were calculated from the remaining trees. In addition, we searched Uab homologues of the 347 eukaryote-specific proteins (ESPs)21 and actin-binding proteins37 in the Saccharomyces Genome Database65 by BLASTp with the cut-off: E-value < 1E–5. The Uab proteins with any hits were re- checked by BLASTp against the NCBI nr database. Molecular phylogenomic analyses of single proteins. For molecular phyloge- netic analyses of single proteins (α-amylase, actin, acyloxyacyl hydrolase (AOAH), phospholipase C (PLC), diacylglycerol acyltransferase (DGAT), carboxypeptidase, DNase I and EPT1), we screened each protein sequence by BLASTp, against the NCBI nr database and constructed the dataset. Datasets were aligned by MAFFT v7.273, and were then manually edited with SeaView version 4.6 or MEGA 7. ARTICLE Phylogenetic analysis was performed as described above using RAxML version 8.2.9. A nonparametric bootstrap analysis with 200 replicates under the LG + gamma + I model was performed. A Bayesian analysis was run using MrBayes 3.2.6 with the same model for 1.0 × 106 generations; lnL values and trees were sampled at 1000-generation intervals. The initial 25% generations with ASDSF values greater than 0.01 were discarded as “burnin”. BPP and branch lengths were calculated from the remaining trees. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. ARTICLE The final alignments consisted of 331 amino acid positions and 17 OTUs for α-amylase, 362 amino acid positions and 103 OTUs for actin, 534 amino acid positions and 28 OTUs for AOAH, 255 amino acid positions and 17 OTUs for PLC, 408 amino acid positions and 17 OTUs for DGAT, 206 amino acid positions and 6 OTUs for g We screened for sequences related to ‘Ca. Uab amorphum’ from Tara Ocean 16S rRNA gene sequences10 using BLASTn, with the ‘Ca. Uab amorphum’ 16S rRNA gene sequence and environmental Uab clade sequences as query. We added sequences of BLASTn hits with sequence similarities above 97% and sequence length over 100 bp to the dataset used in the environmental 16S rRNA gene sequence analysis. Sequences in the dataset were automatically aligned in MAFFT with the G-INS-i algorithm at default settings, and were then manually aligned and trimmed using SeaView. Preliminary maximum likelihood tree was constructed by RAxML with rapid bootstrap analysis (100 replicates) under a GTR + Γ model. We removed environmental sequences that did not form a clade with ‘Ca. Uab amorphum’ from the final dataset. Sequences in the final dataset were aligned and trimmed as shown above; the final alignment consisted of 189 OTUs and 1,509 sites. The ML tree was heuristically searched for using RAxML under the GTR + Γ + I model. Tree searches began with 20 randomized maximum- parsimony trees, and the highest lnL was selected as the ML tree. A nonparametric bootstrap analysis with 1,000 replicates was conducted using RAxML under the GTR + Γ + I model. carboxypeptidase, 173 amino acid positions and 11 OTUs for DNase I and 154 amino acid positions and 10 OTUs for EPT1. ML trees were constructed using IQ- TREE 1.5.5 following the best-fit model, which was chosen in accordance with BIC (WAG + I + G4 for α-amylase, LG + G4 for actin, DGAT and DNase I, LG + I + G4 for AOAH and PLC, WAG + G4 for carboxypeptidase and mtZOA + I + G4 for EPT1) with 200 replicates of nonparametric bootstrap. A Bayesian analysis was run using MrBayes 3.2.6 with the LG + Γ model for the actin dataset. One cold and three heated Markov chain Monte Carlos with default temperatures were run for 1 × 107 generations; lnL values and trees were sampled at 100-generation intervals. Data availability The 16S rRNA gene sequence of ‘Ca. Uab amorphum’ was deposited in DDBJ/GenBank/ ENA with the accession number LC496071. Genome assembly was deposited in DDBJ/ GenBank/ENA with accession number AP019860. Other relevant data are available in this article and its Supplementary Information files, or from the corresponding author upon request. Inference of digestive proteins in the FV. Subcellular localization of proteins was predicted using PSORTb 3.060, Cello v.2.561 and LocTree362 with an organism option: Gram-negative bacteria or bacteria. Proteins were categorized into “secreted” or “extracellular” by at least one of the three prediction programmes that were considered a candidate FV protein. Digestive proteins were identified using the KEGG database. Digestive proteins for DNA/RNA were searched for using GO terms (GO:0004518). Digestive proteins for peptidoglycan were searched for using the HyPe web server63. Received: 1 February 2019; Accepted: 8 November 2019; Received: 1 February 2019; Accepted: 8 November 2019; Received: 1 February 2019; Accepted: 8 November 2019; References 1. Lynch, M. & Conery, J. S. The origins of genome complexity. Science 302, 1401–1404 (2003). References 1. Lynch, M. & Conery, J. S. The origins of genome complexity. Science 302, 1401–1404 (2003). 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Acknowledgements We thank the Ministry of Natural Resources, Environment and Tourism, Palau for providing permits for collecting marine resources (permit number, RE-15-12). We thank Dr. Naoto Hanzawa at the Yamagata University for managing sampling trips. We thank Dr. Hirokazu Sakamoto for proposing the name “Uab”. This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers: 13J00587, 18J02091 and 19K22445. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. ARTICLE Planctomycetes do possess a peptidoglycan cell wall. Nat. Commun. 6, 7116 (2015). 58. Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016). 29. Sanchez-Amat, A. & Torrella, F. Formation of stable bdelloplasts as a starvation-survival strategy of marine bdellovibrios. Appl. Environ. Microbiol. 56, 2717–2725 (1990). 59. Nguyen, L. T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. 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BMC Genomics 17, 411 (2016). Supplementary information is available for this paper at https://doi.org/10.1038/s41467- 019-13499-2. 64. Zhu, Q., Kosoy, M. & Dittmar, K. HGTector: an automated method facilitating genome-wide discovery of putative horizontal gene transfers. BMC Genomics 15, 717 (2014). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13499-2 Competing interests © The Author(s) 2019 The authors declare no competing interests. 11 NATURE COMMUNICATIONS | (2019) 10:5529 | https://doi.org/10.1038/s41467-019-13499-2 | www.nature.com/naturecommunications
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2 Analytical Extension of Graph-Metric Space By following corollary from understanding Fermat’s Last Theorem [7]: g Last Theorem [7]: Let an + bn = cn, a = a1 · i1 + a2 · i2 + · · · + ap · ip, b = b1 · j1 + b2 · j2 + · · · + bq · jq, c = c1 · k1 + c2 · k2 + · · · + cr · kr (a∀x, b∀x, c∀x, p, q, r ∈N+), (i∀x, j∀x, k∀x ∈C) Let ⃗va = (a1, a2, · · · , ap) , ⃗vi = (i1, i2, · · · , ip) , ⃗vb = (b1, b2, · · · , bq) , ⃗vj = (j1, j2, · · · , iq) , ⃗vc = (c1, c2, · · · , cr) , ⃗ck = (k1, k2, · · · , kr) ⇒(⃗va · ⃗vi)n + (⃗vb · ⃗vj)n = (⃗vc · ⃗vk)n ⇒[In size of matrix] ((1 × p) · (p × 1))n + ((1 × q) · (q × 1))n = ((1 × r) · (r × 1))n Let ⃗va = (a1, a2, · · · , ap) , ⃗vi = (i1, i2, · · · , ip) , Let ⃗va = (a1, a2, · · · , ap) , ⃗vi = (i1, i2, · · · , ip) , ⃗vb = (b1, b2, · · · , bq) , ⃗vj = (j1, j2, · · · , iq) , ⃗vc = (c1, c2, · · · , cr) , ⃗ck = (k1, k2, · · · , kr) ⇒(⃗va · ⃗vi)n + (⃗vb · ⃗vj)n = (⃗vc · ⃗vk)n ⇒[In size of matrix] ((1 × p) · (p × 1))n + ((1 × q) · (q × 1))n = ((1 × r) · (r × 1))n ⃗vb = (b1, b2, · · · , bq) , ⃗vj = (j1, j2, · · · , iq) , vc = (c1, c2, · · · , cr) , ck = (k1, k2, · · · , kr) ⇒(va · vi)n + (vb · vj)n = (vc · vk)n ⇒[In size of matrix] ((1 × p) · (p × 1))n + ((1 × q) · (q × 1))n = ((1 × r) · (r × 1))n =⇒n(max p)+(max q) = rn (n ∈N+) ⇒n = 1 or 2 =⇒22 ≤a2 + b2 ≤2a2+b2 This implies a number in any complex field could be rearranged by graph conserving ordinal attribute. 2 Analytical Extension of Graph-Metric Space As atomic energy consists itself by a countable form with keeping their topological boundary, we could apply this implication to the atom which is (would be) a form of quantized energy. 1 Introduction In a perspective of matrix-graph relation, scalar could be represented as two row vector v1, v2 producting as v1 · vT 2 . In sense of keeping ordinal of each particle being countable, this graph-matrix relation could be interpreted in an interaction of particle as a form of energy. Abstract By extending metric space to expression in hash of directed graph, spatiotemporal observation could be described in matrix quantized state. Jihyeon Yoon(Freelancer Programmer, flyingtext@nate.com) December 13, 2022 Descriptive Method in Observation and Its Application Jihyeon Yoon(Freelancer Programmer, flyingtext@nate.com) 2 Analytical Extension of Graph-Metric Space While the idea of contraction in metric space gives a chance of graph being non-loss hashable in format of scalar[3], matrix gives a relation in graph, conserving its ordinal attribute. 4 Extended usage of matrix If scalar could be extended in an expression of vector and matrix as a expression of basis of span, matrix that represents energy itself gains duality in set of construction. In ordinal perspective by given contraction as span ⃗v, interaction itself could be contained in an regularity in given quantized energy amount as E. In cardinal perspective by quantized energy amount as E, next interaction decided by given contraction amount could be spread out. Consequently, by given equation to calculate Total Energy Observed TEO [4]: y q gy , y g Consequently, by given equation to calculate Total Energy Observed TEO [ TEOAO,E = N(AO) X n LFO(AO, OGn, F) · TEOAO,E · PODAO,OGn · (1 −OE(E))(1 −ME(E))(1 −PE(E)) Optimal t∗ wanted = arg max t TEOAO,E Optimal t∗ actual = arg max t (TEOAO,E −CRt + SRt) (CRt > 0, SRt > 0) TEOAO,E = N(AO) X n LFO(AO, OGn, F) · TEOAO,E · PODAO,OGn Optimal t∗ wanted = arg max t TEOAO,E Optimal t∗ actual = arg max t (TEOAO,E −CRt + SRt) (CRt > 0, SRt > 0) ∃E ⇔Optimal t∗ actual = arg max t (TEOAO,E −CRt + SRt) ≈0 ∃E ⇔Optimal t∗ actual = arg max t (TEOAO,E −CRt + SRt) ≈0 ∃E ⇔Optimal t∗ actual = arg max t (TEOAO,E −CRt + SRt) ≈0 =⇒TEO = ∀t X (∥⃗vt∥· TEOAO,E · PODAO,OGn =⇒TEO = ∀t X t (∥⃗vt∥· TEOAO,E · PODAO,OGn · (1 −OE(E))(1 −ME(E))(1 −PE(E)) + SRt −CRt) ⇒ ∀t X t (SRt −CRt SRt · POt · (1 −OE(E))(1 −ME(E))(1 −PE(E)) + SRt −CRt) ⇒ ∀t X t (SRt −CRt SRt · POt · (1 −OE(E))(1 −ME(E))(1 −PE(E)) + SRt −CRt) If limiting ⃗vt three-dimension: If limiting ⃗vt three-dimension: If limiting ⃗vt three-dimension: If limiting ⃗vt three-dimension: g ⃗vt three-dimension: (Decomposing at period t) SR −CR SR = Z  ∇· ∆SR −∆CR SR  dt While calculating each axis of ∇in assumed three vector: While calculating each axis of ∇in assumed three vector: In R3, Z  ∇· ∆SR −∆CR SR  dt = −→ SR −−→1 = −→1 −−→ SR = 1 2(cos 2θ + 1) Note that, this could be interpreted as a curvature in a space which could be another form in Einstein Field Equation. 3 Larger to Deep Field Observation If every state of observation could be quantized in ordinal, relation between observation of larger to deep field could be related within. Continuing from what has been discussed in understanding of observing reality [5]: Assuming Field ∃F at given period t, LFOt(∃F) = SRt −CRt SRt =⇒(Mostly) 0 ≤LFOt(∃F, R) ≤1 1 Note that, by preceding definition, field at period t is related mainly to convergence or divergence of spatiotem- poral when observation distance is far enough. What has been said to be ‘mostly’ is that there is a possibility of distraction or extension when considering additional complex plain. Again, like what said before, LFO could be also expressed into quantized graph by their each observation. no = n (Partial Observation ∀O at time t) LFO =            ⃗v1 ⃗v2 ⃗v3 · · · ⃗ vno            ⇒Partial Observation PO · LFO = Total Energy Observed TEO 4 Extended usage of matrix And this implies correlation in gravity also by: Note that, this could be interpreted as a curvature in a space which could be another form in Einstein Field Equation. And this implies correlation in gravity also by: |EGravity · EElectromagnetic| ≤cos θ |EGravity · EElectromagnetic| ≤cos θ 2 2 0 1 2 3 4 5 6 0 0.2 0.4 0.6 0.8 1 Figure 1: Effect of Einstein field equation in energy perspective Figure 1: Effect of Einstein field equation in energy perspective 5 Phase Transition of Energy-Matter ⇒f(ni) = ln(W) + α  N − X ni  + β  E − X niεi  ⇒(By Stirling′s approximation) ni = arg max ni f(ni) = gi eα+βεi + 1 ⇒(By Stirling′s approximation) ni = arg max ni f(ni) = gi eα+βεi + 1 where W is the number of ways that total of particles can be classified into energy levels according to their energies [2] with a fixed number of particle N and a fixed energy E. So, it would be natural to say that at least both Einstein field equation and conservation of CPT affect phase transition. If then, the model could be rearranged by maximal consideration within given concepts from Fermi-Dirac statistics. 5 Phase Transition of Energy-Matter The result from Einstein field equation affecting energy expression implies amount of energy that added as electromagnetic- gravitational energy causes not only spin of particle to be in specific boundary, but also boundary phase of energy that interacts with. As ordered in fermion spin, matter has distribution of Fermi-Dirac statistics. Assuming that, X i ¯ni = N ⇒¯n(εi) = gi¯ni = gi e(εi−µ)/kBT + 1 X i ¯n(εi) = E ⇒¯n(εi) = gi¯ni = gi e(εi−µ)/kBT + 1 where κB is the Boltzmann constant, T is the absolute temperature, εi is the energy of the single-particle state i, and µ is the total chemical potential [1]. And sum by all states of single-particle, the distribution normalizes. There are three known ways to explain ensembles resulting in phase state. Grand-canonical,canonical, micro- canonical each has their own relation with describing ensemble. Grand-canonical ensemble describes itself by the equation of its own by expectation in given energy within law of conservation. Canonical ensemble describes itself by chaining free energy given by the sum of other ensembles of energy states, which resembles with preceding discussion of Einstein field equation. Zi(N −ni) ≡ X(i) n1,n2,... e−β(n1ε1+n2ε2+··· ) ⇒¯ni = X n1,n2,... ni e−β(n1ε1+n2ε2+···+niεi+··· ) X n1,n2,... e−β(n1ε1+n2ε2+···+niεi+··· ) = 1 [Zi(N)/Zi(N −1)] eβεi + 1 = 1 e(εi−µ)/kBT + 1 ⇒eεi−µ =  1 ¯ni −1 κBT ! Zi(N −ni) ≡ X(i) n1,n2,... e−β(n1ε1+n2ε2+··· ) n1,n2,... ⇒¯ni = X n1,n2,... ni e−β(n1ε1+n2ε2+···+niεi+··· ) X n1,n2,... e−β(n1ε1+n2ε2+···+niεi+··· ) = 1 [Zi(N)/Zi(N −1)] eβεi + 1 = 1 e(εi−µ)/kBT + 1 ⇒eεi−µ =  1 ¯ni −1 κBT ! Micro-canonical ensemble describes itself by Lagrange multipliers α, β [1]: Micro-canonical ensemble describes itself by Lagrange multipliers α, β [1]: 3 W = Y i w(ni, gi) = Y i gi! ni!(gi −ni)! ⇒f(ni) = ln(W) + α  N − X ni  + β  E − X niεi  ⇒(By Stirling′s approximation) ni = arg max ni f(ni) = gi eα+βεi + 1 W = Y i w(ni, gi) = Y i gi! ni!(gi −ni)! 6 Chain of Ensembles with Conservation of CPT By terms of LFO and conservation in SR and CR, assuming that canonical and micro-canonical ensemble has equal conclusion: (LFO Chain) ≡(CPT Conservation) =⇒Zi(N)/Zi(N −1) = e−µ/kBT =  1 ¯ni −1  · 1 eεi/κBT =⇒  1 ¯ni −1  · 1 eεi/κBT − 1 eµ/kBT + 1 = 1 =⇒  1 ¯ni −1  · 1 e(εi−µ)/κBT −1 + eµ/κBT = eµ/κBT =⇒  1 ¯ni −1  · 1 eεi/κBT + 1  · 1 e−µ/κBT −1 = eµ/κBT =⇒  1 ¯ni −1  · 1 eεi/κBT −1  · 1 e−µ/κBT −1 = −eµ/κBT While in CPT conservation table [6]: Optimal t With PE With ME With OE Original (Expectation) E Not specific Not specific Not specific Type 1 SR = CR In symmetry With T asymmetry Not specific Not specific Type 2 SR - CR >0 With P, T asymmetry With P, T asymmetry With P, T asymmetry Not specific Type 3 CR - SR >0 With C, P, T asymmetry With C, P, T asymmetry With C, P, T asymmetry With C, P, T asymmetry Table 1: Asymmetry-error relations Table 1: Asymmetry-error relations Table 1: Asymmetry-error relations 4 TEO = E = ET ype1 + ET ype2 + ET ype3 TEO = E = ET ype1 + ET ype2 + ET ype3 ⇒ET ype1 = LFO · TEO · (1 −PE(E)), ET ype2 = LFO · TEO · (1 −PE(E))(1 −ME(E)) + X SR − X CR, ET ype3 = LFO · TEO · (1 −PE(E))(1 −ME(E))(1 −OE(E)) + X SR − X CR ⇒E = LFO · TEO · ((1 −PE(E)) + (1 −PE(E))(1 −ME(E)) + (1 −PE(E))(1 −ME(E))(1 −OE(E))) Assuming that α = 1 −PE(E), β = 1 −ME(E), and γ = 1 −OE(E): LFO · (α + αβ + αβγ) = 1 α + β = 1 −γ LFO · (α + αβ + αβγ) = 1 α + β = 1 −γ And by definition of PE: 1 −γ = LFO =⇒(α + β)α(1 + β + βγ) = 1 ⇒1 + β + βγ = 1 (α + β)α ⇒1 = 1 (α + β)αβ −1 β −γ =⇒γ = 1 β  1 α(α + β) −1  −1 6 Chain of Ensembles with Conservation of CPT The two perspectives are each related with theory of relativity and CPT conservation. Then why does summation of series product in power of e corresponds to LFO? And why does maximized number of ways in combination of particle corresponds to conservation in SR and CR? References [1] Fermi–Dirac statistics, Oct. 2022. Page Version ID: 1117379295. [1] Fermi–Dirac statistics, Oct. 2022. Page Version ID: 1117379295. [2] Maxwell–Boltzmann statistics, Aug. 2022. Page Version ID: 1102787889. [2] Maxwell–Boltzmann statistics, Aug. 2022. Page Version ID: 1102787889. [3] E. Ameer, H. Aydi, M. Arshad, and M. De la Sen. Hybrid ´Ciri´c Type Graphic ,-Contraction Mappings with Applications to Electric Circuit and Fractional Differential Equations. Symmetry, 12(3):467, Mar. 2020. Number: 3 Publisher: Multidisciplinary Digital Publishing Institute. [3] E. Ameer, H. Aydi, M. Arshad, and M. De la Sen. Hybrid ´Ciri´c Type Graphic ,-Contraction Mappings with Applications to Electric Circuit and Fractional Differential Equations. Symmetry, 12(3):467, Mar. 2020. Number: 3 Publisher: Multidisciplinary Digital Publishing Institute. [4] J. Yoon. General Specification for Simulating Universe, Sept. 2022. [5] J. Yoon. Intuitive Understanding of Observing Reality: With Extension of the Game Theory, Aug. 2022. 6] J. Yoon. Nuclear Fusion Framework for Simulating Universe. Technical report, Zenodo, [7] J. Yoon. Understanding Fermat’s Last Theorem through Matrix-Graph Relation. Technical report, Zenodo, Dec. 2022. 5
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Soil Properties Responding to &lt;em&gt;Pinus tabulaeformis&lt;/em&gt; Forest Thinning in Mountainous Areas, Beijing
Advance journal of food science & technology/Advance journal of food science and technology
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p. Accepted: July 19, 2014 Accepted: July 19, 2014 Published: November 10, 2014 Submitted: June 11, 2014 Submitted: June 11, 2014 Accepted: July 19, 2014 Research Article Soil Properties Responding to Pinus tabulaeformis Forest Thinning in Mountainous Areas, Beijing Kuangji Zhao, Yirong Hao, Zhongkui Jia, Lvyi Ma and Fang Jia Beijing Forestry University, Silviculture and Conservation Key Laboratory of the Ministry of Education, Beijing 100083, China, Beijing Forestry Society, BFS, Beijing 100029, China Abstract: Studied soil physiochemical properties of Pinus tabulaeformis plantations under varying silviculture intensity in shade, slope and thick soil areas in mountain areas of Beijing. The results show that at intensive (50%), medium (45%), weak (33%) thinning intensity compared with the control, (1) soil bulk density was reduced by 7.9, 5.7 and 3.9%, respectively in depth from 0 to 10 cm (layer0-10) and soil properties in layer0-10 were better than in depth of 10-20 cm (layer10-20). (2) Total soil porosity increased by 7.3, 5.4 and 2.9%, respectively; the effects of thinning intensity on soil non-capillary porosity ranked as: weak>medium>intensive>control and the total soil porosity and non-capillary porosity both changed similarly in the two layers. (3) Water infiltration capacity and total soil pondage capacity were enhanced by thinning intensity. (4) Soil organic matter, total Nitrogen and rapidly- available Phosphorus were maximized in the medium thinning plot, while the rapidly-available Potassium was maximized in the intensive intensity plot, demonstrating that medium and intensive thinning can improve the P. tabulaeformis forest soil nutrient level. (5) The effects of thinning intensity on catalase and urease activities ranked as: medium>intensive>control>weak, which revealed that the soil enzyme activity can be improved by forest thinning. In conclusion, to improve soil physiochemical properties of P. tabulaeformis plantation in shade, slope and thick soil areas in mountain areas of Beijing, we prefer intensive thinning management. Keywords: Forest thinning, principal component analysis, soil enzyme activity, soil nutrient level, soil organic matter, soil physical properties Corresponding Author: Zhongkui Jia and Lvyi Ma, Beijing Forestry University, Silviculture and Conservation Key Laboratory of the Ministry of Education, Beijing 100083, China, Beijing Forestry Society, BFS, Beijing 100029, China Corresponding Author: Zhongkui Jia and Lvyi Ma, Beijing Forestry University, Silviculture and Conservation Key Laboratory of the Ministry of Education, Beijing 100083, China, Beijing Forestry Society, BFS, Beijing 100029, China This work is licensed under a Creative Commons Attribution 4.0 International License (URL: http://creativecommons.org/licenses/by/4.0/). 1219 Advance Journal of Food Science and Technology 6(11): 1219-1227, 2014 DOI:10.19026/ajfst.6.188 ISSN: 2042-4868; e-ISSN: 2042-4876 © 2014 Maxwell Scientific Publication Corp. Advance Journal of Food Science and Technology 6(11): 1219-1227, 2014 DOI:10.19026/ajfst.6.188 ISSN: 2042-4868; e-ISSN: 2042-4876 © 2014 Maxwell Scientific Publication Corp. Advance Journal of Food Science and Technology 6(11): 1219-1227, 2014 DOI:10.19026/ajfst.6.188 ISSN: 2042-4868; e-ISSN: 2042-4876 © 2014 Maxwell Scientific Publication Corp. ; © 2014 Maxwell Scientific Publication Corp. METHODOLOGY Setting of sample plots: In April, 2004, the P. tabulaeformis plantation on shady thick soil slope was chosen owing to its representativeness (0.04 hm2) for thinning operation. The target limbs to be cut off were mainly those with serious diseases and insect pests, or the overstocked trees and shrubs interfering with the growth and suitable density of P. tabulaeformis. Thinning intensity was divided into control (0%, C), weak (33%, W), medium (45%, M) and intensive (50%, I). Before the thinning operation, each sample plot was detailed surveyed in details, including breast height diameter of each trees, plot altitude, the position aspect and grade of slope, soil thickness and litter thickness. The statues of sample plot are shown in Table 1. Thinning disturbs surface soil including water, heat and other material redistribution. And soil properties will be changed corresponding to the method, time, intensity of thinning, forest types and other aspects (Li et al., 2003). Pinus tabulaeform is one of the main planting species in Beijing. The existing area is 79,000 hm2, accounting for 25% of the whole forest area in the city; the planted area is 61,000 hm2 with 86% of young and half-mature trees (Ma et al., 2005). These forests play an important role in improving the ecological environment of Beijing. Measurement of soil physical properties and moisture holding capacity: Soil samples were collected before thinning in 2004 and 5 years after thinning in April, 2009, from depth 0-10 cm (layer0-10) and 10-20 cm (layer10-20) by digging out soil profile along the diagonal of each plot, with three repetitions in each sample. The formulas are as follows: This study is focused on a five-year thinning program of P. tabulaeformise stands in mountainous areas in Beijing. We analyzed how soil physical properties changed before and after thinning. The results could help to manage and improve stand quality for young and immature stands in mountainous regions. In addition to potential stand quality improvements it could also allow for the study of protective effects of ecological public welfare forests. INTRODUCTION not significantly different from the control stand (Chi et al., 2006). Soil enzyme activities including catalase, urease, alkaline phosphatase, invertase and polyphenoloxidase were improved and enzyme activities in the A layer were more affected than those in the B and C, based on 5 years of Pinus tabulaeformis forest tending (Guo et al., 2007). The results of natural forest and Korean Pine (Pinus koraiensis) were slightly better than non-thinning area except Mongolian oak forest’s (Quecus mongolica) through studying soil properties of the natural forests under different thinning intensities in east Liaoning Province. The soil chemical properties were improved to different levels and weak thinning intensity stand contained the largest soil microbial quantity (Sun, 2007). The 18 year thinning program in pine forests indicated that with enhanced thinning intensity, the forest soil total porosity, capillary moisture holding capacity, soil nutrient level and enzyme activities were significantly enhanced. It demonstrates that forest thinning can improve the forest soil quality (Yu et al., 2008). Thinning significantly improved soil alkaline phosphatase activity and other Forest soil plays an important role in forest water conservation (Zheng et al., 1997). The effects of silviculture on forest stands, especially forest soils, concern many aspects. Thinning forests will interfere with in different ways and the final conclusions regarding the effects of the thinning are inconsistent among existing studies (Li et al., 2003). For instance, thinning significantly affected soil fertility and increased the contents of soil micro-organisms and soil nutrient (Hu and Zhu, 1999). Soil microbial quantity, enzyme activity, total porosity, available nutrients and soil fertility were improved and soil bulk density were all reduced, after 2 year thinning in Cunninghamia Lanceolata, Pinus massoniana, Fokienia hodginsii, Cryptomeria fortunei and Schima superba (Zhang et al., 2001). The activities of phosphatase, chitinase and phenol oxidase were enhanced significantly in Loblolly Pine (Pinus taeda) forest soil after thinning (Boerner et al., 2006). Weak intensity thinning improved soil physiochemical properties, but under medium or intensive intensity, the soil condition was 1219 Adv. J. Food Sci. Technol., 6(11): 1219-1227, 2014 properties in Platycladus orientalis forests, especially in intensively thinned forests (Xu et al., 2008). average annual evaporation was about three times of precipitation. Soil consisted of the leaching brown soil and mountain brown soil developed from sandstone and shale weathering slope, which were under varying degrees of long-term erosion. INTRODUCTION The soils consisted of coarse osseous characteristics, such as thin soil layers, hypoplasia structure, intensive gravel content and weak water retention capacity. The vegetation there is composed of shrubs, such as chastetree (Vitex negundo var. Heterophylla), wild jujube (Ziziphus jujuba var. Spinosa (Bunge) Hu), shrubby bushclover (Lespedeza bicolor Turcz), Myripnois dioica Bunge, Spiraea (Spiraea trilobata Lindl), patches of Platycladus orientalis and Pinus tabulaeformis plantations. y ( ) However, some research shows thinning did not significantly improve soil quality, even damaging soil structure. For instance, after 5 years of thinning a pine forest in northeast Germany, soil microbial activity was not significantly improved and soil enzyme activities including xylanase, phenol oxidase and peroxidase in both the organic layer and mineral layer were not significantly different from the control stand (Maassen et al., 2006). Forest soil nutrients in Larch-fir forest stands did not significantly change under <40% thinning intensity, indicating that long-term effects of thinning were not significant (Wang et al., 2009). With a 7 years thinning program, the activities of dehydrogenase, catalase, urease and phosphatase in soil were significantly reduced but protease activity did not change (Garcia et al., 1997). In Pinus massoniana forests, thinning reduced soil structure stability, soil total porosity, soil moisture storage capacity and holding capacity, but increased soil volume quality (Jing and Xiao, 1998). METHODOLOGY • Measuring soil bulk density by using loop-cuttig method, soil bulk density (g/cm3) = dried soil weight/volume • Soil total porosity (%) = [1-(soil bulk density/soil specific gravity)] ×100%, soil specific gravity: 2.65 g/cm3 Sample plots: The sample plots were located in Shui Zhang village, Mu Jia Yu town, Miyun district of Beijing (40° 25′N、116° 53′E), with elevation between 220-599 m and average annual temperature of 11.8°C. The annual average precipitation there was 623.0 mm, with 970.1 mm in rainy years and 285.3 mm in dry years and the precipitation of June-September accounted for more than 80% of the year’s rainfall. The g • Maximum moisture holding capacity (t/hm2): Wt = 10000 Pth, Pt is soil total porosity (%); h is soil thickness (m) 2 • Non-capillary moisture holding capacity (t/hm2): W0 = 10000 P0h, P0 is for soil non-capillary porosity (%) 1220 Table 1: Basic characteristics of Pinus tabulaeformis sample plots Plot type Label Age/a Altitude/m Aspect Grade Density before thinning/hm2) Density after thinning (/hm2) Thinning intensity Shady thick soil slope I 38 125 N 27 3556 1734 50 Shady thick soil slope M 38 125 N 31 3467 1956 45 Shady thick soil slope W 38 125 N 27 3416 2289 33 Shady thick soil slope C 38 131 N 27 3378 3378 0 Table 1: Basic characteristics of Pinus tabulaeformis sample plots 1220 Adv. J. Food Sci. Technol., 6(11): 1219-1227, 2014 into a 150-mL triangular flask and add 40 mL of distilled water and 5 mL of 0.3% H2O2. Meanwhile, set a control by injecting 40 mL of distilled water and 5 mL of 0.3% H2O2 into a triangular flask without soil. Plug in the stopper and, put in a Dual-Action shaker at 120 rpm, for 30 min. Then inject 5 mL of 15 mol/L sulfuric acid to terminate reaction and filter the solution using quantitative filter paper. Get 25 mL of filtrate and titrate until slightly red using 0.1 mol/L KMnO4 solution. The KMnO4 solution (mL) represented enzyme activity within a unit weight of soil. Measurement of soil permeability: Soil permeability was measured by Bicyclic infiltration method. Sampling method and time were the same with Measurement of soil physical properties and moisture holding capacity. The calculation methods are: • Initial permeability rate = Initial infiltration amount/initial infiltration time, in this research the initial infiltration time is 2 min. RESULT ANALYSIS Effects of thinning on soil bulk density and porosity: Woodland soil is the main place in the forest to store water and the quantity and mode of soil moisture storage are strongly affected by soil physical properties (Yu and Wang, 1991). For instance, soil bulk density can directly affect the soil moisture storage capacity; soil total porosity can directly affect the soil air permeability and penetrating capacity; the non-capillary porosity can decide soil moisture storage quantity, the main aspect of forest soil moisture conservation and soil/water conservation function. This research determined the effects of different thinning intensities on the above three soil physical properties. METHODOLOGY • Steady infiltration rate is infiltration rate thinning to be stable in a unit time. • Average infiltration rate is the average rate of water infiltration of the soils. Data analysis: A data were analyzed using Least Significant Difference (LSD) multiple comparison on SPSS 16.0 (SPSS Inc., Chicago, US). Principal Component Analysis (PCA) was used to evaluate the comprehensive effect of thinning improvement on soil. • Total amount of infiltration was for comparison. Because each plot had reached steady infiltration before 36, 36 min was taken as unified time. Measurement of soil nutrient level: The measurement methods were: total Nitrogen, Kjeldahl determination; available Phosphorus, NaHCO3 leaching with Molydenum- Antimony- D-iso-Ascorbic- acid- Colorimetry (MADAC); available Potassium, NH4OAC leaching with flame photometer method (CACCSI, 1984). Sampling time was the same with Measurement of soil physical properties and moisture holding capacity and sampling depth was 5-10 cm, with three repetitions in each plot. Measurement of soil organic matter and enzyme activity: Food Sci. Technol., 6(11): 1219-1227, 2014 , 6(11): 1219-1227, 2014 Fig. 3: Diversification of the soil non-porosity of Pinus tabulaeformis before and after thinning Fig. 1: Diversification of the soil bulk density of Pinus tabulaeformis before and after thinning Fig. 2: Diversification of the soil porosity of Pinus tabulaeformis before and after thinning Fig. 1: Diversification of the soil bulk density of Pinus tabulaeformis before and after thinning Fig. 1: Diversification of the soil bulk density of Pinus tabulaeformis before and after thinning Fig. 3: Diversification of the soil non-porosity of Pinus tabulaeformis before and after thinning Fig. 1: Diversification of the soil bulk density of Pinus tabulaeformis before and after thinning increased by 3.0, 5.7 and 10.2% respectively in layer0-10 and by 3.1, 7.2 and 17.8%, respectively in layer10-20. Fig. 2: Diversification of the soil porosity of Pinus tabulaeformis before and after thinning Five years after cutting, the forest soil porosity was higher than before. At intensity W, M and I compared with the control, soil total porosity increased by 2.9, 5.4 and 7.3%, respectively in layer0-10 and by 3.4, 7.1 and 7.9%, respectively in layer10-20. Through investigating the soil porosity 5 years after cutting, we found that stand soil porosity increased after cutting, in positive relation to the thinning intensity: intensive>medium>weak>control. LSD analysis showed that soil total porosity was significantly improved in layer0-10 (p<0.05) after thinning, but not significant in layer10-20, indicating that thinning can improve soil at depth 0-10 cm more obviously than at depth 10-20 cm. Fig. 2: Diversification of the soil porosity of Pinus tabulaeformis before and after thinning Effect of thinning forest on soil non-capillary porosity: To analyze the effect of thinning on the non- capillary porosity, we compared data collected from P. tabulaeformis forests before and 5 years after thinning (Fig. 3). The results showed that the non- capillary porosity was improved as thinning intensity increased. At intensity I, M and W, the non-capillary porosity increased significantly by 26.2, 49.6 and 52.5%, respectively in layer 0-10 (ANOVA, p<0.05); and increased by 2.8, 22.6 and 43.2%, respectively in layer 10-20, with ANOVA showing no significant difference between intensity levels. These results indicated that thinning has its greatest effect on non- capillary soil porosity at soil depths between 0-10 cm. Measurement of soil organic matter and enzyme activity: Organic matter: Potassium dichromate dilution heat method. Soil urease: Indophenol colorimetric method. The steps are: get 10 g of air-dried soil sample screened by a 1- mm sifter, put into a 100-mL volumetric flask, add 2 mL toluene, place at room temperature for 15 min and injected in 10 mL of matrix (10% urea solution) and 20 mL of citrate buffer (pH 6.7). Blended carefully, put the flask into a constant temperature box at 37°C; for each group, set no-matrix soil (10 g soil+10 mL distilled water+and 20 mL citrate buffer) as control; the whole test was using soil solution (0 g soil+10 mL urea solution+and 20 mL citrate buffer) for comparison. After 24 h, add 38°C distilled water to the constant volume of 100 mL (toluene should float above scale line), then filter the solution and save the filtrate. Put 1 mL of filtrate in a 50-mL volumetric flask, immediately add 20 mL of distilled water, 4 mL of phenol sodium solution and 3 mL of sodium hypochlorite solution and shake up the flask. After 20 min when color changes, add to constant volume for colorimetry at 578 nm. Enzyme activity was represented by mg of amino Nitrogen per gram of soil hydrolysis generated. Effects of thinning on soil bulk density: To evaluate the effect of thinning on soil bulk density, we compared data before thinning and five years after thinning of P. tabulaeformis forests (Fig. 1). The results showed that soil bulk density was lower after thinning and decreased with improved intensity. Under thinning intensity W, M and I, the soil bulk density was reduced by 3.2, 6.1 and 10.3%, respectively in layer0-10 and by 3.2, 7.4 and 15.3%, respectively in layer10-20. Five years after cutting, soil bulk density was lower in all the thinned forests. Under thinning intensity W, M and I compared to control, soil bulk density from 0- 10 cm reduced by 3.9, 5.7 and 7.9%, respectively in layer0-10 and by 3.5, 7.2 and 8.1%, respectively in layer10-20. The Analysis of Variance (ANOVA) showed that soil bulk density ranked as intensive<medium<weak <control intensity, which was negatively related with thinning intensity, after 5 years of thinning in the P. tabulaeformis forest. Comparisons of soil bulk density in intensively thinned plots in layer0-10 Soil catalase: Titration method. The steps are: get 5 g of air-dried soil sample screened by the 1 mm sifter, put 1221 Adv. J. Measurement of soil organic matter and enzyme activity: Multiple comparisons showed that the non-capillary porosity in layer0-10 cm was significant (p<0.05), indicating that the non-capillary porosity improved after five years of thinning; but non-capillary porosity increased a little as intensive>medium> weak>control in layer10-20, but not remarkable (p> 0.05). This result indicated that the non-capillary porosity in layer10-20 cm was improved less than in layer0-10. 2.4, 2.3, 2.3, 2.3 and 2.3 times respectively (p<0.05) at Medium thinning (M); no significant difference (p>0.05) was found at weak thinning or in the control. At depth 10-20 cm, all thinned forests were almost the same, without significant difference (p>0.05), which proved that thinning was beneficial for stand surface (0- 10 cm) by improving permeability (Table 2). Effects on soil moisture conservation amount: Woodland soil is the main place to store water. A study showed that the gravity water in the non-capillary pores plays the major role in adjustable storage in saturated moisture holding capacity (Hao and Wang, 1998). Therefore, forest soil moisture holding capacity mainly depends on the soil non-capillary pores and the quantity depends on the soil non-capillary porosity and soil thickness (Liu et al., 1996). Soil total storage is the sum of the water storage capacity of non-capillary and capillary pores. And the maximum of potential soil conservation reflects the potential storage, regulation ability and the soil moisture conservation ability as well (Wei et al., 2008). Effects on soil permeability: Soil infiltration capacity is one major factor of erosion and reflects hydrological evaluation standards of soil/water conservation vegetation and water conservation function (Zhang et al., 2009). The higher soil infiltration capacity means the rainfall can be infiltrated and stored by the soil in wooded areas faster, with less surface runoff and soil erosion become; on the opposite, erosion will be the surface runoff (Luo et al., 2004). In this test, five indexes including initial permeability rate, average infiltration rate, the steady infiltration rate and penetration amount and saturated hydraulic conductivity were used to evaluate soil infiltration capacity. ( ) The resulting effects of 5 years of thinning on the amount of water conservation (non-capillary moisture holding capacity) are shown in Table 3. Water conservation quantities in the samples from thinned forests at depths of 0-10 and 10-20 cm were increased following the thinning. In layer 0-20 cm under thinning intensity by W, M and I, water conservation quantities were generally increased by 12.2, 48.7 and 44.4%, respectively. Measurement of soil organic matter and enzyme activity: were significantly different from weak intensity and control plots (p<0.05), but there was no significant difference among plots in layer10-20 (p>0.05), indicating that soil bulk density was reduced 5 years after cutting, but not remarkably. These results showed that forest thinning significantly reduced the soil bulk density in P. tabulaeformis forests, which effectively improved the surface soil structure especially at depth 0-10 cm. Effect of thinning forest on soil total porosity: To evaluate the effect of thinning on soil total porosity, we compared soil total porosity data collected from the P. tabulaeformis forest before and 5 years after cutting (Fig. 2). The results showed that after 5 years of forest thinning, soil total porosity was higher than before thinning and as the porosity increased more as foster intensity increasing. At intensity W, M and I compared with the control, the forest soil total porosity Meanwhile, comparing data after thinning with control, the non-capillary porosity of each thinning plot was obviously higher. At intensity W, M and I, the non- capillary porosity increased by 26.2, 49.6 and 52.5%, respectively in layer0-10,; and by 3.0, 22.6 and 43.2%, respectively in layer10-20. ANOVA showed that the non-capillary porosity was positively related to the thinning intensity and the 1222 Adv. J. Food Sci. Technol., 6(11): 1219-1227, 2014 Table 2: Soil infiltration of Pinus tabulaeformis before and after thinning Permeability type Depth (cm) Control ----------------------------- Weak ------------------------------ Medium ------------------------------ Intensive --------------------------- Before 5 years After 5 years Before After 5 years Before After 5 years Before After 5 years Initial permeability rate (mm/min) 0-10 3.595 2.167 10.225 4.335 0.515 7.267 2.932 9.945 10-20 3.340 2.550 6.069 3.315 0.459 3.697 0.571 6.120 Steady infiltration rate (mm/min) 0-10 2.448 1.912 4.845 3.825 0.535 6.375 0.969 6.630 10-20 2.703 2.422 4.284 2.805 0.663 3.060 0.490 4.845 Average infiltration rate (mm/min) 0-10 2.864 2.004 6.336 4.060 0.540 6.582 0.969 7.630 10-20 2.991 2.477 4.720 2.996 0.643 3.315 0.490 5.202 Penetration amount (mm) 0-10 95.624 70.124 213.178 143.819 19.344 232.813 56.967 264.688 10-20 101.336 87.974 162.077 104.039 23.256 115.259 17.931 181.559 Saturated hydraulic conductivity (mm/min) 0-10 1.632 1.275 3 .230 2.550 0.357 4.250 0.646 4.420 10-20 1.802 1.615 2.856 1.870 0.442 2.040 0.326 3.230 Table 2: Soil infiltration of Pinus tabulaeformis before and after thinning Control Weak non-capillary porosity increased as intensive>medium >weak>control. Measurement of soil organic matter and enzyme activity: Compared with the control, the total storages after 5 years were higher in a positive correlation with intensity. Specifically, under W, M and I intensity, total storages was increased by 3.1, 6.2 and 7.7%, respectively, with significant level in ANOVA (p<0.05). The intensive and medium cutting effectively improved the amount and the capacity. storage capacities in both layer0-10 and layer10-20 were both higher than before. Total storage capacity under weak, medium and intensive thinning was enhanced by 3.1, 6.5 and 13.9%, respectively. Compared with the control, the total storages after 5 years were higher in a positive correlation with intensity. Specifically, under W, M and I intensity, total storages was increased by 3.1, 6.2 and 7.7%, respectively, with significant level in ANOVA (p<0.05). The intensive and medium cutting effectively improved the amount and the capacity. Changes of soil nutrients: By comparing the pre and post thinning forest soil nutrient content in our plots we were able to see that; soil total N, effective P and K content (p<0.05) was increased in all plots. Five years later, the soil total N, effective P and K contents were improved by 43.1, 172.5 and 59.2%, respectively under Intensive thinning (I) and by 66.0, 63.3 and 41.2%, respectively under Medium thinning (M). Integrated analysis of the above data showed that thinning can enhance forest soil moisture holding capacity, because thinning greatly affected soil porosity. Soil moisture holding capacity and porosity are positively related. In considering thinning intensity levels, the most effective level was I cutting, followed by M cutting. However, only the effective K content (23.224 mg/kg) was significantly increased under weak cutting (17.070 mg/kg) (p<0.05), while the other indicators were not significantly different (p>0.05) (Table 4). Changes of organic matter and enzyme activity in forest soil: Organic matter is an important component of soil, as it affects and restricts soil properties. It is generally thought that soil organic matter content and soil quality are in positive correlation (Shimizu, 1994). Improving soil organic matter content can promote the formation of aggregates, thereby keeping their stability and thus, improving soil moisture holding capacity (Haynes et al., 1991). Organic matter is the energy of soil microbial activities, which can improve the microbial diversity and activity and soil physiochemical properties (Peverill and Judson, 1999). Measurement of soil organic matter and enzyme activity: Compared with the control, the amounts of water conservation were all higher in a positive correlation with intensity. Specifically, under thinning intensity W, M and I the water conservation quantities were increased by 14.4, 36.0 and 47.8%, respectively. ANOVA showed that thinning significantly changed soil moisture conservation quantity, especially after intensive and medium thinning (p<0.05), but not after weak thinning. These results indicated that intensive and medium thinning intensities were an effective measure to improve the soil moisture conservation quantity. Forest soil permeability of each thinning plot was enhanced significantly after thinning (p<0.05). Under Intensive thinning (I), the five indices were improved by 2.4, 5.8, 2.9, 3.6 and 5.8 times, respectively in layer0-10. Under Medium thinning (M), the five indices were improved by 13.1, 10.9, 11.2, 11.0 and 10.9 times respectively; however, no obvious change was observed under Weak thinning (W) or in the Control (C) (p>0.05). At depth 10-20 cm, the five indexces were obviously improved (p>0.05) by 9.7, 8.9, 9.3, 9.1 and 8.9 times, respectively at Intensive thinning (I) and by 7.1, 3.6, 4.2, 4.0 and 3.6 times respectively at Medium thinning (M); but no significant difference was observed at Weak thinning (W) or in the Control (C) (p>0.05). The results among the thinned forests showed that: at layer0-10, the five indices were higher than Control (C) by 3.6, 2.5, 2.8, 2.8 and 2.5 times, respectively at Intensive thinning (I) and were significantly higher by The resulting effect 5 years of thinning had on total soil moisture storage is showed in Table 3. Soil total 1223 Adv. J. Food Sci. Technol., 6(11): 1219-1227, 2014 Table 5: The organic matter and enzyme activity of Pinus tabulaeformis plantation before and after thinning Control ---------------------------------- Weak -------------------------------- Medium --------------------------------- Intensive ---------------------------------- 5 years before 5 years after cutting Before 5 years after cutting Before 5 years after cutting Before 5 years after cutting Organic matter (g/kg) 34.676 34.383 12.759 32.243 28.099 57.72 33.445 47.185 Urease (mg/g) 0.780 0.752 0.423 0.468 0.764 2.482 0.744 1.175 Catalase (mg/g) 0.488 0.603 0.398 0.563 0.437 0.643 0.474 0.910 available K were chosen to reflect the soil nutrient statuses before and after forest thinning. storage capacities in both layer0-10 and layer10-20 were both higher than before. Total storage capacity under weak, medium and intensive thinning was enhanced by 3.1, 6.5 and 13.9%, respectively. Measurement of soil organic matter and enzyme activity: The averages of the indicators from different soil layers were used for analysis, including 16 indicators: density, total porosity, non-capillary porosity, hydraulic conductivity, initial permeability rate, steady infiltration rate, average penetration rate, 36 min infiltration quantity, water conservation quantity, total storage capacity, total N, available P, available K, urease content, catalase content and organic matter. The results are shown below. nutrient transformation and directly affect the growth of vegetation, which is of great significance to the maintenance of soil fertility. The soil organic matter contents (g/kg) before and after thinning showed that forest soil organic matter content increased by 41.1% from 33.445 to 47.185 g/kg after Intensive thinning (I), by 105.4 and 152.7% under Medium (M) and Weak (W) thinning respectively, without significant change in control plot (p>0.05) (Table 5). In Table 6 cumulate contribution rate of the first two principal components (F1 and F2) reached 96.853%, indicating that calculation of F1 and F2 was quite reasonable, F1 contributed more to soil physical properties and F2 did more to soil chemical properties such as total N, available P, urease content and organic matter. Change of soil enzyme activity: Soil enzyme activity is an important indicator of soil fertility. Its change to a certain extent can reflect the evolution trend of forest soil quality and the effects of forest management measures on soil quality. This can provide certain guidance for maintaining forest health and formulating management methods (Geng et al., 2008). The experiment was based on the activities of urease and catalase, so as to know the changes before and 5 years after the forest thinning. Urease as a hydrolytic enzyme, can decompose organic matter and promote the hydrolysis of ammonia nitrogen and carbon dioxide, among which ammonia nitrogen is the direct source of trees (Bai et al., 2005). Catalase is widely distributed in soil and its activities can be used to analyze harm of H2O2 to plant growth (Dai and Wang, 1994). Its activity Using F1 and F2, weighted calculation of each stand integrated score was L = 0.84019 * F1 + 0.12833 * F2, where the weight was the contribution to each main factor (Table 7). According to Table 8 the integrated scores of P. tabulaeformis forest were 0.93, 0.26, 0.09 and 1.11, under intensity control, weak, medium and intensive respectively. Clearly, after thinning P. Measurement of soil organic matter and enzyme activity: Meanwhile, organic matter can affect the quantity and speed of Effect on soil nutrients: Thinning is an important measure to adjust forest stand soil nutrients. Thinning can change forest community structure, species composition habitat conditions and affect the productivity and soil fertility status (Zhang et al., 2001). Soil nutrients levels are important indicators of the soil fertility, while understanding the soil nutrient element contents after thinning will be meaningful to promote benign nutrient circulation and maintain forest productivity (Fang et al., 1998). In this experiment, three indexes including total N, available P and 1224 Adv. J. Food Sci. Technol., 6(11): 1219-1227, 2014 Table 6: Initial eigen values Principal component Eigen value Contribution rate/% Cumulate contribution rate/% F1 13.443 84.019 84.019 F2 2.053 12.833 96.853 F3 0.504 3.147 100.000 Table 7: Component matrix Variable The first component F1 The second component F2 Density -0.978 0.091 Total porosity 0.978 -0.094 Non-capillary porosity 0.993 -0.093 Hydraulic conductivity 0.987 -0.143 Initial permeability rate 0.961 -0.273 Steady infiltration rate 0.987 -0.143 Average penetration rate 0.978 -0.203 36 min infiltration quantity 0.980 -0.194 Water conservation quantity 0.993 -0.093 Total storage capacity 0.978 -0.094 Total N 0.788 0.587 Available P 0.768 0.609 Available K 0.941 -0.190 Urease 0.637 0.766 Catalase 0.793 -0.396 Organic matter 0.824 0.567 is related to soil respiration intensity and can reflect microbial activity intensity (Dai and Bai, 1995). Comparing both urease and catalase contents before and after cutting, we found that: under Intensive thinning (I), soil urease and catalase contents increased by 57.9% from 0.744 to 1.175 mg/g and by 92.0% from 0.474 to 0.910 mg/g, respectively; under Medium (M), thinning two contents rose by 224.9 and 47.1% respectively; under Weak thinning (W) and in the Control (C), the two indicators did not change significantly (p>0.05). Integrated evaluation of thinning effects on forest soil improvement: Because different thinning intensities affected the soil in many ways, the effects of thinning on soil improvement can be clarified should be studied in greater detail in order to maximize its efficiency. To do so it would be necessary to analyze the data of all indicators after 5 years thinning. This study chose SPSS 16.0 and PCA. CONCLUSION Boerner, R.E.J., T.A. Waldrop and V.B. Sheldune, 2006. Wildfire mitigation strategies affect soil enzyme activity and soil organic carbon in loblolly pine (Pinus taeda forests). Can. J. Forest Res., 36: 3148-3154. Thinning improves soil physical properties: Thinning effectively reduced the soil bulk density and increased soil porosity and structure in P. tabulaeformis forest, especially in surface soil (0-10 cm) following intensive and medium thinning. Decreased forest canopy density, led to improved lighting conditions and light intensity in forested stands. This also led directly to speed up litter decomposition, resulting in increased soil humus content, which promoted the surface soil formation crumb structure and the soil’s air permeability. CACCSI (Committee of Agriculture Chemistry of Chinese Soil Institute), 1984. Conventional Analysis Method of Soil Agriculture Chemistry. Science Press, Beijing. Chi, D.X., M.G. Liu, F.L. Su et al., 2006. The analysis effect of thinning in natural shaw. J. Anhui Agric. Sci., 34: 1869-1870. In addition, thinning measures also greatly improved the soil permeability and water retention capacity of P. tabulaeformis forested land (similarly, better in 0-10 cm), which was helpful to reduce surface runoff, increase infiltration of rainfall to soil and improve the ability of water conservation. Dai, W. and B. Wang, 1994. Correlations of soil catalase activity and soil property of red and yelweak soils in dagangshan of Jiangxi province. Forest Sci. Technol., 17: 17-18. Dai, W. and H.Y. Bai, 1995. Correlations of soil catalase activity and it’s kinetic characteristic with some soil properties. J. Beijing Forest. Univ., 17: 37-41. Thinning effectively improves the soil nutrients: The thinning effects on P. tabulaeformis soil nutrient content were different among the thinning intensities: effective P and K contents increased the most under intense thinning and total N content increased the most under medium thinning. Soil nutrients did not improve much as under weak thinning. Fang, H.B., D.L. Tian and W.X. Kang, 1998. Nutrient study of understory vegetation in the thinned chinese fir plantation II. Contents of nutrient in the soil and accumulation index of plants to the soil. J. Cent. South Forest. Univ., 18: 92-95. Garcia, C., T. Hernandez, A. Roldan and J. Albaladejo, 1997. Biological and biochemical quality of a semiarid soil after induced devegetation. J. Environ. Qual., 26: 1116-1122. Soil organic matter content rose the highest 5 years of weak thinning (up to 152.7%) by 41.1% after intensive thinning and by 105.4% after medium thinning. ACKNOWLEDGMENT Haynes, R.J., R.S. Swift and R.C. Stephen, 1991. Influence of mixed cropping rotations (Pasture- arable) on organic matter content, water stable aggregation and clod porosity in a group of soils. Soil Till. Res., 19: 77-87. This study was supported by grants from service for science and technologies in Beijing the Forestry Service Industry (No. 201004021), service for science and technologies in Beijing Forestry University (No. TD2011-08), service for science and technologies in Beijing Forestry University (No. TD2011-08), the Special Research Funds for Fundamental Research at the Central Universities (No. BLJD200904), Cooperation of experimental monitoring about forest health in China and America (2009DFA92900). Hu, J.W. and C.Q. Zhu, 1999. The impacts of intermediate felling on forest environment. J. Northeast Forest. Univ., 27: 65-67. Jing, Y. and H.S. Xiao, 1998. Study of soil moisture physical character change before and after wood harvesting. East China Forest Manage., 12: 63-67. CONCLUSION g Furthermore, soil urease content and catalase content were increased the most by Medium thinning (M) and Intensive thinning (I) respectively. Geng, Y.Q., W. Dai, X.X. Yu et al., 2008. Research advances for the effects of forest management on soil enzyme activity. J. Beijing Forest. Univ., 30: 132-138. Integrated evaluation of thinning effects on forest soil improvement: Following 5 years of varied intensity thinning on P. tabulaeform is forested stands; intensive thinning (50%) led to the greatest improvement of the forests’ soil physiochemical properties. Guo, B., Y. Liu, G.L. Li, J. Gan and Y. Xu, 2007. Response of soil enzyme activity to thinning intensity of aerial seeding pinus tabulaeformis stands. Sci. Silvae Sin., 43: 128-134. Hao, Z.Q. and L.H. Wang, 1998. Water conservaion capacities of soils with major forest types in mountainous regions of east Liaoning Province. Chinese J. Appl. Ecol., 9: 237-241. Measurement of soil organic matter and enzyme activity: tabulaeformis forest, the soil improvement was higher than the control, especially after intensive and medium thinning. Thus, it is better to use intensive cutting to improve soil for P. tabulaeformis forest. 1225 Table 8: Principle components estimation of factor score Score --------------------------------------------------------------------------------------------------------------------------------------- Intensity The first component F1 The second component F2 Integrated score Order Control -1.068060 -0.266510 -0.93339630 4 Weak -0.168320 -0.938500 -0.26214730 3 Medium -0.112090 1.415573 0.08729141 2 Intensive 1.348467 -0.210560 1.10825221 1 Table 8: Principle components estimation of factor score Score Adv. J. Food Sci. Technol., 6(11): 1219-1227, 2014 REFERENCES Li, C.M., J.S. Du and H.R. Zhang, 2003. The effects of thinning on forest growth and model study. Forest Res., 16: 636-64. Bai, C.X., Y.Q. Geng and W. Lang, 2005. Studies on soil urease activity of major forest types in badaling of Beijing. J. Sichuan Agric. Univ., 23: 424-481. Liu, S.R., Y.G. Wen, B. Wang and G.Y. Zhou, 1996. 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Effects of thermal treatments on microstructure and mechanical properties of a Co–Cr–Mo–W biomedical alloy produced by laser sintering
Journal of the mechanical behavior of biomedical materials/Journal of mechanical behavior of biomedical materials
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This is the peer reviewd version of the followng article: This is the peer reviewd version of the followng article: Effects of thermal treatments on microstructure and mechanical properties of a Co-Cr-Mo-W biomedical alloy produced by laser sintering / Mengucci, P; Barucca, G.; Gatto, Andrea; Bassoli, Elena; Denti, Lucia; Fiori, F.; Girardin, E.; Bastianoni, P.; Rutkowski, B.; Czyrska Filemonowicz, A.. - In: JOURNAL OF THE MECHANICAL BEHAVIOR OF BIOMEDICAL MATERIALS. - ISSN 1751-6161. - STAMPA. - 60:(2016), pp. 106- 117. [10.1016/j.jmbbm.2015.12.045] Terms of use: The terms and conditions for the reuse of this version of the manuscript are specified in the publishing policy. For all terms of use and more information see the publisher's website. 24/10/2024 06:02 (Article begins on next page) Author’s Accepted Manuscript Effects of thermal treatments on microstructure and mechanical properties of a Co-Cr-Mo-W biomedical alloy produced by laser sintering P. Mengucci, G. Barucca, A. Gatto, E. Bassoli, L. Denti, F. Fiori, E. Girardin, P. Bastianoni, B. Rutkowski, A. Czyrska-Filemonowicz www.elsevier.com/locate/jmbbm PII: S1751-6161(16)00002-3 DOI: http://dx.doi.org/10.1016/j.jmbbm.2015.12.045 Reference: JMBBM1760 To appear in: Journal of the Mechanical Behavior of Biomedical Materials Author’s Accepted Manuscript Effects of thermal treatments on microstructure and mechanical properties of a Co-Cr-Mo-W biomedical alloy produced by laser sintering P. Mengucci, G. Barucca, A. Gatto, E. Bassoli, L. Denti, F. Fiori, E. Girardin, P. Bastianoni, B. Rutkowski, A. Czyrska-Filemonowicz PII: S1751-6161(16)00002-3 DOI: http://dx.doi.org/10.1016/j.jmbbm.2015.12.045 Reference: JMBBM1760 PII: S1751-6161(16)00002-3 DOI: http://dx.doi.org/10.1016/j.jmbbm.2015.12.045 Reference: JMBBM1760 To appear in: Journal of the Mechanical Behavior of Biomedical Materials Received date: 4 November 2015 Revised date: 29 December 2015 Accepted date: 30 December 2015 Cite this article as: P. Mengucci, G. Barucca, A. Gatto, E. Bassoli, L. Denti, F. Fiori, E. Girardin, P. Bastianoni, B. Rutkowski and A. Czyrska-Filemonowicz, Effects of thermal treatments on microstructure and mechanical properties of a Co-Cr-Mo-W biomedical alloy produced by laser sintering, Journal of the Mechanical Behavior of Biomedical Materials, http://dx.doi.org/10.1016/j.jmbbm.2015.12.045 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Kraków, Poland. Effects of thermal treatments on microstructure and mechanical properties of a Co-Cr-Mo-W biomedical alloy produced by laser sintering P. Mengucci1*, G. Barucca1, A. Gatto2, E. Bassoli2, L. Denti2, F. Fiori3, E. Girardin3, P. Bastianoni1, B. Rutkowski4, A. Czyrska-Filemonowicz4 1 Dipartimento SIMAU, Università Politecnica delle Marche, 60131 Ancona, Italy. 1 Dipartimento SIMAU, Università Politecnica delle Marche, 60131 Ancona, Italy. 2 Dipartimento DIEF, Università di Modena e Reggio Emilia, Via Vivarelli 10, 41125 Modena, Italy. 2 Dipartimento DIEF, Università di Modena e Reggio Emilia, Via Vivarelli 10, 41125 Modena, Italy. 3 Dipartimento DISCO, Università Politecnica delle Marche, 60131 Ancona, Italy. 4 International Centre of Electron Microscopy for Material Science & Faculty of Metals Engineering and Industrial Computer Science, AGH University of Science and Technology, Al. A. Mickiewicza 30, 30-059 Kraków, Poland. 4 International Centre of Electron Microscopy for Material Science & Faculty of Metals Engineering and Industrial Computer Science, AGH University of Science and Technology, Al. A. Mickiewicza 30, 30-059 Kraków, Poland. * Corresponding author: Paolo Mengucci Paolo Mengucci Dip. SIMAU, Università Politecnica delle Marche I-60131 Ancona Italy Dip. SIMAU, Università Politecnica delle Marche I-60131 Ancona l y E-mail: p.mengucci@univpm.it 1 1 Abstract Direct Metal Laser Sintering (DMLS) technology based on a layer by layer production process was used to produce a Co-Cr-Mo-W alloy specifically developed for biomedical applications. The alloy mechanical response and microstructure were investigated in the as-sintered state and after post- production thermal treatments. Roughness and hardness measurements, and tensile and flexural tests were performed to study the mechanical response of the alloy while X-ray diffraction (XRD), electron microscopy (SEM, TEM, STEM) techniques and microanalysis (EDX) were used to investigate the microstructure in different conditions. Results showed an intricate network of -Co (hcp) lamellae in the γ-Co (fcc) matrix responsible of the high UTS and hardness values in the as- sintered state. Thermal treatments increase volume fraction of the -Co (hcp) martensite but slightly modify the average size of the lamellar structure. Nevertheless, thermal treatments are capable of producing a sensible increase in UTS and hardness and a strong reduction in ductility. These latter effects were mainly attributed to the massive precipitation of an hcp Co3(Mo,W)2Si phase and the contemporary formation of Si-rich inclusions. nclusions. Keywords: Co alloys; laser sintering; heat treatments; mechanical properties; structure characterisation. Co alloys; laser sintering; hea ion. ion. 2 2 1. Introduction In medical applications such as knee and hip joint replacement, Co alloys are widely used due to biocompatibility, high strength, excellent wear and corrosion resistance (Balagna et al. 2012; Hiromoto et al. 2005; Saldivar-Garcia and Lopez 2005; Yamanaka et al. 2013). In dentistry, Co alloys are used in the production of metal frames, porcelain fused to metal (PFM) crowns and removable partial dentures (Craig et al. 2004; Yamanaka et al. 2015; Wataha 2002). Generally, in the conventional production processes based on casting, plastic deformation and cutting the physical and mechanical properties of the Co alloys, such as high melting point, limited ductility and high hardness values, may constitute factors that increase the total cost of the biomedical devices. On the other hand, proper operation and long life expectancy of biomedical devices require biocompatibility, good mechanical properties and high corrosion resistance. Proposed solutions to these problems concern the manufacturing technology. In particular, additive manufacturing (AM) technologies based on a layer-by-layer production process assisted by a computer aided design (CAD) software are receiving growing attention (Harris 2012; Mumtaz et al. 2008; Stamp et al. 2009; Ucar et al. 2009). Direct metal laser sintering (DMLS, Power Bed Fusion according to ASTM F42 committee) is an AM technology based on a laser beam that locally melts a metal powder according to the CAD model (Bassoli et al. 2013; Simchi 2006). The DMLS technology allows obtaining finished devices in a single production process, thus contributing to save time and improve efficiency. Furthermore, DMLS is particularly suitable for prosthetic applications in orthopaedics and dentistry where complex geometries, low volumes and strong individualization are required. 3 At times, there is a lack of knowledge regarding the mechanical performances of components produced by laser sintering due to the complex physical processes associated to laser sintering that include heat transfer and melting of powder (Simchi 2006). In other observations, parts produced by DMLS are even stronger than obtained by traditional processes, but they may suffer from lower reliability (Bassoli et al. 2013; Frazier 2014). In general, AM generates highly distinctive 3 In this study, a Co-Cr-Mo-W alloy specifically developed for biomedical applications was produced by the DMLS technology. After production, the alloy was submitted to shot-peening, stress relieving and firing cycle in order to simulate a complex post-production treatment. Mechanical tests and structural characterisation were carried out on as-sintered samples as well as on samples submitted to post-production treatments. Results of the structural characterisation performed on the as-sintered sample were already published in a previous paper (Barucca et al. 2015). In this paper, results obtained from tensile and flexural tests on the post-processed samples are discussed in function of the structural properties of the alloy investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission and scanning transmission electron microscopy (TEM, STEM) and energy dispersive X-ray microanalysis (EDX). Results evidenced strong modifications of microstructure and mechanical behaviour of the alloy as a consequence of the post-production treatments. In particular, changes in the mechanical response were mainly correlated to the formation and growth of coarse and elongated precipitates induced by the post- production treatment. To our best knowledge, the presence of coarse and elongated precipitates is reported here for the first time. 2. Material and methods 2.1 Material composition and sintering parameters 2.1 Material composition and sintering parameters EOS CobaltChrome SP2 powder was submitted to the action of a laser beam in the EOSINT M270 system to produce flexural and tensile specimens by using the standard sintering parameters shown in Table 1. microstructures, very dissimilar to those observed elsewhere, which turns into an urgent need for better knowledge of the expected performances. microstructures, very dissimilar to those observed elsewhere, which turns into an urgent need for better knowledge of the expected performances. High surface roughness, porosity, heterogeneous microstructure and thermal residual stresses are typically the most limiting problems that may affect the sintered products (Bassoli et al. 2012; Simchi 2006). In many cases, improvement of the mechanical response was obtained by post- production treatments (Balagna et al. 2012; Giacchi et al. 2011, 2012; Saldivar-Garcia et al. 1999a; Sanz and Navas 2013; Yamanaka et al. 2015; Zangeneh et al. 2010). Nevertheless, the development of high-strength materials is still the basic requirement for biomedical applications of Co alloys. Typically, a strength increase results in an improved mechanical behaviour of biomedical devices based on Co alloys (Lu et al. 2015; Takaichi et al. 2013; Yoda et al. 2012). Several papers in literature report on the problem of biocompatibility of Co-based materials produced by conventional and AM techniques mainly because the corrosion susceptibility and the extent of metal release strongly depend on the alloy microstructure developed during the fabrication procedure or after post-production treatments (Antunes et al. 2012; Hedberg et al. 2014; Madl et al. 2015; Mostardi et al. 2010; Xin et al. 2012). Post-production treatments can be quite complex as for example in dental applications where for aesthetic reasons restorations are veneered with dental porcelain in a firing process that include heating and cooling cycles. The heating temperatures in the firing cycle for Co alloys can reach values as high as 950 °C causing possible microstructural modifications of the material (Al Jabbari et al. 2014; Xin et al. 2014). Although a large body of published papers is present in literature dealing with problems linked to applications of laser sintered Co alloys to the production of biomedical devices, a limited number of information are available on the structural modifications induced by post-production treatments performed to improve the alloy mechanical response and corrosion resistance. 4 4 Table 2 about here Table 2 about here Material properties claimed by the producer after stress relieving (see the complete sequence of the thermal stress relieving treatment reported below), oxide fire simulation (5 min. at 950 °C) and ceramic fire simulation (4 x 2 min. at 930 °C) procedures according to EN ISO 22674, are listed in Table 3. Table 3 about here 2.2 Thermal treatments and mechanical tests Table 3 about here 2.2 Thermal treatments and mechanical tests Table 3 about here Flexural specimens (FLEX) were produced according to ASTM B528-05 (Standard Test Method for Transverse Rupture Strength of Metal Powder Specimens) and B 925–08 (Standard Practices for Production and Preparation of Powder Metallurgy (PM) Test Specimens). Tensile specimens (TENS) were built following ASTM E8M:11 (Standard Test Methods for Tension Testing of Metallic Materials). Figure 1 shows geometry and dimensions of FLEX (Figure 1a) and TENS (Figure 1b) samples, respectively. In both drawings, Z is the direction of growth in the AM machine, and XY is the plane of powder deposition; X is the slide direction of the recoater. Table 1 about here 5 The material is a Co-Cr-Mo-W alloy (Type 4 according to standard DIN EN ISO 22674:2006) for biomedical applications. The nominal powder composition provided by the manufacturer (EOS 5 GmbH Electro Optical System) is reported in Table 2. The powder is free of Ni, Be and Cd in accordance to EN ISO 22674. Figure 1 about here 6 After additive fabrication, parts were shot-peened following the datasheet recommendations, using fine spherical ceramic media (70% ZrO2 - 30% SiO2, 125 - 250µm), at 3 bar pressure. Shot-peening 6 6 produces a compressive residual stress layer (Sanz and Navas 2013). A thermal stress relieving treatment was then performed under argon atmosphere, with the following sequence: - Heat furnace to 450 °C in 60 minutes. - Heat furnace to 450 °C in 60 minutes. - Hold temperature for 45 minutes. - Heat furnace to 750 °C in 45 minutes. - Heat furnace to 750 °C in 45 minutes. - Hold temperature for 60 minutes. - Switch furnace heating off. When temperature dropped down to approx. 600 °C, open the furnace door. From now on, the stress relieving thermal treatment just described above is referred to as RHT. In dental restorations, after the RHT process the metal structure is then veneered with dental ceramic material during a firing operation, which includes five steps. The standard five-steps firing cycle, compliant to EN ISO 22674, adopted in this work for the samples already submitted to the RHT process, is outlined in Figure 2. Starting from a pre-heating temperature of 450 °C, five steps simulate the firing of oxide and ceramic layers. From now on, samples or groups of samples submitted to both the RHT treatment and the firing cycle are labelled as CHT. d as CHT. Figure 2 about here In order to evaluate the effects of the stress relieving heat treatment (RHT) and the firing cycle (CHT) on mechanical properties and microstructure of the investigated alloy, a number of tensile (TENS) and flexural (FLEX) specimens in different conditions were studied. For each condition a number of 6 samples were investigated. The sample group in the as-sintered (AS) condition was named TENSAS. Details of the mechanical tests performed on the different groups of samples are summarised in the following: 7 7 - Tensile tests (crosshead speed 5 mm/min, load cell capacity 250 kN); - Three point bending tests (support width 25.4 mm, crosshead speed 2 mm/min, load cell capacity 30 kN). - Tensile tests (crosshead speed 5 mm/min, load cell capacity 250 kN); - Three point bending tests (support width 25.4 mm, crosshead speed 2 mm/min, load cell capacity 30 kN). Figure 1 about here Although it is known that parts are weaker when built in the Z direction, we decided to measure the performances in X direction because that reproduces the orientation in which dental structures are built. Any dental prostheses, for example a bridge, would be laid in the XY plane, in order to reduce building time and costs and to allow for optimal support positioning. Therefore, the bridge can be considered as a beam built along the X-axis, or in the XY plane without substantial changes, and subject to flexure. Possible variation of the mechanical properties with specimen orientation cannot be excluded. 2.3 Hardness measurements 2.3 Hardness measurements Based on the recommendations of ISO 4498:2010 (Sintered metal materials, exclud Based on the recommendations of ISO 4498:2010 (Sintered metal materials, excluding hard metals Based on the recommendations of ISO 4498:2010 (Sintered metal materials, excluding hard metals -- Determination of apparent hardness and microhardness), Rockwell A hardness test was chosen, and performed by means of an ERNST NR3D Hardness tester, following the specifications of ISO -- Determination of apparent hardness and microhardness), Rockwell A hardness test was chosen, and performed by means of an ERNST NR3D Hardness tester, following the specifications of ISO 6508-1:2015 (Metallic materials -- Rockwell hardness test). -- Determination of apparent hardness and microhardness), Rockwell A hardness test was chosen, and performed by means of an ERNST NR3D Hardness tester, following the specifications of ISO 6508-1:2015 (Metallic materials -- Rockwell hardness test). s Statistical tests were performed in order to assess whether the heat treatments cause significant changes in the mechanical response. Possible significant differences between the groups were checked by means of the t-test for independent samples by using a software tool for statistical analysis (Statistica 8, Statsoft). The test can be used to determine if two sets of data are significantly different from each other. It results in probability values (p-values): when lower than 0.05 they can be taken as a decision to reject the null hypothesis of equality between two groups of samples. In this study, when for example tensile strengths of TENSAS and TENSRHT are compared by means of a t-test, a p-value lower than 0.05 allows rejecting the hypothesis that the relief treatment leaves strength unchanged, that is to say it allows to admit a significant effect of the heat treatment on strength. 2.5 Structural characterisation 2.4 Roughness measurements Roughness measurements adhering to EN 10049:2013 were carried out by means of a DIAVITE DH-5 stylus tester, by adopting a travel length lt of 4.8 mm, with a cut-off filter of 0.8 mm, giving an evaluation length lm of 4 mm. Five measurements per sample were recorded for statistical reasons, taken on XZ faces along the X direction. The standard deviations (SD) associated to the roughness value were estimated from the five measurements. 2.4 Statistical tests 8 8 2.5 Structural characterisation Rupture surfaces of one sample per group were observed by an environmental scanning electron microscope (ESEM) FEI Quanta 200, operating in low-vacuum mode (0.5 Torr) at 25 kV to relate the mechanical properties to failure mechanisms in the micro-scale Furthermore, in order to correlate the mechanical behaviour of the several sample groups to the microstructure developed during the production process and the different thermal treatments, two groups of samples were structurally characterized. The first group (named AS) was in the as sintered condition, while samples belonging to the second group (named CHT) were firstly submitted to the stress relieving treatment and then to the firing cycle. Independently on the sample group (AS or CHT), samples structurally characterised by the analytical techniques listed below, were not submitted to either tensile or flexural mechanical tests. Structural characterizations were carried out by X-ray diffraction (XRD), transmission (TEM) and scanning transmission (STEM) electron microscopy techniques and energy dispersive X-ray microanalysis (EDX). 9 9 For XRD measurements a Bruker D8 Advance diffractometer operating at V= 40kV and I= 40 mA in the angular range 2θ=10 - 80° was used. XRD measurements were carried out by Cu-Kα radiation. For XRD measurements a Bruker D8 Advance diffractometer operating at V= 40kV and I= 40 mA in the angular range 2θ=10 - 80° was used. XRD measurements were carried out by Cu-Kα radiation. TEM analyses were performed by a Philips CM200 electron microscope and JEOL JEM-2010 ARP microscope equipped with an Oxford Inca energy dispersive X-ray microanalysis, both operating at 200 kV. Analytical high resolution microscopy was conducted by a probe Cs-corrected FEI Titan3 G2 60-300 equipped with ChemiSTEM technology (X-FEG field-emission gun and Super-X EDX detector system) developed at FEI (FEI application note AN002707-2010. Available from: www.fei.com). TEM bright-field imaging and STEM imaging using high-angle annular-dark-field (HAADF) contrast as well as EDX mapping and high resolution TEM (HRTEM) imaging were used for characterization of the samples micro/nanostructure and chemical composition of phases down to the nano-scale. Phase identification was performed by selected area electron diffraction (SAED), STEM-EDX and fast Fourier transform (FFT) of HRTEM images. The diffraction patterns and HRTEM images were interpreted with the JEMS software (Stadelman 2007). Samples for TEM observations were mechanically prepared by grinding on abrasive papers and diamond pastes. Disks with a 3 mm diameter were cut from the bulk material by an ultrasonic cutter (Gatan). 3.1 Roughness measurements 3.1 Roughness measurements Roughness measurements provided values in the same range for all the specimens. The average value of the roughness Ra was 2.2 µm, with a standard deviation (SD) of 0.4 µm. As maximum roughness Rmax, an average value of 15 μm (SD = 2 μm) was obtained. 3.2 Hardness and mechanical tests The results of tensile and hardness tests on the tensile specimens are shown in Table 4, separately for the different groups (TENSAS, TENSRHT, TENSCHT). The average value (AV) is reported with the corresponding standard deviation (SD). Tensile strength just after building (TENSAS) is around 1340 MPa, it increases after the stress relieving treatment (TENSRHT) and is maintained by the firing cycle (1440 MPa for TENSCHT). Accordingly, hardness increases from about 73 HRA, for the TENSAS group, to about 76 HRA for the TENSCHT group (Table 4). On the contrary, the elongation at break (εb) shows the highest value for the TENSAS group, decreases after the RHT process and reaches its lowest value of 4.7 % for the TENSCHT group of samples (Table 4). Table 4 about here Results for the flexural specimens are listed in Table 5. Transverse rupture strength in the CHT state (FLEXCHT) is as high as 2500 MPa, just slightly lower than after the sole relieving treatment (FLEXRHT). 2.5 Structural characterisation In order to reduce time of ion milling, in the last step of the mechanical thinning procedure, each 3 mm disk was mechanically thinned in a central area by a Dimple Grinder (Gatan). Final thinning was carried out by an ion beam system (Gatan PIPS) using Ar ions at 5 kV. Samples (lamellae) for STEM, HRTEM and EDX analyses were prepared by Focused Ion Beam (FIB) technique by the ZEISS NEON CrossBeam 40EsB microscope. Before milling, a layer of Pt was deposited at the place of cutting in order to protect the thin sample against heavy Ga ions during the preparation. Final milling was performed with the 4 keV Ga+ beam. 3. Results 10 (TENSAS – TENSRHT). On the other hand, the firing cycle causes significant increase of hardness and loss of ductility, but leaves the alloy strength unchanged (TENSCHT – TENSRHT). (TENSAS – TENSRHT). On the other hand, the firing cycle causes significant increase of hardness and loss of ductility, but leaves the alloy strength unchanged (TENSCHT – TENSRHT). Comparison of the results obtained for tensile and flexural tests, reported in the last rows of Table 6, evidences an almost identical behaviour. Comparison of the results obtained for tensile and flexural tests, reported in the last rows of Table 6, evidences an almost identical behaviour. 3.3 Rupture surfaces Figure 3 allows comparing the rupture surfaces of TENSAS samples with samples submitted to stress relieving treatment and firing cycle (TENSCHT). Signs of ductile failure modes can be observed in the TENSAS sample (Figure 3a). Areas of ductile failure surround “quasi-cleavage” facets to which are added, on the facets themselves, surface irregularities that can be ascribed to shear failure (Figure 3a). Ductile failure modes disappear after the heat treatments (Figure 3b), where “quasi-cleavage” mechanisms take up the whole surface, together with many cracks (dotted lines in Figure 3b). Figure 3 about here Figure 3 about here 3.3 X-ray Diffraction (XRD) 3.3 X-ray Diffraction (XRD) Table 5 about here 11 Results of statistical tests relating mechanical properties of TENS and FLEX groups of samples are reported in Table 6 in terms of p-value, estimated by the statistical software, as described above. It is evident from Table 6 that the relieving treatment makes the alloy stronger and more fragile 11 11 In Figure 4 XRD patterns of as sintered (AS) and heat-treated (CHT) samples are reported for comparison. Both samples are formed of a mixture of the ε-Co (hcp, a=0.25031 nm, c=0.40605 nm, ICDD card n. 5-727) and γ-Co (fcc, a=0.35447, ICDD card n. 15-806) phases. However, in the XRD pattern of the CHT sample in addition to the diffraction peaks of the ε-Co and γ-Co phases, low intensity peaks (evidenced by full dots in Figure 4) are visible. 3.3 X-ray Diffraction (XRD) In order to follow the structural modifications induced on the material by the production process and the subsequent heat treatments, XRD investigations were carried out on the following samples: a) the metallic EOS CobaltChrome SP2 powder, b) the as sintered (AS) sample and c) the sintered sample submitted to the relieving and firing thermal treatments (CHT). Results of XRD measurements performed on the metallic powder have been already discussed in a previous paper (Barucca et al. 2015). Metallic powder is entirely composed of γ-Co (fcc) phase with an estimated lattice parameter a=0.3586±0.0002 nm (Barucca et al. 2015). 12 12 Figure 4 about here Quantitative information on the crystallographic structure of the ε-Co and γ-Co phases as well as their relative volume fraction were obtained by peak analysis of the two XRD patterns reported in Figure 4. The results of peak analysis are summarised in Table 7, where the experimental results obtained for the metallic powder, already discussed in our previous paper (Barucca et al. 2015), are reported for comparison. Table 7 about here The lattice parameters of the ε-Co and γ-Co phases were estimated from the angular position of the diffraction peaks. On the contrary, the volume fraction of the hcp phase (fhcp) was calculated from the integrated intensities of the γ (200) and ε (101) peaks by using the method by Saude and Gillaud (Balagna et al. 2012). The experimental uncertainties in Table 7 were calculated from the statistical errors provided by the peak analysis software. The peak analysis of the low intensity peaks (evidenced by full dots in Figure 4) in the CHT pattern of Figure 4 suggest the formation of an hcp compound with estimated lattice parameters a=0.4734±0.0002 nm and c=0.7661±0.0003 nm. 3.4 Transmission electron microscopy (TEM, STEM, HRTEM) and microanalysis (EDX) 13 TEM observations of the AS and CHT samples confirm the simultaneous presence of the ε-Co and γ-Co phases. In both samples, the two phases give rise to a peculiar structure formed of alternating ε-Co and γ-Co lamellae. Figure 5 shows the lamellar structure in both samples. Figure 5 about here Figure 5a is a TEM bright field image of the AS sample taken in <110>γ zone axis orientation. Lamellae are parallel to each other with a width ranging from 5 to 100 nm. The corresponding selected area electron diffraction (SAED) pattern of the AS sample is shown in the inset of Figure 5a. The lattice cells due to the fcc γ-phase (dashed line) and to the hcp ε-phase (dotted line) are evidenced in the SAED pattern in Figure 5a. Relative orientation of the two lattice cells allows deducing the following Shoji-Nishiyama (Nishiyama 1978) orientation relationships between the ε- Co and γ-Co lamellae: {001}ε // {111}γ <100>ε // <1-10>γ For comparison, an HRTEM image of the CHT sample is shown in Figure 5b. Also in this case, the lamellar structure is clearly visible. A Fourier analysis (FFT) performed on different regions (squares in Figure 5b) of two adjacent lamellae allowed obtaining the diffraction patterns reported in the insets of Figure 5b. The inset in the upper left corner shows the diffraction pattern of the fcc γ-Co phase (lattice cell evidenced by dashed line). On the contrary, the inset in the lower right corner of Figure 5b is the diffraction pattern of the hcp ε-Co phase with the lattice cell evidenced by dotted line. From the two diffraction patterns, one can conclude that the orientation relationships between the ε-Co and γ-Co adjacent lamellae in the CHT sample are unaltered with respect to the AS sample. Concerning average size of lamellae, TEM observations evidenced a slightly increase in the CHT sample with width now ranging from 10 to 120 nm. 14 14 The most evident effect induced by the thermal treatments performed on the Co alloy under study regard number and density of precipitates. Figure 6 shows the precipitates distribution in the AS (Figure 6a) and CHT (Figure 6b) samples, respectively. Figure 6 about here From the STEM - HAAD images of Figure 6 can be appreciated the larger amount of precipitates present in the CHT sample (Figure 6b) in comparison to the AS one (Figure 6a). In Figure 6a (AS sample) two main types of precipitates are visible: coarse (bright) and small spherical (dark) precipitates. On the contrary, in Figure 6b (CHT sample) three different shapes can be distinguished: coarse (bright), small spherical (dark) and elongated (bright) precipitates. In the AS sample (Figure 6a) size of coarse precipitates ranges from 40 to 100 nm, while spherical dark ones have an average size of 10 nm and are often associated to coarse precipitates. In the CHT sample (Figure 6b) coarse precipitates range from 60 to 250 nm, elongated precipitates have length between 125 and 440 nm and the average size of small spherical ones is 13 nm. Small spherical precipitates also in the CHT sample are often associated to coarser ones. EDX analysis performed during TEM and STEM observations allowed studying the chemical composition of the different precipitates. An elemental mapping analysis of the CHT sample performed by EDX signals of Si, Cr, Co, Mo and W is reported in Figure 7. Figure 8 about here Figure 8a shows a TEM bright field image of a coarse precipitate with the corresponding SAED pattern (inset). On the contrary, the HRTEM image of a coarse precipitate is reported in Figure 8b together with the FFT pattern (inset) obtained from the area of sample enclosed in the square. Crystallographic information deduced from SAED and FFT patterns in Figure 8 suggest an hcp structure of the coarse precipitate. Similar results obtained also for the elongated precipitates suggest a substantial identical crystallographic structure (hcp) for differently shaped precipitates. Small spherical precipitates, often associated to coarser ones, are Si-rich inclusions already reported in literature (Giacchi et al. 2012). Figure 7 about here It is evident from Figure 7 that coarse and elongated precipitates have almost the same composition while small spherical precipitates are mainly composed of Si even when they are associated to coarser ones. With respect to the Co matrix, coarse and elongated precipitates reveal a higher concentration of Mo and W. 15 Quantitative results of the EDX analysis performed on matrix and on a large number of coarse, elongated and spherical precipitates are shown in Table 8, where the average experimental concentration value for each element is reported with the corresponding standard deviation. Table 8 about here In order to further investigate the nature of coarse and elongated precipitates, SAED analyses and HRTEM observations were carried out on a number of different precipitates. The results obtained are summarised in Figure 8. Figure 8 about here Figure 8 about here 4. Discussion The Co-based material considered in this study is a typical low carbon alloy specifically developed by EOS GmbH Electro Optical System for biomedical applications (Table 2). 16 The metallic powder used as raw material for production of samples is entirely composed of the γ- Co (fcc) phase in form of spherical particles with size ranging from 4 to 80 m (Barucca et al. 2015). The laser sintering process, carried out by using the sintering parameters shown in Table 1, 16 melts the metallic powder and induces the martensitic γ-Co (fcc)  -Co (hcp) transformation (Saldivar-Garcia et al. 1999a, 1999b; Song et al. 2006). After solidification, the as-sintered (AS) sample is compact without any visible surface porosity. It is worth to note that the average roughness of all analysed samples shows an unexpected constant value, independently on the post- production treatment. (AS) In terms of microstructure, the AS sample shows a mixture of martensitic -Co (hcp) inside the metastable γ-Co (fcc) phase (Figure 4), with a volume fraction of the -Co (hcp) phase around 49% (Table 7). Lattice parameters of γ-Co (fcc) and -Co (hcp) phases in metal powder and AS sample (Table 7), estimated from the XRD patterns, are in close agreement with values reported in literature for similar compositions (Saldivar-Garcia and Lopez 2004). Furthermore, TEM observation of the AS sample shows -Co (hcp) phase forming an intricate network of small lamellae in γ-Co (fcc) matrix (Figure 5a), while SAED pattern (inset in Figure 5a) confirms the Shoji-Nishiyama (SN) orientation relationships between the two Co phases (Nishiyama 1978). The particular orientation relationships (SN) developed are responsible of the mechanical properties observed in the AS sample (Table 4) (Barucca et al. 2015). The SN orientation relationships are typically observed in Co alloys submitted to fatigue deformation where the strain-induced γ-Co (fcc) → -Co (hcp) martensitic transformation plays a prominent role (Koizumi et al. 2013; Mitsunobu et al. 2014; Yamanaka et al 2014). The stress relieving thermal treatment (RHT) carried out on AS samples just after the shot peening process induces modifications in the mechanical response of samples. From Table 4 it is evident that the alloy becomes harder and less ductile, with elongation at break decreasing from 9 to 6 %. Results of the t-test, listed in Table 6, further confirm the significant effect of the relieving treatment in making the alloy stronger and more fragile. 4. Discussion 17 Further modifications in the mechanical behaviour of the alloy are evident after the firing cycle. Although the firing operation leaves strength unchanged, it causes additional significant rise in 17 17 hardness and loss in ductility (Tables 4 and 6). Nevertheless, the final properties are, overall, well above those expected from the datasheet, in terms of both UTS and εb (compare Tables 3 and 4). Flexural tests of CHT and RHT samples show a slightly different behaviour with respect to tensile specimens (Table 5). In fact, while tensile strength is substantially unaffected by the firing cycle (Table 4), flexural strength decreases, by approximately 7% (Table 5), with a statistical significance just barely below the 0.05 threshold (Table 6). The last rows in Table 6 refer to t-test for hardness values, when tensile and flexural specimens subjected to the same treatment are compared. Results for p-values high above the level of 0.05 prove that tensile and flexural specimens are undifferentiated. Modifications of the mechanical behaviour are strictly linked to structural changes induced by RHT and CHT thermal treatments. As shown in Figure 4 and quantitatively reported in Table 7, heat treatments tend to stabilize the amount of martensitic -Co (hcp) phase (volume fraction increases from about 50% in the SA sample to 63% in the CHT state) as well as to induce formation of a hexagonal compound not present in the AS condition. Increase of total amount of martensitic -Co (hcp) phase in the CHT sample is accompanied by a limited increase of the size of -Co lamellae while the orientation relationships between -Co and γ- Co lamellae remain unaltered (Figure 5). Furthermore, XRD investigations (not reported here) performed in the unstrained shoulder and in proximity of highly strained gage region of the TENSAS sample evidenced a volume fraction increase of the martensitic -Co (hcp) phase from about 50% to about 65%, totally ascribable to strain induced martensitic transformation (Koizumi et al. 2013; Mitsunobu et al. 2014). It is worth to note that this latter result spans the same interval values of the martensitic transformation induced by heat treatments. Thus, volume fraction increase of the martensitic phase as well as slight size increase of its lamellar structure cannot fully justify the observed variations of mechanical response between AS and CHT samples. 18 18 On the other side, TEM/STEM observations evidenced in the CHT sample a massive formation of coarse and elongated precipitates (Figure 6b) having identical chemical composition (Table 8) independently of their own shape. The uniformity of composition is further confirmed in the EDX elemental mapping of Figure 7 where differently shaped precipitates show the same contrast level (same colour). Furthermore, HRTEM investigations evidenced an identical hcp crystallographic structure for both coarse and elongated precipitates (Figure 8). As far as the chemical composition of both coarse and elongated precipitates is concerned, it must be stressed that, with respect to matrix, the Mo and W content in the precipitates highly increases while the Si amount undergoes a more limited variation (Table 8). Chemical databases such as International Centre for Diffraction Data (The International Centre for Diffraction Data (ICDD), 12 Campus Boulevard, Newtown Square, PA 19073-3273 U.S.A., http://www.icdd.com) and Mat Navi (NIMS Materials Database – Inorganic Materials Database, Atom Work) (Xu et al. 2011) report two possible compounds compatible with the above crystallographic structure and elements content: a) a molybdenum-containing phase Co3Mo2Si, hcp with a=0.47 nm and c=0.767 nm (ICDD card n. 30- 449), and b) a tungsten-containing phase WCo1.5Si0.5, hcp with a=0.4718 nm and c=0.76 nm (Atom Work). Therefore, crystallographic and compositional results suggest that the post-production treatments are responsible of formation and growth of coarse and elongated precipitates having an hcp structure, with lattice parameters a=0.4734±0.0002 nm and c=0.7661±0.0003 nm and a composition resembling Co3(Mo,W)2Si, with possible interchange of Mo and W atoms in the crystallographic lattice. To our better knowledge, the presence of such coarse and elongated precipitates is reported here for the first time. 19 Actually, a number of paper in literature report the presence of precipitates with various morphology (blocky, star-like, lamellar) in biomedical Co-Cr alloys produced by conventional processes. In particular, the contemporary presence of differently shaped precipitates was clearly evidenced while chemical extraction techniques allowed their detailed crystallographic and compositional characterisation (Mineta et al. 2010; Alfirano et al. 2011). A complete review of 19 contrary, total amount and size of martensitic lamellae seem to have a limited influence on modifications of the alloy mechanical response observed between AS and CHT states. In this case, the main variations of the mechanical behaviour can be ascribed to formation and growth of coarse and elongated precipitates that takes place during the stress relieving heat treatment and the subsequent firing cycle. morphology and composition of precipitates observed thus far in Co-Cr biomedical alloys is reported in the paper by Narushima et al. (2013) where the influence of alloying elements such as carbon, nitrogen, silicon and manganese is also investigated. Always from this paper, it follows that precipitates with identical or similar composition can occur in the same alloy with different morphologies. On the contrary, the chemical composition and the crystallographic structure of the coarse and elongated precipitates observed here do not match any known phase already reported in literature for biomedical Co-Cr-W systems (Narushima et al. 2013). Although this latter result can be almost completely ascribed to the chemical composition of our alloy (Table 2), it cannot be excluded some influence of the laser sintering production process. The presence of isolated Si inclusions or associated to larger precipitates, typically carbides in carbon containing alloys, is well known in literature (Yamanaka et al. 2015; Giacchi et al. 2011, 2012). As evident in Figures 6 and 7, in our case Si inclusions, the small dark spherical particles, form as isolated precipitates or, more frequently, associated to both coarse and elongated precipitates. It cannot be excluded that Si inclusions can have a role in nucleation and growth of coarse and elongated precipitates. The rupture surfaces shown in Figure 3 contribute to explain the change in the mechanical response due to the heat treatments, towards a stronger and more fragile behaviour. Final microstructure exhibits clear signs of fragile failure modes and diffused cracks (Figure 3b), which is consistent with precipitates acting as stress intensifiers and crack initiators. In addition, the decrease of γ-Co (fcc) lattice parameters from AS to CHT condition (Table 7) suggests that the contraction trend of the matrix may be hampered by the presence of precipitates. Consequently, this would cause a tensile stress state in the matrix. 20 Summarising the complete set of results, one can conclude that high strength and hardness values measured in AS condition are mainly due to the intricate network of -Co (hcp) lamellae formed in the γ-Co (fcc) matrix during sintering and solidification processes (Barucca et al. 2015). On the 20 reported here for the first time, and small spherical Si-rich inclusions. Quasi-cleavage mechanisms and cracks are evident from the rupture surfaces;  XRD investigations carried out in conjunction with TEM/STEM/HRTEM observations and EDX analysis show that the coarse and elongated precipitates observed here for the first time have a hcp structure with lattice parameters a=0.4734±0.0002 nm and c=0.7661±0.0003 nm and a possible composition resembling Co3(Mo,W)2Si.  XRD investigations carried out in conjunction with TEM/STEM/HRTEM observations and EDX analysis show that the coarse and elongated precipitates observed here for the first time have a hcp structure with lattice parameters a=0.4734±0.0002 nm and c=0.7661±0.0003 nm and a possible composition resembling Co3(Mo,W)2Si. Complex post-production treatments eventually associated to strain-induced γ-Co (fcc) → -Co (hcp) martensitic transformation can be used to induce structural modifications capable of producing controlled changes in the mechanical response of laser sintered Co-Cr-Mo-W biomedical alloys. Acknowledgements The research leading to these results has received funding from the European Union Seventh Framework Programme under grant agreement 312483 - Esteem2 (integrated infrastructure initiative–I3). The authors kindly acknowledge MSc. Adam Gruszczynski (AGH-UST) and A. Di Cristoforo (UNIVPM) for FIB lamellae and TEM sample preparation. Conclusions A Co-Cr-Mo-W alloy specifically developed for biomedical applications has been produced by Direct Metal Laser Sintering (DMLS). In order to investigate the effects of complex post- production treatments, the alloy was submitted to shot-peening, stress relieving and firing treatments. Temperature values as high as 950 °C were reached during the thermal treatments causing modifications of alloy microstructure and mechanical behaviour. The main results obtained in this study can be summarised as follows:  samples are compact without any visible porosity with an average surface roughness value independent on the post-production treatment;  the as-sintered sample is formed of an intricate network of -Co (hcp) lamellae in the γ-Co (fcc) matrix responsible of the high UTS and hardness values. Rupture surfaces evidence ductile failure modes;  the stress relieving treatment (RHT) increases both UTS and hardness while decreases ductility;  the firing cycle performed after the RHT treatment produces a further loss in ductility and a significant hardness increase while leaving strength unchanged. The alloy microstructure is still formed of slightly grown -Co (hcp) lamellae in the γ-Co (fcc) matrix to which superimposes a massive precipitation of coarse and elongated precipitates, which are 21 21 Balagna, C., Spriano, S., Faga, M.G., 2012. 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Mat. 21, 67–76. 25 Figure and table captions Table 1 Standard sintering parameters used for DMLS Table 2 Nominal powder composition (wt%) Table 3 Physical and mechanical properties in the material datasheet, after stress relieving, oxide fire simulation (5 min at 950 °C) and ceramic fire simulation (4 x 2 min at 930 °C) procedures according to EN ISO 22674. Table 4 Average value (AV) and standard deviation (SD) of ultimate tensile strength (UTS), strain at break (εb) and Rockwell A hardness (HRA) of the tensile specimens (TENS) under different condition. Table 5 Average value (AV) and standard deviation (SD) of Rockwell A hardness (HRA) and transverse rupture strength (TRS) of the flexural specimens. Table 6 p-values resulting from the t-test for UTS, εb, HRA and TRS among the groups AS, RHT and CHT. Records below the level of significance of 0.05 are underlined. Table 7 Experimental results of the peak analysis performed on the XRD spectra of Figure 4. 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A 527, 6494-6500. 26 26 Figure and table captions Thermal cycle used for fire simulation. The cooling phase between each firing step is not represented. Figure 3 SEM images of rupture surface: a) TENSAS, b) TENSCHT. SEM images of rupture surface: a) TENSAS, b) TENSCHT. Figure 4 XRD patterns of the as sintered (AS) and heat treated (CHT) samples. 27 27 Table 1 Table 1 Table 1 Standard sintering parameters used for DMLS laser power 200W laser spot diameter 0.200 mm Scan speed up to 7.0 m/s Building speed 2-20 mm3/s Layer thickness 0.020 mm Protective atmosphere max 1.5% oxygen Table 1 Standard sintering parameters used for DMLS laser power 200W laser spot diameter 0.200 mm Scan speed up to 7.0 m/s Building speed 2-20 mm3/s Layer thickness 0.020 mm Protective atmosphere max 1.5% oxygen Figure 5 TEM images taken in <110>γ zone axis orientation showing the lamellar structure of samples: a) AS sample – inset: corresponding SAED pattern, b) CHT sample – insets: Fourier analysis of the squared regions. Figure 6 STEM images of the samples microstructure: a) AS condition, b) CHT condition. Figure 7 STEM-EDX elemental mapping obtained with signals from Si, Cr, Co, Mo and W. Figure 8 Coarse precipitates: a) TEM bright field and corresponding SAED pattern (inset), b) HRTEM image and corresponding FFT (inset) of the squared region. Figure 5 TEM images taken in <110>γ zone axis orientation showing the lamellar structure of samples: a) AS sample – inset: corresponding SAED pattern, b) CHT sample – insets: Fourier analysis of the squared regions. Figure 5 TEM images taken in <110>γ zone axis orientation showing the lamellar structure of samples: a) AS sample – inset: corresponding SAED pattern, b) CHT sample – insets: Fourier analysis of the squared regions. Figure 6 STEM images of the samples microstructure: a) AS condition, b) CHT condition. Figure 7 STEM-EDX elemental mapping obtained with signals from Si, Cr, Co, Mo and W. Figure 8 Coarse precipitates: a) TEM bright field and corresponding SAED pattern (inset), b) HRTEM image and corresponding FFT (inset) of the squared region. 28 28 Table 1 Standard sintering parameters used for DMLS Table 1 Standard sintering parameters used for DMLS laser power 200W laser spot diameter 0.200 mm Scan speed up to 7.0 m/s Building speed 2-20 mm3/s Layer thickness 0.020 mm Protective atmosphere max 1.5% oxygen 29 29 Table 2 Table 2 Nominal powder composition (wt%) Co Cr Mo W Si Fe Mn 63.8 24.7 5.1 5.4 1.0 < 0.5 < 0.1 30 30 Table 3 Table 3 Physical and mechanical properties in the material datasheet, after stress relieving, oxide fire simulation (5 min at 950 °C) and ceramic fire simulation (4 x 2 min at 930 °C) procedures according to EN ISO 22674. Coefficient of thermal expansion [K-1] 14.3·10-6 Density [kg/dm3] 8.5 UTS [MPa] 1350 Rp0.2 [MPa] 850 Elongation at break 3% Young’s Modulus [GPa] 200 HV10 420 Coefficient of thermal expansion [K-1] 14.3·10-6 Density [kg/dm3] 8.5 UTS [MPa] 1350 Rp0.2 [MPa] 850 Elongation at break 3% Young’s Modulus [GPa] 200 HV10 420 31 31 Table 4 SD 1.8 1.8 1.5 Table 5 Table 5 Table 4 Table 4 Average value (AV) and standard deviation (SD) of ultimate tensile strength (UTS), strain at break (εb) and Rockwell A hardness (HRA) of the tensile specimens (TENS) under different condition. UTS (MPa) εb (%) HRA AV SD AV SD AV SD TENSAS 1340 20 9.0 0.8 72.8 1.8 TENSRHT 1430 10 6.2 0.4 74.2 1.8 TENSCHT 1440 20 4.7 0.3 75.8 1.5 UTS (MPa) εb (%) HRA AV SD AV SD AV SD TENSAS 1340 20 9.0 0.8 72.8 1.8 TENSRHT 1430 10 6.2 0.4 74.2 1.8 TENSCHT 1440 20 4.7 0.3 75.8 1.5 32 32 Table 5 Table 5 Average value (AV) and standard deviation (SD) of Rockwell A hardness (HRA) and transverse rupture strength (TRS) of the flexural specimens. HRA TRS (MPa) AV SD AV SD FLEXRHT 73.2 0.4 2700 25 FLEXCHT 76.0 0.5 2500 157 HRA TRS (MPa) AV SD AV SD FLEXRHT 73.2 0.4 2700 25 FLEXCHT 76.0 0.5 2500 157 HRA TRS (MPa) AV SD AV SD FLEXRHT 73.2 0.4 2700 25 FLEXCHT 76.0 0.5 2500 157 33 33 Table 6 Table 6 p-values resulting from the t-test for UTS, εb, HRA and TRS among the groups AS, RHT and CHT. Records below the level of significance of 0.05 are underlined. Table 6 p-values resulting from the t-test for UTS, εb, HRA and TRS among the groups AS, RHT and CHT. Records below the level of significance of 0.05 are underlined. p-values UTS (MPa) εb (%) HRA TRS TENSAS -TENSRHT 0.00 0.00 0.01 - TENSAS - TENSCHT 0.00 0.00 0.00 - TENSCHT -TENSRHT 0.13 0.00 0.00 - FLEXCHT -FLEXRHT - - 0.00 0.04 TENSRHT - FLEXRHT - - 0.28 - TENSCHT – FLEXCHT - - 0.77 - 34 34 Table 7 Table 7 Experimental results of the peak analysis performed on the XRD spectra of Figure 4. The estimated lattice parameter of the γ-Co phase in the powder is reported for comparison. Sample Lattice parameter (fcc) γ-Co (nm) Lattice parameters (hcp) ε-Co (nm) Volume fraction hcp fhcp Powder a= 0.3586±0.0002 - - AS a= 0.3589±0.0001 a= 0.2539±0.0002 c= 0.415±0.003 c/a= 1.636 0.49±0.03 CHT a= 0.3574±0.0006 a= 0.2534±0.0004 c= 0.408±0.002 c/a= 1.612 0.625±0.005 35 35 Table 8 Table 8 Results of STEM-EDX analysis performed on matrix and precipitates. Element concentration in wt%. Co Cr Mo W Si Matrix 64.7 ± 0.6 23.3 ± 0.2 3.2 ± 0.2 5.7 ± 0.5 3.16 ± 0.06 Coarse and elongated precipitates 36 ± 2 13± 2 21 ± 4 26 ± 3 5 ± 2 Spherical precipitates 24.8 ± 0.8 15.1 ± 0.6 1.7 ± 0.3 1.7 ± 0.1 56.7 ± 0.6 Table 8 Results of STEM-EDX analysis performed on matrix and precipitates. Element concentration in wt%. Co Cr Mo W Si Matrix 64.7 ± 0.6 23.3 ± 0.2 3.2 ± 0.2 5.7 ± 0.5 3.16 ± 0.06 Coarse and elongated precipitates 36 ± 2 13± 2 21 ± 4 26 ± 3 5 ± 2 Spherical precipitates 24.8 ± 0.8 15.1 ± 0.6 1.7 ± 0.3 1.7 ± 0.1 56.7 ± 0.6 Table 8 Results of STEM-EDX analysis performed on matrix and precipitates. Element concentration in wt%. Co Cr Mo W Si Matrix 64.7 ± 0.6 23.3 ± 0.2 3.2 ± 0.2 5.7 ± 0.5 3.16 ± 0.06 Coarse and elongated precipitates 36 ± 2 13± 2 21 ± 4 26 ± 3 5 ± 2 Spherical precipitates 24.8 ± 0.8 15.1 ± 0.6 1.7 ± 0.3 1.7 ± 0.1 56.7 ± 0.6 Table 8 Results of STEM-EDX analysis performed on matrix and precipitates. Element concentration in wt%. 36 36 a) FLEX b) TENS Figure 1 Figure 1 Geometry of samples for mechanical tests: a) flexural (FLEX), b) tensile (TENS). Figure 1 a) FLEX b) TENS Figure 1 Geometry of samples for mechanical tests: a) flexural (FLEX), b) tensile (TENS). 37 37 Figure 2 Figure 2 Thermal cycle used for fire simulation. The cooling phase between each firing step is not represented. Figure 2 Figure 2 Thermal cycle used for fire simulation. The cooling phase between each firing step is not represented. 38 38 he squared regions. Figure 3 Figure 3 Figure 3 SEM images of rupture surface: a) TENSAS, b) TENSCHT. Figure 3 SEM images of rupture surface: a) TENSAS, b) TENSCHT. Figure 3 SEM images of rupture surface: a) TENSAS, b) TENSCHT. 39 39 Figure 4 Figure 4 XRD patterns of the as sintered (AS) and heat treated (CHT) samples. Figure 4 XRD patterns of the as sintered (AS) and heat treated (CHT) samples. 40 Figure 5 Figure 5 Figure 5 TEM images taken in <110>γ zone axis orientation showing the lamellar structure of samples: a) AS sample – inset: corresponding SAED pattern, b) CHT sample – insets: Fourier analysis of the squared regions. 41 41 Figure 6 Figure 6 Figure 6 STEM images of the samples microstructure: a) AS condition, b) CHT condition. Figure 6 STEM images of the samples microstructure: a) AS condition, b) CHT condition. Figure 6 STEM images of the samples microstructure: a) AS condition, b) CHT condition. Figure 6 STEM images of the samples microstructure: a) AS condition, b) CHT condition. 6 STEM images of the samples microstructure: a) AS condition, b) CHT condition. 42 Figure 7 re 7 STEM-EDX elemental mapping obtained with signals from Si, Cr, Co, Mo and W. Figure 7 Figure 7 STEM-EDX elemental mapping obtained with signals from Si, Cr, Co, Mo and W. Figure 7 STEM-EDX elemental mapping obtained with signals from Si, Cr, Co, Mo and W. Figure 7 STEM-EDX elemental mapping obtained with signals from Si, Cr, Co, Mo and W. 43 43 Figure 8 Figure 8 Coarse precipitates: a) TEM bright field and corresponding SAED pattern (inset), b) HRTEM image and corresponding FFT (inset) of the squared region. Figure 8 Coarse precipitates: a) TEM bright field and corresponding SAED pattern (inset), b) HRTEM image and corresponding FFT (inset) of the squared region. Figure 8 Coarse precipitates: a) TEM bright field and corresponding SAED pattern (inset), b) HRTEM image and corresponding FFT (inset) of the squared region. Figure 8 Coarse precipitates: a) TEM bright field and corresponding SAED pattern (inset), b) HRTEM image and corresponding FFT (inset) of the squared region. HRTEM image and corresponding FFT (inset) of the squared region. Highlights  Co-based alloy with improved mechanical properties are produced by DMLS  A network of  lamellae in γ matrix is present in the as-sintered sample  Thermal treatments increase both UTS and hardness while decreasing ductility  Mechanical response is influenced by the massive precipitation of Co3(Mo,W)2Si phase 44 44 As sintered Thermal treatments induce precipitation of Si-rich inclusions (arrowed) and hcp Co3(Mo,W)2Si phase that reduce ductility and increase strength and hardness. As sintered Heat-treated Heat-treated As sintered As sintered Heat-treated As sintered As sintered Thermal treatments induce precipitation of Si-rich inclusions (arrowed) and hcp Co3(Mo,W)2Si phase that reduce ductility and increase strength and hardness. 45
https://openalex.org/W4391785892
https://www.qeios.com/read/JF6SCT/pdf
English
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Review of: "Growing Confidence and Remaining Uncertainty About Animal Consciousness"
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REVIEW OF THE PAPER: Growing Confidence and Remaining Uncertainty About Animal Consciousness Animal Consciousness This paper is well written and shows that the author knows the literature about the question well. The aim of the paper is also well defined and interesting. Below, I will review some of the points that, in my opinion, present problems. Below, I will review some of the points that, in my opinion, present problems. 1. The author says explicitly that he agrees with the 4E approach (p 4), but he focuses only on the structure of the nervous system (analyzing and comparing the types of brains of different animal taxa) and ignores all along the paper the relation between the brain and the body in each case. What can be inferred from this is that for him, this relationship is not relevant to explain the emergence and evolution of consciousness. 1. The author says explicitly that he agrees with the 4E approach (p 4), but he focuses only on the structure of the nervous system (analyzing and comparing the types of brains of different animal taxa) and ignores all along the paper the relation between the brain and the body in each case. What can be inferred from this is that for him, this relationship is not relevant to explain the emergence and evolution of consciousness. In p 8, for example, the author says that “Different theories of consciousness propose different mechanisms for raising the primary NCCs to conscious awareness (…) All three were proposed in the context of mammalian (and largely human) neuroanatomy, but their characteristics are generalizable enough to be applicable to any animal with a sufficiently complex nervous system” (the italics are mine). This is, in my view, a very hasty conclusion because it neglects the highly specific relation between the brain and body of the mammal class (and probably the whole vertebrate phylum), focusing exclusively on the neural/brain structure. And this is surprising, given that the author himself seems to claim an embodied view of the brain in p 4. It is significant that the author seems to reduce the neurophysiological domain to “the bioelectrical and neurochemical dynamics of the relevant circuits” (p 9). Now, if one thing is clear from what we know of human and mammal C, it is that it involves (and requires) a continuous, fine-tuned, and highly integrative control of the body. 1. Qeios, CC-BY 4.0 · Review, February 13, 2024 Alvaro Julian Moreno Bergareche1 Alvaro Julian Moreno Bergareche1 1 Universidad del País Vasco Potential competing interests: No potential competing interests to declare. Review of: "Growing Confidence and Remaining Uncertainty About Animal Consciousness" Alvaro Julian Moreno Bergareche1 REVIEW OF THE PAPER: In p 5, the author defends the idea that “consciousness is an evolved product of complex brains in complex bodies, so it is an emergent feature of a complex physical system (…) animals with sensory hierarchies can be conscious if they have four or more levels of neurons projecting to (and including) the highest processing area, or in some cases, just three levels (because in this type of hierarchical structure) … neural circuits combine diverse details and types of sensory input into one unified experience”. Yet, this argument sounds vague: how does this hierarchy work to generate a globally unified sense of self? How is this process related to the physiology of the animal? Which –if any— is the role Qeios ID: JF6SCT · https://doi.org/10.32388/JF6SCT 1/4 Qeios, CC-BY 4.0 · Review, February 13, 2024 of the radical architectural difference between vertebrate and invertebrate brains and –also- the different ways these brains are embodied? And in the different taxa that share the same number of neural levels, which –if any—is the role of the enormous differences in the size and the type of organization of the brain? None of these important questions is addressed. 2. In p 6, the author says that “Subsequent evolution relocated details of sensory perception and motor control to higher brain centers, now considered a neuroanatomical necessity for animals that perceive qualia at a high level of resolution and manage motion with fine motor control”. Yet, there is not a detailed explanation of what is specifically meant by a “high” sensorimotor control center. The majority of nervous systems have some form of sensorimotor control center, so terms like “high”, when presented as a fundamental substrate for consciousness (henceforth, C), need to be carefully explained. 2. In p 6, the author says that “Subsequent evolution relocated details of sensory perception and motor control to higher brain centers, now considered a neuroanatomical necessity for animals that perceive qualia at a high level of resolution and manage motion with fine motor control”. Yet, there is not a detailed explanation of what is specifically meant by a “high” sensorimotor control center. The majority of nervous systems have some form of sensorimotor control center, so terms like “high”, when presented as a fundamental substrate for consciousness (henceforth, C), need to be carefully explained. 3. Another problem is how the author approaches the evolutionary dimension of C in animals. REVIEW OF THE PAPER: If in different animal taxa the evolutionary history of C has been so different in terms of complexification and diversification of C, why not discuss and analyze this question? For example, the evolutionary history of amniotes has shown a very rich diversification in the forms and degrees of complexity of C, whereas that of coleoid cephalopods has shown a comparatively poor diversification and complexification. After all, comparing the diverse evolutionary paths of complexification and diversification of C in the different taxa is easy, and it is reasonable to hypothesize that there should be a relation between a specific form of neurophysiological structure and its evolutionary unfolding. If during hundreds of millions of years nothing qualitatively relevant happens in terms of complexification of C in a given phylum, while in another phylum the evolutionary history shows a powerful process of complexification of C, one can suspect that some important neurophysiological difference exists ab initio. And this testable fact could be a cue that relevant neurophysiological differences concerning C in the different taxa or phyla. Although the author deals specifically with the question of the evolution of C in pp 6-7 (SECTION: “The Evolutionary Imperative”), he does not address this important point. In fact, what he does is just to mention the different authors that claim the existence of C in certain groups of arthropods, cephalopods, and lower vertebrates. 4. It is surprising that, after having presented a whole set of arguments to show that there is a growing consensus in identifying a certain neurological structure as the basis of C, the author admits that much more work is necessary to clarify this question. As he says, “the gap between the phenomenological experience of consciousness and its neurological correlates remains controversial.” If so, how can it be said that “but plausible explanations relating mechanism to phenomenology are arising as the neuroscientific study of consciousness matures”? These different statements seem somewhat contradictory. 5. Another important question that the author seems to neglect is the different meanings of the term C in the literature of animal C. Actually, there is an increasing association of a minimal form of “sentience” with the existence of a “subjective experience”. Hence, it is important to discuss the distinction between C as a cognitive representational phenomenon, and (a much simpler concept of) C, as just a feeling of pain. REVIEW OF THE PAPER: As said, in the recent literature about animal C, there is an increasing tendency to use the term “sentience” for referring to the most basic form of C (see, for example, Damasio 1999; Denton et al. 2009; Godfrey-Smith 2016, Birch et al. 2021, to cite but a few). An example of basic sentience would be what Denton and colleagues (op cit) call "primordial emotions" – bodily feelings which 5. Another important question that the author seems to neglect is the different meanings of the term C in the literature of animal C. Actually, there is an increasing association of a minimal form of “sentience” with the existence of a “subjective experience”. Hence, it is important to discuss the distinction between C as a cognitive representational phenomenon, and (a much simpler concept of) C, as just a feeling of pain. As said, in the recent literature about animal C, there is an increasing tendency to use the term “sentience” for referring to the most basic form of C (see, for example, Damasio 1999; Denton et al. 2009; Godfrey-Smith 2016, Birch et al. 2021, to cite but a few). An example of basic sentience would be what Denton and colleagues (op cit) call "primordial emotions" – bodily feelings which Qeios ID: JF6SCT · https://doi.org/10.32388/JF6SCT 2/4 Qeios, CC-BY 4.0 · Review, February 13, 2024 register important metabolic conditions such as thirst, the need for salt, or the feeling of not having enough air; or pain, which for Godfrey-Smith (op cit) is perhaps the best case for motivating a divide between a broad sense of subjective experience and full-blown C. This last sense corresponds essentially to the concept of C presented in the list of seven characteristics that, according to Brofman and colleagues (2016), reflect the most accepted use of the term C among neurologists, cognitive psychologists, and philosophers. The sense used by the author in the 1-5 features of the abstract and in pp 2-4. This difference in the meaning of the term “subjective experience” is important because Brofman and colleagues and the author’s sense of C implies a cognitively more complex dimension than that of basic sentience. For example, Edelman’s (and Dehaene’s) concept of a “scene” implies a representational dimension that is lacking in Denton’s concept of basic sentience. Hence, this second sense of C is more basic and likely evolutionarily more primitive than the former. REVIEW OF THE PAPER: Now, this distinction is relevant for the discussion of what is the neurophysiological substrate of C. With the exception of coleoid cephalopods, a review of the literature shows that the more evolutionarily removed from mammals the examples are, the more imprecise and speculative the neurophysiological data supporting evidence for subjective experience (if this last term is understood in a full-blown sense). This is probably one of the reasons why the term “sentience” is increasingly used when looking for minimal forms of C (in lower vertebrates and/or certain invertebrates) where it is much more difficult to assess objectively the presence of any form of “representational” subjective experience than in animals evolutionarily closer to us. In any case, this search would probably be less difficult if we understand the concept of minimal C in the less demanding sense of Denton, Godfrey-Smith, and Birch, namely, as synonymous with sentience. As for the case of coleoid cephalopods, the number and detail of studies (both at the neurophysiological and at the behavioral level) supports a growing certainty that these animals can have some kind of subjective experience. But researchers are quite divided about what type of “consciousness” they can have, and more specifically, to what extent they can have a "unified self" (i.e., Carls-Diamante 2022). In fact, the author himself mentions this question. In sum, it is much more difficult to argue for the existence of this strong sense of C in evolutionarily distant animals (and even worse if they have very small brains). 1. Another surprising fact is the use of phrases at the end of the section that relativize and weaken the main argument developed by the author himself. For example, in p. 9, at the end of the section, the author says that “the applicability of currently proposed NCCs to animals with neural architectures very different from those on which most models are based will remain in question until mechanisms compatible with the diversity of nervous systems over a broader range of the animal kingdom are explored.” If so, why has he said that “their characteristics (of these models) are generalizable enough to be applicable to any animal with a sufficiently complex nervous system” just a few paragraphs before (p. 8)? It is really contradictory. And this contradiction concerns his main message (namely, that there is a growing consensus about the empirical distribution of C). the one hand what he has given with the other. Hence, I don’t see that it makes a significant contribution to the debate on this important issue. My recommendation is not publication. Qeios ID: JF6SCT · https://doi.org/10.32388/JF6SCT REVIEW OF THE PAPER: In sum, Even acknowledging that the article is well written and shows that the author knows the literature, it does not really offer a conclusive argument in support of the message contained in the abstract. In several places, the author takes back with Qeios ID: JF6SCT · https://doi.org/10.32388/JF6SCT 3/4 Qeios, CC-BY 4.0 · Review, February 13, 2024 Qeios, CC-BY 4.0 · Review, February 13, 2024 the one hand what he has given with the other. Hence, I don’t see that it makes a significant contribution to the debate on this important issue. My recommendation is not publication. the one hand what he has given with the other. Hence, I don’t see that it makes a significant contribution to the debate on this important issue. My recommendation is not publication. Qeios ID: JF6SCT · https://doi.org/10.32388/JF6SCT 4/4
https://openalex.org/W3039712416
https://link.springer.com/content/pdf/10.1007/s00440-020-00984-9.pdf
English
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On the number of excursion sets of planar Gaussian fields
Probability theory and related fields
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Dmitry Beliaev1 · Michael McAuley1 · Stephen Muirhead2,3 Received: 25 October 2018 / Published online: 6 July 2020 © The Author(s) 2020 B Michael McAuley mcauley@maths.ox.ac.uk Probability Theory and Related Fields (2020) 178:655–698 https://doi.org/10.1007/s00440-020-00984-9 Probability Theory and Related Fields (2020) 178:655–698 https://doi.org/10.1007/s00440-020-00984-9 2 Department of Mathematics, King’s College London, London, UK Abstract The Nazarov–Sodin constant describes the average number of nodal set components of smooth Gaussian fields on large scales. We generalise this to a functional describing the corresponding number of level set components for arbitrary levels. Using results from Morse theory, we express this functional as an integral over the level densities of different types of critical points, and as a result deduce the absolute continuity of the functional as the level varies. We further give upper and lower bounds showing that the functional is at least bimodal for certain isotropic fields, including the important special case of the random plane wave. Keywords Gaussian fields · Nodal set · Level sets · Critical points Mathematics Subject Classification 60G60 · 60G15 · 58K05 3 Present Address: School of Mathematical Sciences, Queen Mary University of London, London, UK 1 Mathematical Institute, University of Oxford, Oxford, UK Mathematics Subject Classification 60G60 · 60G15 · 58K05 Mathematics Subject Classification 60G60 · 60G15 · 58K05 Dmitry Beliaev was supported by the Engineering & Physical Sciences Research Council (EPSRC) Fellowship EP/M002896/1. Stephen Muirhead was supported by the EPSRC Grant EP/N009436/1 “The many faces of random characteristic polynomials”. 2 Department of Mathematics, King’s College London, London, UK 123 D. Beliaev et al. 656 1.1 The Nazarov–Sodin constant Let f : R2 →R be a continuous stationary planar Gaussian field normalised to have zero mean and unit variance. The nodal set of f is the random set N =  x ∈R2 : f (x) = 0  . Let κ : R2 →[−1, 1] denote the covariance kernel of f , i.e. κ(x) = E[ f (x) f (0)]. We assume throughout that κ is C4+, which ensures that almost surely f is C2+. Since κ is positive definite, continuous and κ(0) = 1, by Bochner’s theorem there exists a probability measure ρ such that κ(x) =  R2 ei⟨t,x⟩dρ(t); (1) (1) this is known as the spectral measure of f , and must be Hermitian (that is, ρ(−A) = ρ(A) for all Borel sets A). Since the distribution of f is uniquely determined by its covariance function (Kolmogorov’s theorem), (1) shows that the distribution of f is uniquely determined by ρ. The geometric properties of N are of interest, in part, because in the case that f is a random eigenfunction of the Laplacian they relate to a significant conjecture in the physics literature: the Berry conjecture [4]. A summary of this conjecture and other research on this topic may be found in [17]. One of the main analytical results concerning this set, due to Nazarov and Sodin [13,15], states that the number of components of N in a large domain scales like the area of the domain. Specifically, if NR denotes the number of components of N inside the centred ball of radius R > 0, then provided f is ergodic, there exists a constant cLS = cLS(ρ) ≥0 such that NR/(π R2) →cLS as R →∞, where convergence occurs almost surely and in L1. Nazarov–Sodin also obtained analogous results in higher dimensions and for Gaussian ensembles on manifolds [13]. In the case that f is not ergodic, it has been shown [10] (under the additional assumption that ρ has compact support) that the expected number of nodal components, scaled by the area, still converges, i.e. E[NR]/(π R2) →cLS as R →∞. Further, in [10] it was also shown that among fields with compactly supported spectral measures, the constant cLS varies continuously with ρ (in the weak- ∗topology). as R →∞. 1.1 The Nazarov–Sodin constant Further, in [10] it was also shown that among fields with compactly supported spectral measures, the constant cLS varies continuously with ρ (in the weak- ∗topology). 123 123 12 On the number of excursion sets of planar Gaussian fields 657 Theorem 1.2 Let f satisfy Conditions 1.1. For each ℓ∈R, there exists cLS(ρ, ℓ) ≥0 such that E[NLS,R(ℓ)] = cLS(ρ, ℓ) · π R2 + O (R) as R →∞. The constant implied by the O(·) notation may depend on ρ but is independent of ℓ. If f is also ergodic, then NLS,R(ℓ)/(π R2) →cLS(ρ, ℓ) 1.2 The main results The first contribution of this paper is to extend the results of Nazarov–Sodin and [10] to arbitrary levels. For u ∈R2 and R > 0 let B(u, R) be the ball of radius R centred at u and B(R) := B(0, R). Let Nℓ= {x ∈R2 : f (x) = ℓ} denote a level set of f and let NLS,R(ℓ) be the number of components of Nℓcontained in B(R) (i.e. those which intersect B(R) but not ∂B(R)). We consider fields satisfying the following assumptions: Conditions 1.1 The Gaussian field f satisfies: Conditions 1.1 The Gaussian field f satisfies: Conditions 1.1 The Gaussian field f satisfies: 1. For some ν > 0, f ∈C2+ν loc (R2) almost surely; 1. For some ν > 0, f ∈C2+ν loc (R2) almost surely; 2. ∇2 f (0) is a non-degenerate Gaussian vector (here, and later on, we treat ∇2 f a a vector of distinct partial derivatives ( fxx, fxy, fyy)); y yy 3. For any t ∈R2, if f (t) −f (0) is a non-degenerate Gaussian variable then ( f (t) −f (0), ∇f (t), ∇f (0)) is a non-degenerate Gaussian vector. y yy 3. For any t ∈R2, if f (t) −f (0) is a non-degenerate Gaussian variable then ( f (t) −f (0), ∇f (t), ∇f (0)) is a non-degenerate Gaussian vector. We note that these assumptions are quite minimal. The first condition holds if the covariance function κ is C4+ϵ for some ϵ > 2ν, or equivalently when  R2|λ|4+ϵ dρ(λ) < ∞. The second condition is equivalent to the support of ρ not being contained in the union of two lines through the origin. The third condition holds provided that the support of ρ is not too degenerate; in particular it holds if the support of ρ contains an open set. Theorem 1.2 Let f satisfy Conditions 1.1. For each ℓ∈R, there exists cLS(ρ, ℓ) ≥0 such that almost surely and in L1. Remark 1.4 Our use of the domain B(R) in the definitions of NLS,R(ℓ) and NES,R(ℓ) is mainly for simplicity, and after minor modifications the proofs of Theorems 1.2 and 1.3 go through equally well for rescaled copies of any bounded reference domain Ω ⊂R2, provided that Ω is convex and ∂Ω is piecewise smooth (and probably more generally as well). Moreover the limiting constants cLS and cES do not depend on Ω (after replacing the scaling factor π R2 by Area(Ω)R2). Remark 1.5 Theorem 1.3 can also be applied to lower excursion sets (the components of {x ∈R2 : f (x) ≤ℓ}) since f has symmetric distribution and {x ∈R2 : f (x) ≤ ℓ} = {x ∈R2 : −f (x) ≥−ℓ}. Theorem 1.3 can then be used to prove Theorem 1.2 by making use of Euler’s formula to show that the number of level set components NLS,R(ℓ) is equal to the number of upper and lower excursion set components in B(R) and a bounded error term (see the proof of Lemma 2.5 for details of this argument). The symmetry of f along with the observations in the previous remark immediately give the following corollary. Corollary 1.6 Let f satisfy Conditions 1.1. Then (1) cLS(ℓ) = cLS(−ℓ) for all ℓ∈R, (2) cLS(ℓ) = cES(ℓ) + cES(−ℓ) for all ℓ∈R, (3) cLS(0) = 2cES(0). Corollary 1.6 Let f satisfy Conditions 1.1. Then (1) cLS(ℓ) = cLS(−ℓ) for all ℓ∈R, (2) cLS(ℓ) = cES(ℓ) + cES(−ℓ) for all ℓ∈R, (3) cLS(0) = 2cES(0). Corollary 1.6 Let f satisfy Conditions 1.1. Then (1) cLS(ℓ) = cLS(−ℓ) for all ℓ∈R, (2) cLS(ℓ) = cES(ℓ) + cES(−ℓ) for all ℓ∈R, (3) cLS(0) = 2cES(0). Theorems 1.2 and 1.3 are, in isolation, only a modest improvement on previous results; they could be proven by slightly adapting the analysis in [10,13]. The main novelty of our work is to relate the functionals cLS and cES to the density of critical points of f of different types and at different levels. To state the relationship, we shall require, in particular, a classification of the saddle points of the field into two types: Definition 1.7 Let x0 be a saddle point of a function g : R2 →R such that there are no other critical points at the same level as x0. 1 A Gaussian field is said to be isotropic if its covariance function κ(x) can be expressed as a function of |x| where |·| denotes the Euclidean norm. almost surely and in L1. There is also interest in studying the number of excursion sets of Gaussian fields. Let NES,R(ℓ) denote the number of components of {x ∈R2 : f (x) ≥ℓ} contained in B(R). Theorem 1.3 Let f satisfy Conditions 1.1. For each ℓ∈R, there exists cES(ρ, ℓ) ≥0 such that Theorem 1.3 Let f satisfy Conditions 1.1. For each ℓ∈R, there exists cES(ρ, ℓ) ≥0 such that E[NES,R(ℓ)] = cES(ρ, ℓ) · π R2 + O (R) as R →∞. The constant implied by the O(·) notation may depend on ρ but is independent of ℓ. If f is also ergodic, then NES,R(ℓ)/(π R2) →cES(ρ, ℓ) 123 123 D. Beliaev et al. 658 almost surely and in L1. almost surely and in L1. We say that x0 is an upper connected saddle if it is in the closure of only one component of {x ∈R2 : g(x) > g(x0)}. Similarly, x0 is said to be lower connected if it is in the closure of only one component of {x ∈R2 : g(x) < g(x0)}. We say that a Gaussian field f satisfying Conditions 1.1 is periodic if there exists x ̸= 0 with κ(x) = 1 and is aperiodic otherwise. A Gaussian field which is aperiodic almost surely has no two critical points at the same level (see Lemma 2.4), and we will show that for such fields, all saddle points of f satisfy exactly one of the two conditions in Definition 1.7. In the case that f is periodic, we will require a more general definition for classifying saddle points as upper or lower connected, which is given in Sect. 3. Previous work has shown that, for sufficiently regular isotropic1 Gaussian fields, the expected number of local maxima, local minima or saddle points with value in a certain interval can be expressed as the integral of an explicit density function [5,6]. 123 12 On the number of excursion sets of planar Gaussian fields 659 In Sect. 3 we prove the following version of this result, which applies to more general Gaussian fields and also isolates the upper and lower connected saddles, but does not explicitly identify the densities. This proposition uses Definition 3.1 for upper and lower connected saddle points (which coincides with Definition 1.7 for aperiodic fields). Proposition 1.8 Let f satisfy Conditions 1.1. Then there exist non-negative functions pm+, pm−, ps+, ps−and ps on R such that the following holds. Let Ω ⊂R2 be compact and ∂Ω have finite Hausdorff-1 measure. Let ℓ∈R and let Nm+(ℓ), Nm−(ℓ), Ns+(ℓ), Ns−(ℓ) and Ns(ℓ) denote the number of local maxima, local minima, upper connected saddles, lower connected saddles and saddles of f in Ω with level above ℓrespectively. Then E[Nh(ℓ)] = Area(Ω)  ∞ ℓ ph(x) dx for h = m+, m−, s+, s−, s. Furthermore, these functions can be chosen to satisfy the relations pm+(x) = pm−(−x), ps+(x) = ps−(−x) and ps−+ ps+ = ps, and such that pm+, pm−and ps are continuous. for h = m+, m−, s+, s−, s. almost surely and in L1. Furthermore, these functions can be chosen to satisfy the relations pm+(x) = pm−(−x), ps+(x) = ps−(−x) and ps−+ ps+ = ps, and such that pm+, pm−and ps are continuous. The main theorem of the paper gives an explicit expression for the functionals cLS and cES in terms of the densities introduced in Proposition 1.8. As a result, we deduce the absolute continuity of these functionals as the level varies. In the case of spectral measures with compact support, we also show the joint continuity of these functionals with respect to both the level and the spectral measure. For each f satisfying Conditions 1.1 we can associate a spectral measure ρ via (1). Let Pc denote the collection of such measures that are supported in the closure of B(1). By rescaling the axes, the results we state for Pc can be shown to hold for any spectral measures with compact support. Theorem 1.9 Let f satisfy Conditions 1.1 and let pm+, pm−, ps+, ps−denote the densities specified in Proposition 1.8. Then Theorem 1.9 Let f satisfy Conditions 1.1 and let pm+, pm−, ps+, ps−denote the densities specified in Proposition 1.8. Then cLS(ρ, ℓ) =  ∞ ℓ pm+(x) −ps−(x) + ps+(x) −pm−(x) dx (2) (2) and cES(ρ, ℓ) =  ∞ ℓ pm+(x) −ps−(x) dx (3) (3) (3) and hence cLS and cES are absolutely continuous in ℓ. In addition cLS and cES are jointly continuous in (ρ, ℓ) ∈Pc × R where Pc is given the weak-∗topology. Remark 1.10 Theorem 1.9 provides a new tool with which to analyse the Nazarov– Sodinconstant.Sincethedensities pm+, pm−and ps areinprincipleknownbytheKac– Rice formula, our result demonstrates that the study of the Nazarov–Sodin constant can be reduced to an analysis of the density ps−(or, equivalently, ps+ = ps −ps−), which may be an easier quantity to handle. 123 D. Beliaev et al. 660 x1 {f = ℓ} {f = ℓ+ ϵ} x2 {f = ℓ} {f = ℓ−ϵ} Fig. 1 The number of excursion set components increases by one on passing through the lower connected saddle x1 and is constant on passing through the upper connected saddle x2 x1 {f = ℓ} {f = ℓ+ ϵ} {f = ℓ} Fig. almost surely and in L1. 1 The number of excursion set components increases by one on passing through the lower connected saddle x1 and is constant on passing through the upper connected saddle x2 Remark 1.11 Theorems 1.2, 1.3 and 1.9 can be generalised to many examples of non- Gaussian stationary random fields, since our proof requires only that the field satisfies certaintopologicalpropertiesalmostsurelyandissufficientlyregulartoapplytheKac– Riceformula(seethemoregeneralversionofourmainresultsstatedinPropositions2.6 and 2.7 below). We also believe Theorems 1.2, 1.3 and 1.9 could be generalised to higher dimen- sions, with the analogues of (2) and (3) still valid once the saddle points defining ps+ and ps−are replaced with critical points of index 1 and d −1 respectively (using a more general definition for upper and lower connected saddles). However, since some of the topological arguments in the proof increase in complexity in higher dimensions, in the interest of simplicity we do not pursue this generalisation here. Let us mention the key intuition behind the proof of Theorem 1.9. This theorem is based on a deterministic relationship between the excursion sets and critical points of sufficiently regular planar functions which is closely related to Morse theory. The excursion set of such a function above a level deforms continuously as the level increases, provided it does not pass through a critical point. In particular, there is no change in the number of components of the excursion set. When passing through a critical point, the topology of the excursion set changes in a way that is predicted by Morse theory and depends on the index of the critical point. For local maxima and local minima, the number of components of the excursion set changes in a consistent way. For saddle points, the change in the number of components is determined by whether it is upper connected or lower connected (see Fig. 1). Ultimately, Theorem 1.9 is a probabilisitic expression, in the setting of Gaussian fields, of this deterministic relationship between excursion set components and critical points of various types. We also briefly discuss the assumptions required for our results (i.e. Conditions 1.1). The assumption that f is almost surely C2+ν is necessary to apply the topological arguments that we borrow from Morse theory. The non-degeneracy assumptions on ∇2 f (0) and ( f (t) −f (0), ∇f (t), ∇f (0)) are used with the Kac–Rice theorem (see Sect. 2) to show certain non-degeneracy properties of f , which are again necessary for our topological arguments. 123 1.3 Bounds on cLS and cES It is possible to bound the expected number of excursion sets or level sets of stationary Gaussian fields using local estimates (i.e. estimates which depend only on the deriva- tives of κ at the origin). Here we outline how these estimates apply to the functionals cES and cLS and how they can be better characterised by making use of our results. Corollary 1.12 Let f be a Gaussian field satisfying Conditions 1.1 with covariance function κ. Then for ℓ∈R cES(ℓ) −cES(−ℓ) =  det ∇2κ(0) ℓ 2π φ(ℓ) (4) (4) almost surely and in L1. It is natural, therefore, to ask whether our results still apply when these assumptions are weakened. While we suspect that this is true, we are not able to show it with our methods. On the other hand, for a certain special case of non-trivial degenerate field – namely, the case where the spectral measure ρ is supported on at most five points – 123 On the number of excursion sets of planar Gaussian fields 661 we are able to give a complete description of cLS and cES, which in particular shows that the main results hold also in this case (see Sect. 1.4 below). we are able to give a complete description of cLS and cES, which in particular shows that the main results hold also in this case (see Sect. 1.4 below). where φ denotes the standard normal probability density function. It can also be shown that, for planar isotropic fields, if λ = √ 2 then κ(x) = J0(  8/η2 |x|), where J0 is the 0-th Bessel function. When η2 = 8, this particular field is known as the random plane wave (hereafter abbreviated to RPW), and is an object of great interest as it is the subject of the Berry conjecture [17]. Proposition 1.15 ([16]) Let f be an isotropic Gaussian field satisfying Conditions 1.1 with covariance function κ(x) = K(|x|). Then for ℓ≥0 cLS(ℓ) ≤λ2 πη2 φ(ℓ) 2 √ 3 −λ2 λ φ  λℓ/  3 −λ2  + ℓ  2Φ  λℓ/  3 −λ2  −1  . (6) where φ and Φ denote the standard normal probability density and cumulative density functions respectively. f p y In particular, if λ2 > 6e 2e+π ≈1.9 then by Corollary 1.12, cLS(0) < cLS(1) so that cLS is at least bimodal (that is, it has at least two local maxima). Remark 1.16 This result is proven in [16] using the method of ‘flip points’. Specifically, for any fixed direction u, the number of level set components at level ℓin a finite region is bounded above by half the number of points t in the region such that f (t) = ℓand ∂u f (t) = 0 where ∂u denotes the partial derivative in the direction u. The expected number of such points can be computed using the Kac–Rice formula. For isotropic fields, the choice of direction u is irrelevant. This method could also be applied to non-isotropic fields, and the bound could be optimised over the direction u, but we omit this for simplicity. It is also possible to construct an upper bound on cLS using the inequality 0 ≤ ps−≤ps and the densities pm+, ps and pm−, which are explicitly known for isotropic fields. However this bound is larger than the bound in (6) at all levels ℓ. Remark 1.17 Figure 2 shows lower and upper bounds for cES and cLS for the RPW based on (4), (6) and the equality cLS(ℓ) = cES(ℓ) + cES(−ℓ) (recall that λ = √ 2 and η2 = 8 in this case). Although these bounds are not particularly tight for ℓclose to 0, they quickly become accurate as |ℓ| increases. where φ denotes the standard normal probability density function. where φ denotes the standard normal probability density function. Remark 1.13 Since −∇2κ(0) is the covariance matrix of ∇f (0), this result implies that the difference cES(ℓ) −cES(−ℓ) depends only on the covariance of ∇f (0) and not on the covariance of higher order derivatives of f . Remark 1.14 This result was proven for isotropic fields in [16] using a winding number calculation. We prove this result using Theorem 1.9 which simplifies the calculation in the non-isotropic case and also highlights an interesting identity. Specifically, the proof is as follows: substituting (3) into the left hand side of (4) and using the symmetries pm+(x) = pm−(−x), ps+(x) = ps−(−x) and ps−+ ps+ = ps, we see that cES(ℓ) −cES(−ℓ) =  ∞ ℓ pm+(x) −ps(x) + pm−(x) dx = E(Nm+(ℓ) −Ns(ℓ) + Nm−(ℓ)) where Nm+(ℓ), Ns(ℓ) and Nm−(ℓ) are the number of critical points above level ℓas defined in Proposition 1.8 for Ω = B(1/√π). Lemma 11.7.1 of [1] states that this alternating sum is precisely the right hand side of (4). The alternating sum of critical points of a function of different indices above a certain level can be used to calculate the Euler characteristic of the excursion set of the function above the level (see Chapter 9 of [1]). When working on finite subsets of the plane, boundary effects must be considered, but these become negligible as the area of the subset increases. Formally, if we let ϕ(A) denote the Euler characteristic of a set A, then Theorem 11.7.2 of [1] gives an expression for the expected Euler characteristic of an excursion set on a cube (including boundary effects) which implies that cES(ℓ) −cES(−ℓ) = lim R→∞ 1 R2 E  ϕ  {x ∈[0, R]2 : f (x) ≥ℓ}  123 662 D. Beliaev et al. Corollary 1.12 immediately gives a lower bound for cES(ℓ) since cES(−ℓ) ≥0. We now consider an upper bound on cLS. This is most easily formulated when f is an isotropic Gaussian field. In this case its covariance function may be expressed as κ(x) = K(|x|). It is shown in [5] that λ := − √ 3K (2)(0)  K (4)(0) ∈  0, √ 2 and η2 := −6K (2)(0) K (4)(0) ∈[0, ∞) (5) (5) parameterise the critical point densities pm+, pm−and ps of isotropic fields in any dimension. where φ denotes the standard normal probability density function. For ℓ≥1 both the upper and lower bounds on cLS(ℓ) are within 5.1% of the true value (since the upper bound is within 5.1% of the lower bound) while for ℓ≥1.5 both bounds are within 0.6% of the true value. 123 On the number of excursion sets of planar Gaussian fields 663 Fig.2 Subfigures a and b show lower bounds (solid) and upper bounds (dashed) for cES(ρ, ℓ) and cLS(ρ, ℓ) respectively in the RPW case, for which λ = √ 2 and η2 = 8 Fig.2 Subfigures a and b show lower bounds (solid) and upper bounds (dashed) for cES(ρ, ℓ) and cLS(ρ, ℓ) respectively in the RPW case, for which λ = √ 2 and η2 = 8 Fig.3 Subfigures a and b show lower bounds (solid) and upper bounds (dashed) for cES(ρ, ℓ) and cLS(ρ, ℓ) respectively, where ρ is the density of a standard two-dimensional Gaussian random vector (i.e. the spectral measure of the Bargmann–Fock field), for which λ = 1 and η2 = 2 Fig.3 Subfigures a and b show lower bounds (solid) and upper bounds (dashed) for cES(ρ, ℓ) and cLS(ρ, ℓ) respectively, where ρ is the density of a standard two-dimensional Gaussian random vector (i.e. the spectral measure of the Bargmann–Fock field), for which λ = 1 and η2 = 2 For fields with lower values of λ, the percentage difference between the upper and lower bounds on cLS(ℓ) is a bit larger. Figure 3 shows the corresponding bounds on cES and cLS for the Bargmann-Fock field: the centred, planar Gaussian field with covariance kernel κ(x) = exp(−|x|2/2), for which λ = 1 and η2 = 2. In this case for ℓ≥1 the upper and lower bounds on cLS(ℓ) are within 40% of the true value, and the corresponding accuracies for ℓ≥1.5, ℓ≥2 and ℓ≥2.5 are 14%, 5% and 2% respectively. Remark 1.18 Corollary1.12statesthatcES(ρ, ℓ) > 0forallℓ> 0,andsocLS(ρ, ℓ) > 0 for all ℓ̸= 0, however the positivity of cLS(ρ, 0) is not addressed by this method. In [13] Nazarov and Sodin provide sufficient conditions for the positivity of cLS(ρ, 0) in terms of the spectral measure which cover almost all non-degenerate cases. Remark 1.18 Corollary1.12statesthatcES(ρ, ℓ) > 0forallℓ> 0,andsocLS(ρ, ℓ) > 0 for all ℓ̸= 0, however the positivity of cLS(ρ, 0) is not addressed by this method. 1.4 A special class of degenerate fields As mentioned above, our methods do not cover the case in which ρ is supported on the union of two lines through the origin. In this section we consider a certain special class of such fields for which we can give a more or less complete description of cLS and cES. If the spectral measure ρ is supported on a single line through the origin, it is well known that f is almost surely constant in one direction and so in particular has no compact level domains. In this case, all of our results hold trivially with cLS, cES and the critical point densities identically equal to zero. The simplest non-trivial case of degenerate ρ are spectral measures which are supported on four or five points. In this case, we can compute cES and cLS explicitly. Recall that a random variable Y is said to be Rayleigh distributed with parameter σ 2 > 0, denoted Y ∼Ray(σ), if P(Y ≤x) = 1 −e−x2/(2σ 2) for all x ≥0. Proposition 1.20 Let f be the Gaussian field with spectral measure Proposition 1.20 Let f be the Gaussian field with spectral measure ρ = αδ0 + β 2 (δK + δ−K ) + γ 2 (δL + δ−L) where β, γ > 0, α = 1 −β −γ ≥0 and K, L ∈R2 are linearly independent. Then E[NES,R(ℓ)] = cLS(ℓ) · π R2 + O(R) where β, γ > 0, α = 1 −β −γ ≥0 and K, L ∈R2 are linearly independent. Then E[NES,R(ℓ)] = cLS(ℓ) · π R2 + O(R) where β, γ > 0, α = 1 −β −γ ≥0 and K, L ∈R2 are linearly independent. Then E[NES,R(ℓ)] = cLS(ℓ) · π R2 + O(R) E[NES,R(ℓ)] = cLS(ℓ) · π R2 + O(R) and and and E[NLS,R(ℓ)] = cES(ℓ) · π R2 + O(R) where cLS(ℓ) = |K × L| · P (|Y1 −Y2| ≤ℓ+ X0 ≤Y1 + Y2) , cES(ℓ) = |K × L| · P (|Y1 −Y2| ≤|ℓ+ X0| ≤Y1 + Y2) , × denotes the cross product, X0 ∼N(0, α), Y1 ∼Ray(√β), Y2 ∼Ray(√γ ) and X0, Y1, Y2 are independent. Moreover the constants implied by the O(·) notation in these expressions are independent of ℓ. × denotes the cross product, X0 ∼N(0, α), Y1 ∼Ray(√β), Y2 ∼Ray(√γ ) and X0, Y1, Y2 are independent. where φ denotes the standard normal probability density function. In [13] Nazarov and Sodin provide sufficient conditions for the positivity of cLS(ρ, 0) in terms of the spectral measure which cover almost all non-degenerate cases. The only current quantitative lower bound for cLS(ρ, 0) has been proven for the RPW. In this case it has been shown that cLS(0) ≥1.1 × 10−5 (see [9]) although numerical simulations suggest that cLS(0) ≈0.0589/(4π) ≈0.00469 (see [3] and references therein). Although the bounds in the previous two corollaries can be proven by local methods (i.e. without using our analysis of saddle points) there is no scope for improving these estimates using the same methods. However it may be possible to get tighter bounds on cLS and cES through Theorem 1.9 and a better characterisation of ps−. Using the explicitly known densities pm+, pm−and ps along with Theorem 1.9 allows us to derive some monotonicity properties of cES and cLS. 123 664 D. Beliaev et al. Corollary 1.19 Let f satisfy Conditions 1.1 and λ be defined as above. Then cES(ρ, ℓ) and cLS(ρ, ℓ) are strictly decreasing in ℓfor ℓ> √ 2/λ, so that any local maxima of cLS must be contained in [− √ 2/λ, √ 2/λ] and any local maxima of cES must be contained in (−∞, √ 2/λ]. If λ = √ 2, then cES(ℓ) is non-decreasing on (−∞, 0] and strictly decreasing on [1, ∞), so that any strict local maxima of cES must be contained in [0, 1]. If λ = √ 2, then cES(ℓ) is non-decreasing on (−∞, 0] and strictly decreasing o [1, ∞), so that any strict local maxima of cES must be contained in [0, 1]. 1.4 A special class of degenerate fields Moreover the constants implied by the O(·) notation in these expressions are independent of ℓ. So in particular, cLS(ℓ) = 0 if and only if ℓ= α = 0. If cLS(ℓ) ̸= 0 then NLS,R(ℓ)/(π R2) converges in L1 to a non-constant random variable and hence 123 On the number of excursion sets of planar Gaussian fields 665 does not converge a.s. to a constant, and this statement also holds for cES and NES,R(ℓ)/(π R2). does not converge a.s. to a constant, and this statement also holds for cES and NES,R(ℓ)/(π R2). Furthermore there exist functions pm+, ps−, pm−, ps+ and ps satisfying the con- clusions of Proposition 1.8, and these are defined by pm+(x) = pm−(−x) = |K × L| · pX0+Y1+Y2(x) ps−(x) = ps+(−x) = |K × L| · pX0+|Y1−Y2|(x) where pZ denotes the probability density of a random variable Z. Therefore the equal- ities in the conclusion of Theorem 1.9 hold for f . where pZ denotes the probability density of a random variable Z. Therefore the equal- ities in the conclusion of Theorem 1.9 hold for f . Remark 1.21 A stationary centred continuous Gaussian field is ergodic if and only if its spectral measure has no atoms [12, Section 6.1], and so the fields considered in this proposition are not ergodic. The fact that NLS,R(ℓ)/(π R2) does not generally converge to cLS(ℓ) for these fields shows that the ergodicity requirement in the second part of Theorems 1.2 and 1.3 cannot be entirely relaxed. Remark 1.22 It has previously been shown that if spt(ρ) has precisely four points then cLS(0) = 0 (see [10]). Proposition 1.20 shows that cLS(0) > 0 when spt(ρ) has five points, and that cLS(ℓ) > 0 for ℓ̸= 0 when spt(ρ) has four or five points. Remark 1.23 Figures 4 and 5 show cES and cLS for different values of α, β, γ when K = (1, 0) and L = (0, 1). Figure 4 suggests that cLS is bimodal whenever α = 0 (we know that it is at least bimodal, since cLS(0) = 0 and cLS(ℓ) > 0 for ℓ̸= 0 in this case), however for α > 0, cLS can be bimodal or unimodal. Combining this observation with Corollary 1.12 raises the interesting question of determining for which Gaussian fields cLS(ℓ) is bimodal. 1.4 A special class of degenerate fields Remark 1.24 Figures 4 and 5 also suggest that the derivatives of cES and cLS need not be continuous at zero when α = 0. This can be verified analytically since c′ ES(ℓ) = ps−(ℓ) −pm+(ℓ) and these densities are explicitly known. In particular, it can be shown that pY1+Y2 is everywhere continuous, whereas p|Y1−Y2| has a jump discontinuity at zero (but is continuous elsewhere). So c′ ES is continuous except at zero. An analogous calculation permits the same conclusion for c′ LS. On the other hand, when α ̸= 0, cES and cLS are continuously differentiable everywhere. A major unresolved question arising from our work is to determine whether cLS and cES are continuously differentiable more generally (outside degenerate cases). 1.5 Summary of the rest of the paper The remainder of the paper is organised as follows. In Sect. 2 we state the main (completely deterministic) topological lemma underlying our results (Lemma 2.5) and combine this with Proposition 1.8 to prove Theorems 1.2, 1.3 and 1.9 and their 123 D. Beliaev et al. 666 Fig. 4 The functions cES(ℓ) (left) and cLS(ℓ) (right) with α = 0 for β −γ = 0 (solid), β −γ = 0.5 (dashed) and β −γ = 0.9 (dotted) respectively Fig. 4 The functions cES(ℓ) (left) and cLS(ℓ) (right) with α = 0 for β −γ = 0 (solid), β −γ = 0.5 (dashed) and β −γ = 0.9 (dotted) respectively Fig. 5 The functions cES(ℓ) (left) and cLS(ℓ) (right) with β = γ for α = 0.1 (solid), α = 0.3 (dashed) and α = 0.6 (dotted) respectively Fig. 5 The functions cES(ℓ) (left) and cLS(ℓ) (right) with β = γ for α = 0.1 (solid), α = 0.3 (dashed) and α = 0.6 (dotted) respectively corollaries. In Sect. 3 we give the full definition of upper/lower connected saddle points and prove Proposition 1.8. In Sect. 4 we prove Lemma 2.5 via a series of steps, and also prove Proposition 1.20, thereby completing the proof of all results in the paper. corollaries. In Sect. 3 we give the full definition of upper/lower connected saddle points and prove Proposition 1.8. In Sect. 4 we prove Lemma 2.5 via a series of steps, and also prove Proposition 1.20, thereby completing the proof of all results in the paper. 2 Proof of the main results The first step in proving Theorems 1.2, 1.3 and 1.9 is to translate our assumptions on the Gaussian field f into topological properties; these properties differ slightly depending on whether or not f is periodic. Definition 2.1 Let g : R2 →R be a function which is not constant in any direction; that is, there is no v ∈R2\{0} such that for all x ∈R2 anda ∈R, g(x) = g(x+av). We say that g is doubly periodic if there exist two linearly independent vectors y, z ∈R2 such that for all x ∈R2, g(x + y) = g(x) = g(x + z). We say that g is singly periodic if it is not doubly periodic and there exists y ∈R2\{0} such that for all x ∈R2, g(x) = g(x + y). If neither of these conditions holds we say that g is aperiodic. We note that this definition applies to deterministic functions. We say that a random field f is doubly periodic if there exist linearly independent vectors y, z ∈R2 such that with probability one, f (x + y) = f (x) = f (x + z) for all x ∈R2. We make an analogous definition for singly periodic fields, and we say that a random field is aperiodic if it is neither doubly periodic nor singly periodic. The Gaussian field with spectral measure ρ is doubly periodic if and only if there exists A ∈GL(2, R) such 123 On the number of excursion sets of planar Gaussian fields 667 that {Ax : x ∈spt(ρ)} ⊂Z2, and similarly is singly periodic if and only if it is not doubly periodic and there exists A ∈GL(2, R) such that {Ax : x ∈spt(ρ)} ⊂Z×R. For our purposes, it is more natural to consider periodic functions as being defined on the torus or cylinder, and so we will specify the topological properties of Gaussian fields in terms of these domains. For any doubly periodic function g : R2 →R we can choose two linearly inde- pendent vectors y, z ∈R2 satisfying the conditions in Definition 2.1 with minimum distance to the origin, and g is then specified entirely by its values on the parallelogram P :=  x ∈R2 : x = ty + sz, t, s ∈[0, 1)  . 2 Proof of the main results We call this the associated parallelogram for g and call y, z periodic vectors for g (they are not unique since we could also choose −y, −z). By identifying P with the 2-dimensional torus T, we let gT be g defined on the torus. By the choice of y, z above, when g is a stationary random field there are no two distinct points in P on which g is almost surely equal. We call this the associated parallelogram for g and call y, z periodic vectors for g (they are not unique since we could also choose −y, −z). By identifying P with the 2-dimensional torus T, we let gT be g defined on the torus. By the choice of y, z above, when g is a stationary random field there are no two distinct points in P on which g is almost surely equal. If g is singly periodic then we can choose y ∈R2\{0} satisfying the conditions in Definition 2.1 with minimum distance to the origin; we call this a periodic vector for g. By rotating the axes we may assume that g is periodic in the direction of the x-axis (i.e. y = (y1, 0)) so that g is specified entirely by its values on [0, y1] × R. This strip can be identified with the infinite cylinder C := S1 ×R under the quotient relationship that x ∼z if and only if x −z = (my1, 0) for some m ∈Z. We will often work with the compact subset C(n) := [0, y1] × [−n, n] under the same quotient relationship. We let gC and gC(n) denote g restricted to these surfaces under the previous identification. We next introduce some elements of Morse theory, in particular the concept of a Morse function on a manifold; here we follow [8]. We emphasise that, unless explicitly stated, a manifold M may or may not have a boundary ∂M. We also emphasise that we will only ever work with M being the plane R2, the closure of the disc B(R), the torus T, or the cylinders C and C(n), and so it is sufficient to bear these examples in mind. Definition 2.2 Let M be an n-dimensional Riemannian manifold and f ∈C2 loc(M). 2 Proof of the main results (If ∂M ̸= ∅, we take this to mean that for any coordinate chart x, the function f ◦x−1 can be extended to a C2 function on an open subset of Rn. In particular this means that f |∂M is twice continuously differentiable.) We say that f is Morse if the following hold (with any condition depending on ∂M holding implicitly if M has no boundary): 1. The critical points of f and f |∂M are non-degenerate; 2. None of the critical points of f are contained in ∂M; 3. If ∇f is tangent to ∂M at p and u is a unit vector normal to ∂M at p, then ∂∇f (p)∂u f (p) ̸= 0 where ∂∇f (p) and ∂u denote the directional derivatives in the directions ∇f (p) and u respectively; 4. The critical points of f and f |∂M all occur at distinct levels. If M is a manifold without boundary this simplifies to the requirement that all critical points of f are non-degenerate and occur at distinct levels. If M is a manifold without boundary this simplifies to the requirement that all critical points of f are non-degenerate and occur at distinct levels. 123 668 D. Beliaev et al. We note that in the above definition f |∂M is viewed as a function on the (n −1)- dimensional manifold ∂M so that a critical point of f |∂M is not necessarily a critical point of f . Using the definition of a Morse function, we next introduce a set of assumptions that a stationary random field must satisfy in order for our main results to hold. In the lemma that immediately follows, we will claim in particular that these assumptions are satisfied for Gaussian fields satisfying Conditions 1.1, but we have chosen to isolate these assumptions to illustrate the limited role of Gaussianity in the proof of the main results. We say that a point t ∈∂M is a tangent point of f if t is a critical point of the restricted function f |∂M (the name comes from the fact that the level set { f = f (t)} will be tangent to ∂M at t). We will also use the term ‘tangent point’ to describe a local extremum of the restriction of f to a finite union of line segments. 2 Proof of the main results Let Ntang(A) denote the number of tangent points of f in A (where A is the boundary of a manifold or a finite union of line segments) and Ncrit(M) the number of critical points of f in M. We will only use these terms when A and M are compact, so that by the first point in the definition of a Morse function, the number of critical and tangent points will be finite. Conditions 2.3 A stationary random field f satisfies the following: 1. If f is aperiodic, then for each R > 0, f |B(R) is almost surely Morse (taking a union over R ∈N, this implies also that f is almost surely Morse on R2); 2. If f is doubly periodic, then fT is almost surely Morse; 3. If f is singly periodic, then for each n ∈N, fC(n) is almost surely Morse (taking a union over N, this implies that fC is almost surely Morse); 3. If f is singly periodic, then for each n ∈N, fC(n) is almost surely Morse (taking a union over N, this implies that fC is almost surely Morse); 4. If f is periodic and L ⊂R2 is a line segment of length R, then E(Ntang(L)) = cθ for a constant cθ > 0 that depends only on the direction of L; 4. If f is periodic and L ⊂R2 is a line segment of length R, then E(Ntang(L)) = cθ R for a constant cθ > 0 that depends only on the direction of L; 4. E(Ncrit(B(R))) = cR2 for a constant c > 0; 5. E(Ntang(∂B(R))) = O(R) as R →∞. Lemma 2.4 Let f satisfy Conditions 1.1. Then f satisfies Conditions 2.3. Proof All six conditions are established using variations of the Kac–Rice formula. This is an important tool in the study of Gaussian fields, and random functions more gener- ally, which relates moments of the number of zeroes of a field to conditional moments of its gradient/derivative. See [14] for an introduction to the Kac–Rice formula or [1, Chapter 11] for a rigorous derivation. 2 2 (1)–(3). We first note that since f ∈C2 loc(R2) almost surely by assumption, f |M ∈ C2 loc(M) almost surely whenever M is B(R), T or C(n). Corollary 11.3.5 of [1] states that f |M has the first two properties of a Morse function if there exists a countable atlas {xi}i∈I such that for each i ∈I, the covariance kernel of f (i) := f |M ◦x−1 i is C4+ (on its domain of definition) and the Gaussian vectors (∂x f (i)(0), ∂xx f (i)(0), ∂xy f (i)(0)) and (∂y f (i)(0), ∂yy f (i)(0), ∂xy f (i)(0)) are non-degenerate (where ∂x denotes taking the derivative with respect to the first variable, ∂xx denotes taking the second derivative with respect to the first variable and we make analogous definitions for ∂y, ∂yy and ∂xy). For each choice of M that we consider (i.e. B(R), T or C(n)) it is clear that we (1)–(3). We first note that since f ∈C2 loc(R2) almost surely by assumption, f |M ∈ C2 loc(M) almost surely whenever M is B(R), T or C(n). Corollary 11.3.5 of [1] states that f |M has the first two properties of a Morse function if there exists a countable atlas {xi}i∈I such that for each i ∈I, the covariance kernel of f (i) := f |M ◦x−1 i is C4+ (on its domain of definition) and the Gaussian vectors (∂x f (i)(0), ∂xx f (i)(0), ∂xy f (i)(0)) and (∂y f (i)(0), ∂yy f (i)(0), ∂xy f (i)(0)) are non-degenerate (where ∂x denotes taking the derivative with respect to the first variable, ∂xx denotes taking the second derivative with respect to the first variable and we make analogous definitions for ∂y, ∂yy and ∂xy). For each choice of M that we consider (i.e. B(R), T or C(n)) it is clear that we On the number of excursion sets of planar Gaussian fields 669 θ 0 ∇f(p) p u ∂B(R) Fig. Lemma 2.4 Let f satisfy Conditions 1.1. Then f satisfies Conditions 2.3. 6 If p = (R cos(θ), R sin(θ)) and ∇f (p) is orthogonal to u := (cos(θ), sin(θ)), we require that ∂∇f (p)∂u f (p) ̸= 0 ∇f(p) Fig. 6 If p = (R cos(θ), R sin(θ)) and ∇f (p) is orthogonal to u := (cos(θ), sin(θ)), we require that ∂∇f (p)∂u f (p) ̸= 0 can choose a finite atlas of charts xi and that f |M ◦x−1 i will be a translation of f (restricted to some open set) for each such chart. Therefore the covariance kernel of f (i) is a restriction of κ, which is C4+ by assumption, and all that remains is to show that (∂x f (t), ∂xx f (t), ∂xy f (t)) and (∂y f (t), ∂yy f (t), ∂xy f (t)) are non-degenerate Gaussianvectorsforanyt ∈R2.Byassumption∇f (t)and∇2 f (t)arenon-degenerate Gaussian vectors for any t ∈R2. It is a standard fact, for Gaussian fields with constant variance, that ∇f (t) and ∇2 f (t) are independent for fixed t (see e.g. [1, Chapter 5]) and this proves the necessary non-degeneracy. (This part of the result does not depend on whether f is periodic.) We note that f |B(R) has the third property of a Morse function provided that there is no θ ∈[0, 2π] such that 0 = g1(θ) := cos(θ) sin(θ) T ∇f (p) 0 = g2(θ) := cos(θ) sin(θ) T ∇2 f (p) −sin(θ) cos(θ) where p = (R cos(θ), R sin(θ)) (see Fig. 6). If we assumed that f is C3, then Bulinskaya’s lemma would imply that no such θ exists almost surely. Since we assume only that f is C2+ν, we use a different argument which is adapted from the proof of [1, Lemma 11.2.11]. Since f ∈C2+ν loc (R2) almost surely, g2 is almost surely ν-Hölder continuous. Therefore for each δ > 0 we can find Cδ, Dδ > 0 such that, with probability at least 1 −δ, on ∂B(R) the ν-Hölder norm of g2 is at most Cδ and each element of ∇2 f is bounded in absolute value by Dδ; we denote this event by Aδ. Now let Imj be a collection of open intervals in R such that [0, 2π] ⊂∪j Imj for each m,  j Length(Imj) →2π as m →∞, and Length(Imj) →0 uniformly in j as m →∞. Lemma 2.4 Let f satisfy Conditions 1.1. Then f satisfies Conditions 2.3. Furthermore let θmj be the midpoint of each Imj and define Emj =  ∃θ ∈Imj : g1(θ) = g2(θ) = 0  . 123 123 3 670 D. Beliaev et al. On the event Aδ ∩Emj we note that On the event Aδ ∩Emj we note that event Aδ ∩Emj we note that |g1(θmj)| ≤c1DδLength(Imj) and |g2(θmj)| ≤c1CδLength(Imj)ν for some universal constant c1 > 0. Therefore |g1(θmj)| ≤c1DδLength(Imj) and |g2(θmj)| ≤c1CδLength(Imj)ν |g1(θmj)| ≤c1DδLength(Imj) and |g2(θmj)| ≤c1CδLength(Imj)ν for some universal constant c1 > 0. Therefore P(Aδ ∩Emj) ≤  |x|≤c1 DδLength(Imj ) P  |g2(θmj)| ≤c1CδLength(Imj)νg1(θmj) = x  pg1(θmj )(x) dx =  |x|≤c1 DδLength(Imj ) P  |g2(θmj)| ≤c1CδLength(Imj)ν pg1(θmj )(x) dx (7) (7) (7) where pg1(θmj) is the probability density function of g1(θmj) and we have used the fact that g1(θmj) is independent of g2(θmj). Each gi(θ) is a linear combination of elements of a non-degenerate Gaussian vector and so has non-zero variance, therefore on the compact region [0, 2π] these variances are bounded away from zero. Since these are Gaussian fields, their univariate densities are bounded above by a constant times the inverse of their standard deviations, so we see that the density of each gi(θmj) is bounded above uniformly in m and j. We can therefore sum the expression on either side of (7) over j. Since Length(Imj) converges to zero uniformly in j, so does the integrand in each term of the sum, and so we conclude that  j P(Aδ ∩Emj) →0 as m →∞. Since this is true for any δ > 0 and P(Aδ) ≥1−δ we conclude that there is almost surely no θ ∈[0, 2π] such that g1(θ) = g2(θ) = 0, and hence f almost surely has the third property of a Morse function. The proof that fC(n) satisfies the third property of a Morse function is near identical. The argument above can be repeated for the boundary S1×{−n, n}. Finally, fT trivially satisfies the third property of a Morse function. Next we consider the fourth property of a Morse function. We first suppose f is aperiodic and define h1 : Tk →R5 where Tk =  (s, t) ∈B(R) 2  |s −t| ≥1/k  by h1(s, t) = ⎛ ⎝ ∇f (s) ∇f (t) f (s) −f (t) ⎞ ⎠. Lemma 2.4 Let f satisfy Conditions 1.1. Then f satisfies Conditions 2.3. Bulinskaya’s lemma (Lemma 11.2.10 of [1]) states that P(h−1 1 (0) = ∅) = 1 provided that h1 ∈C1(Tk) almost surely and the univariate densities of h1 are bounded uni- formly in Tk. By assumption h1 ∈C1 loc(R2) and so, since h1 is Gaussian, we need only show that the determinant of the covariance matrix of (∇f (s), ∇f (t), f (s)−f (t)) is bounded away from zero on Tk. Since f is aperiodic, f (s) −f (t) is a non-degenerate Gaussian variable for all s ̸= t, and so by Conditions 1.1 (in particular using station- arity of f ), the determinant of this covariance matrix is non-zero for all s ̸= t. Since 123 On the number of excursion sets of planar Gaussian fields 671 this determinant is continuous in (s, t) it is bounded away from zero on the compact set Tk. Taking the countable union of Tk for k ∈N shows that almost surely there are no points s, t ∈B(R) with s ̸= t such that ∇f (s) = ∇f (t) = 0 and f (s) = f (t). this determinant is continuous in (s, t) it is bounded away from zero on the compact set Tk. Taking the countable union of Tk for k ∈N shows that almost surely there are no points s, t ∈B(R) with s ̸= t such that ∇f (s) = ∇f (t) = 0 and f (s) = f (t). Let f∂(θ) be a parametrisation of f |∂B(R). To show that f has no two tangent points at the same level and no tangent points at the level of any critical point, we apply identical arguments to the following two functions: h2(θ, ω) = ⎛ ⎝ f ′ ∂(θ) f ′ ∂(ω) f∂(θ) −f∂(ω) ⎞ ⎠ and h3(t, θ) = ⎛ ⎝ ∇f (t) f ′ ∂(θ) f (t) −f∂(θ) ⎞ ⎠. This completes the proof that f |B(R) is a Morse function when f is aperiodic. When f is doubly periodic, we can repeat these arguments with This completes the proof that f |B(R) is a Morse function when f is aperiodic. Lemma 2.4 Let f satisfy Conditions 1.1. Then f satisfies Conditions 2.3. We now explicitly parametrise f |∂B(R) as g(θ) := f (R cos(θ), R sin(θ)) and note that g′(θ) = −R sin(θ)∂x f (p) + R cos(θ)∂y f (p) g′(θ) = −R sin(θ)∂x f (p) + R cos(θ)∂y f (p) g′′(θ) = −R cos(θ)∂x f (p) −R sin(θ)∂y f (p) g f p y f p +R2 sin2(θ)∂xx f (p) −2R2 sin(θ) cos(θ)∂xy f (p) + R2 cos2(θ)∂yy f (p) +R2 sin2(θ)∂xx f (p) −2R2 sin(θ) cos(θ)∂xy f (p) + R2 cos2(θ)∂yy f (p) (8) where p = (R cos(θ), R sin(θ)). Since ∇f (p) and ∇2 f (p) are independent, a simple calculation shows that (g′(θ), g′′(θ)) has non-degenerate distribution, so we can apply a Kac–Rice formula (specifically, Corollary 11.2.2 of [1]) to show that E(Ntang(∂B(R))) = E  #{θ ∈[0, 2π]  g′(θ) = 0}  =  2π 0 E  |g′′(θ)|  g′(θ) = 0  pg′(θ)(0) dθ. (9) (9) Since f is stationary Var  g′(θ)  = R2E  −sin (θ) ∂x f (0) + cos (θ) ∂y f (0) 2 . The expectation on the right side of this equation is non-zero for each θ, since ∇f (0) is non-degenerate, and by compactness is bounded away from zero uniformly in θ. Therefore, by considering the density of a Gaussian random variable, there exists c0 > 0 such that pg′(θ)(0) ≤c0/R for all θ ∈[0, 2π] and R > 0. 2 g ( ) Using this observation, along with the fact that ∇f (0) is independent of ∇2 f (0), and substituting (8) into (9), we see that there exists a constant c1 > 0 independent of θ and R such that E(Ntang(∂B(R))) ≤c1R  2π 0 E  |∂xx f (0)| + |∂yy f (0)| + |∂xy f (0)|  dθ + c1  2π 0 E  |∂x f (0)| + |∂y f (0)|  sin(θ)∂x f (p) = cos(θ)∂y f (p)  dθ = O(R). ⊓⊔ We now introduce the deterministic relationship between level sets and critical points that is the foundation of our results. Lemma 2.4 Let f satisfy Conditions 1.1. Then f satisfies Conditions 2.3. When f is doubly periodic, we can repeat these arguments with Tk =  (s, t) ∈P2 k : |s −t| ≥1/k  where Pk = {ay + bz : a, b ∈[0, 1 −1/k]} and y, z are the periodic vectors of f , to show that fT is Morse. Finally in the singly periodic case we consider Ck = [0, y1 −1/k] × [−n, n] and Tk =  (s, t) ∈C2 k : |s −t| ≥1/k  where y = (y1, 0) is the periodic vector of f . In both of these cases, the choice of domain ensures that f (t)−f (s) is non-degenerate, so that Conditions 1.1 can be used. This completes the proof of the first three points of the lemma. (4). Let L be a line segment of length R and u a unit length vector parallel to L. Applying a Kac–Rice formula (for the particular form, see Corollary 11.2.2 of [1]) to f |L and using the stationarity of f along with the independence of ∇f (t) and ∇2 f (t), we see that E(Ntang(L)) =  L E(|∂uu f (t)| | ∂u f (t) = 0)p∂u f (t)(0) dt = R p∂u f (0)(0)E(|∂uu f (0)|) =: cθ R where p∂u f (0) is the probability density of ∂u f (0), and cθ < ∞since ∂uu f (0) can be expressed as a linear combination of components of the non-degenerate Gaussian vector ∇2 f (0) and so has finite moments of all orders. where p∂u f (0) is the probability density of ∂u f (0), and cθ < ∞since ∂uu f (0) can be expressed as a linear combination of components of the non-degenerate Gaussian vector ∇2 f (0) and so has finite moments of all orders. (5). Applying the same Kac–Rice formula to f , and using the same arguments as above, shows that E(Ncrit(B(R))) =  B(R) E det ∇2 f (t)   ∇f (t) = 0  p∇f (t)(0) dt = π R2 p∇f (0)(0)E det ∇2 f (0)   =: cR2. 123 672 D. Beliaev et al. (6). 123 Lemma 2.4 Let f satisfy Conditions 1.1. Then f satisfies Conditions 2.3. We denote the number of critical points of f in B(R) of type h with level in [ℓ1, ℓ2] by Nh,R[ℓ1, ℓ2], where h = m+, m−, s+, s− denotes local maxima, local minima, upper connected saddles and lower connected saddles respectively (for an aperiodic function that is Morse on R2, Definition 1.7 defines the upper/lower connected saddle points; the definitions in the general case will be given in Sect. 3). Let us also generalise our earlier notation: for a deterministic, planar function f , we denote the number of components of { f = ℓ} and { f ≥ℓ} in B(R) by NLS,R(ℓ) and NES,R(ℓ) respectively. 123 673 On the number of excursion sets of planar Gaussian fields In the case of a singly periodic function f : R2 →R we also need to define internal and external rectangular tilings of B(x, R) which have a finite number of horizontal and vertical line segments as boundary. Let (y1, 0) be the periodic vector of f , and let S(n1, n2) = [n1y1, (n1+1)y1]×[n2, n2+1]wheren1, n2 ∈Z.Wedefine Bint(x, R)to be the union over n1, n2 ∈Z of all S(n1, n2) contained in B(x, R). Similarly we define Bext(x, R) to be the union over n1, n2 ∈Z of all S(n1, n2) which intersect B(x, R). Lemma 2.5 Let f : R2 →R be a deterministic function. Suppose first that f is ape- riodic and assume that f and f |B(R) are Morse. Then there exists c > 0, independent of f , such that for each ℓ∈R, NLS,R(ℓ) = Nm+,R[ℓ, ∞) −Ns−,R[ℓ, ∞) + Ns+,R(ℓ, ∞) −Nm−,R(ℓ, ∞) + ηℓ,R (10) NLS,R(ℓ) = Nm+,R[ℓ, ∞) −Ns−,R[ℓ, ∞) + Ns+,R(ℓ, ∞) −Nm−,R(ℓ, ∞) + ηℓ,R (10) (10) and and NES,R(ℓ) = Nm+,R[ℓ, ∞) −Ns−,R[ℓ, ∞) + γℓ,R (11) (11) where where max{|ηℓ,R|, |γℓ,R|} ≤cNtang(∂B(R)). (12) (12) Suppose instead that f : R2 →R is doubly periodic and assume that fT is Morse and f |∂P has a finite number of local extrema. Then there exists a constant c f > 0 depending only on P, such that, for each R > 0 and ℓ∈R, (10) and (11) hold with (12) replaced by max{|ηℓ,R|, |γℓ,R|} ≤c f ·  R + Ntang(∂B(R))  . max{|ηℓ,R|, |γℓ,R|} ≤c f ·  R + Ntang(∂B(R))  . max{|ηℓ,R|, |γℓ,R|} ≤c f ·  R + Ntang(∂B(R))  . Finally, suppose that f : R2 →R is singly periodic with periodic vector (y, 0) and assume that fC(n) is Morse for each n ∈Z and that Ntang({0} × [−2R, 2R]) < ∞. Then (10) and (11) still hold with (12) replaced by max{|ηℓ,R|, |γℓ,R|} ≤c f Ntang(∂Bint(0, R)) + Ntang(∂Bext(0, R)) + Ncrit(B(R + r f )\B(R −r f )) max{|ηℓ,R|, |γℓ,R|} ≤c f Ntang(∂Bint(0, R)) + Ntang(∂Bext(0, R)) + Ncrit(B(R + r f )\B(R −r f )) where c f and r f are constants that depend only on the periodic vector y = (y1, 0) of f (so in particular, they are independent of ℓand R). where c f and r f are constants that depend only on the periodic vector y = (y1, 0) of f (so in particular, they are independent of ℓand R). The proof of Lemma 2.5 is contained in Sect. 4. The error term in the above estimate can be intuitively understood as the result of boundary effects from working with the domain B(R) in the definition of NLS,R and NES,R, and although it appears quite complicated (especially in the case of singly periodic functions), when applied to the Gaussian field f it is bounded in expectation by O(R) as a result of Lemma 2.4. 123 674 D. Beliaev et al. We are now ready to state the core technical results of the paper, which are versions of Theorems 1.2, 1.3 and 1.9 that hold for arbitrary random fields (i.e. not necessarily Gaussian) using only the properties contained in Conditions 2.3. Proposition 2.6 Let f be a stationary random field satisfying Conditions 2.3. For each ℓ∈R E  NLS,R(ℓ)  = cLS(ℓ) · π R2 + O(R) as R →∞, where cLS(ℓ) := 1 π E  Nm+,1[ℓ, ∞) −Ns−,1[ℓ, ∞) + Ns+,1(ℓ, ∞) −Ns−,1(ℓ, ∞)  . The constant implied by the O(·) notation may depend on the distribution of f but is independent of ℓ. If in addition f is ergodic, then 1 π R2 NLS,R(ℓ) L1,a.s. −−−−→cLS(ℓ). Proposition 2.7 Let f be a stationary random field satisfying Conditions 2.3. For each ℓ∈R E  NES,R(ℓ)  = cES(ℓ) · π R2 + O(R) as R →∞, where as R →∞, where cES(ℓ) := 1 π E(Nm+,1[ℓ, ∞) −Ns+,1[ℓ, ∞)). The constant implied by the O(·) notation may depend on the distribution of f but is independent of ℓ. max{|ηℓ,R|, |γℓ,R|} ≤c f ·  R + Ntang(∂B(R))  . If in addition f is ergodic, then The constant implied by the O(·) notation may depend on the distribution of f but is independent of ℓ. If in addition f is ergodic, then 1 π R2 NES,R(ℓ) L1,a.s. −−−−→cES(ℓ). Remark 2.8 Since f is stationary, we could equivalently define cLS(ℓ) in Proposi- tion 2.6 as cLS(ℓ) = 1 π R2 E  Nm+,R[ℓ, ∞) −Ns−,R[ℓ, ∞) + Ns+,R(ℓ, ∞) −Ns−,R(ℓ, ∞)  for any R > 0 or make an analogous definition for cES(ℓ) in Proposition 2.7. for any R > 0 or make an analogous definition for cES(ℓ) in Proposition 2.7. Together with Lemma 2.4 and Proposition 1.8, these propositions imply Theo- rems 1.2 and 1.3, and also the first two parts of Theorems 1.9. 123 On the number of excursion sets of planar Gaussian fields 675 Proof (Proposition 2.6) As f is stationary, the expected number of critical points of f of a particular type in a domain is proportional to the area of the domain. Since the field satisfies Conditions 2.3 we can apply Lemma 2.5, and taking expectations yields Proof (Proposition 2.6) As f is stationary, the expected number of critical points of f of a particular type in a domain is proportional to the area of the domain. Since the field satisfies Conditions 2.3 we can apply Lemma 2.5, and taking expectations yields E(NLS,R(ℓ)) = π R2cLS(ℓ) + O(R). Sending R →∞proves the first two statements of the proposition. Sending R →∞proves the first two statements of the proposition. The remainder of the proof follows the general roadmap of the original derivation of the existence of the Nazarov–Sodin constant in [13]. Suppose that f is ergodic and let N (u) h,R denote the number of critical points of type h in B(u, R) with level in [ℓ, ∞) or (ℓ, ∞) for h = m+, s+ and h = m−, s−respectively. The ‘sandwich estimate’ of [13] (Lemma 1) can be slightly altered to show that for any r ∈(0, R) 1 π R2  B(R−r) N (u) h,r πr2 du ≤ N (0) h,R π R2 ≤ 1 π R2  B(R+r) N (u) h,r πr2 du. Applying Wiener’s ergodic theorem (see [13, Section 6.1]) both of these integrals con- verge almost surely and in L1 to the limit E  N (0) h,1  /π. max{|ηℓ,R|, |γℓ,R|} ≤c f ·  R + Ntang(∂B(R))  . So in particular, N (0) h,R/(π R2) has the same limit, and Applying Wiener’s ergodic theorem (see [13, Section 6.1]) both of these integrals con- verge almost surely and in L1 to the limit E  N (0) h,1  /π. So in particular, N (0) h,R/(π R2) has the same limit, and 1 π R2  N (0) m+,R + N (0) s+,R −N (0) s−,R −N (0) m−,R  L1 −→cLS(ℓ). Applying Lemma 2.5 with the bound on E(|ηℓ,R|) implied by Conditions 2.3 shows that 1 π R2 E NLS,R(ℓ) −N (0) m+,R −N (0) s+,R + N (0) s−,R + N (0) m−,R   = o(1) as R →∞. Combining these results completes the proof of L1 convergence. We now extend our notation by defining N (u) LS,r to be the number of components of { f = ℓ} in B(u,r). The original ‘sandwich estimate’ of [13] states that We now extend our notation by defining N (u) LS,r to be the number of components of = ℓ} in B(u,r). The original ‘sandwich estimate’ of [13] states that 1 π R2  B(R−r) N (u) LS,r πr2 du ≤ N (0) LS,R π R2 ≤ 1 π R2  B(R+r) ˜N (u) LS,r πr2 du where ˜N (u) LS,r is the number of components of { f = ℓ} which intersect B(u,r). Since where ˜N (u) LS,r is the number of components of { f = ℓ} which intersect B(u,r). Since ˜N (u) LS,r = N (u) LS,r + O (#{x ∈∂B(u,r) | f (x) = ℓ}) 123 D. Beliaev et al. 676 we can rearrange this estimate as  N (0) LS,R π R2 − 1 π R2  B(R) N (u) LS,r πr2 du  ≤ 1 π R2  B(R+r)\B(R−r) N (u) LS,r πr2 du + c0 π R2  B(R+r) #{x ∈∂B(u,r) | f (x) = ℓ} πr2 du (13) (13) for some universal constant c0 > 0. Applying Lemma 2.5 inside the integral term on the left hand side of (13) shows that for some universal constant c0 > 0. max{|ηℓ,R|, |γℓ,R|} ≤c f ·  R + Ntang(∂B(R))  . Applying Lemma 2.5 inside the integral term on the left hand side of (13) shows that  N (0) LS,R π R2 − 1 π R2  B(R) N (u) m+,r πr2 + N (u) s+,r πr2 − N (u) s−,r πr2 − N (u) m−,r πr2 du  ≤ 1 π R2  B(R+r)\B(R−r) N (u) LS,r πr2 du + c0 π R2  B(R+r) #{x ∈∂B(u,r) | f (x) = ℓ} πr2 du + c f π R2  B(R) r + Ntang(B(u,r)) πr2 du + c f π R2  B(R) Ntang(Bint(u,r)) + Ntang(Bext(u,r)) πr2 du + c f π R2  B(R) Ncrit(B(u,r + r f )\B(u,r −r f )) πr2 du. (14)  N (0) LS,R π R2 − 1 π R2  B(R) N (u) m+,r πr2 + N (u) s+,r πr2 − N (u) s−,r πr2 − N (u) m−,r πr2 du  π R2  B(R+r)\B(R−r) πr2 + c0 π R2  B(R+r) #{x ∈∂B(u,r) | f (x) = ℓ} πr2 du + c f π R2  B(R) r + Ntang(B(u,r)) πr2 du + c f π R2  B(R) Ntang(Bint(u,r)) + Ntang(Bext(u,r)) πr2 du + c f π R2  B(R) Ncrit(B(u,r + r f )\B(u,r −r f )) πr2 du. (14 (14) (The upper bound here is a result of adding the upper bounds on the error terms in Lemma 2.5 in the cases that f is aperiodic, doubly periodic and singly periodic respectively, so that we can deal with all three cases at once.) From Wiener’s ergodic theorem, the integral term within the absolute value signs will converge almost surely to cLS(ℓ) and the first integral term on the right hand side will converge almost surely to zero. Applying the same argument to the remaining integral terms shows that they will each converge to a constant, Φ1(r), Φ2(r), Φ3(r) and Φ4(r) respectively, where Φ1(r) = c0 πr2 E (#{x ∈∂B(r) | f (x) = ℓ}) , Φ2(r) = c f πr + c f πr2 E  Ntang(B(r))  , Φ3(r) = c f πr2  E  Ntang(Bint(0,r))  + E  Ntang(Bext(0,r))  , Φ4(r) = c f πr2 E  Ncrit(B(u,r + r f )\B(u,r −r f ))  . max{|ηℓ,R|, |γℓ,R|} ≤c f ·  R + Ntang(∂B(R))  . 123 123 On the number of excursion sets of planar Gaussian fields 677 We now fix r > 0 and take the limit superior of (14) as R →∞to show that lim sup R→∞  N (0) LS,R π R2 −cLS(ℓ)  ≤Φ1(r) + Φ2(r) + Φ3(r) + Φ4(r) (15) (15) almost surely. Since the number of boundary points of f at level ℓis deterministically bounded above by a constant times the number of tangent points, we see that Φ1(r) ≤ c1Φ2(r) for some c1 > 0. Conditions 2.3 imply that Φ2(r)+Φ3(r)+Φ4(r) = o(1) as r →∞. Therefore we may take a countable sequence rn →∞, such that (15) holds almost surely for each rn and the right hand side of (15) becomes arbitrarily small, to show that N (0) LS,R/(π R2) converges almost surely to cLS(ℓ). ⊓⊔ ⊓⊔ Proof (Proposition 2.7) This follows the proof of Proposition 2.6 almost exactly. Tak- ing expectations of the second part of Lemma 2.5, using the stationarity of f and the bound on E(Ntang(∂B(R))) implied by Conditions 2.3 proves the first two statements of the theorem. Proof (Proposition 2.7) This follows the proof of Proposition 2.6 almost exactly. Tak- ing expectations of the second part of Lemma 2.5, using the stationarity of f and the bound on E(Ntang(∂B(R))) implied by Conditions 2.3 proves the first two statements of the theorem. The proof of the remainder of the theorem follows identically by defining N (u) ES,r as the number of components of { f ≥ℓ} contained in B(u,r), ˜N (u) ES,r as the number of components of { f ≥ℓ} intersecting B(u,r) and noting that the difference in these two terms is also bounded above by the number of tangent points of ∂B(u,r). ⊓⊔ To complete the proof of Theorem 1.9, it remains only to show the joint continuity of cLS and cES with respect to the level and spectral measure. Proof (Theorem 1.9 - Joint continuity) By Prokhorov’s theorem the weak-∗topology on Pc is metrisable and so we can use the sequential definition of continuity for cLS and cES. Let (ρn, ℓn) be a sequence in Pc × R converging to (ρ, ℓ). By the triangle inequality |cLS(ρn, ℓn) −cLS(ρ, ℓ)| ≤|cLS(ρn, ℓn) −cLS(ρn, ℓ)| + |cLS(ρn, ℓ) −cLS(ρ, ℓ)|. (16) |cLS(ρn, ℓn) −cLS(ρ, ℓ)| ≤|cLS(ρn, ℓn) −cLS(ρn, ℓ)| + |cLS(ρn, ℓ) −cLS(ρ, ℓ)|. max{|ηℓ,R|, |γℓ,R|} ≤c f ·  R + Ntang(∂B(R))  . (16) (16) Theorem 1.3 of [10] states that the second term on the right hand side converges to 0 with n in the special case ℓ= 0. However the proof of this theorem can be repeated verbatim replacing the field fρ with fρ −ℓto show that |cLS(ρn, ℓ)−cLS(ρ, ℓ)| →0. Now assume that ℓn ≤ℓ. By the first part of Theorem 1.9 we see that Theorem 1.3 of [10] states that the second term on the right hand side converges to 0 with n in the special case ℓ= 0. However the proof of this theorem can be repeated verbatim replacing the field fρ with fρ −ℓto show that |cLS(ρn, ℓ)−cLS(ρ, ℓ)| →0. N h ℓ ℓB h fi f Th 1 9 h ρ ρ Now assume that ℓn ≤ℓ. By the first part of Theorem 1.9 we see that |cLS(ρn, ℓn) −cLS(ρn, ℓ)| ≤(4/π)Eρn  N (n) crit[ℓn, ℓ]  (17) (17) where N (n) crit[ℓn, ℓ] denotes the number of critical points of fn (the Gaussian field with spectral measure ρn) in the circle of radius 1 with level in [ℓn, ℓ]. By the Kac–Rice theorem (Corollary 11.2.2 of [1]) where N (n) crit[ℓn, ℓ] denotes the number of critical points of fn (the Gaussian field with spectral measure ρn) in the circle of radius 1 with level in [ℓn, ℓ]. By the Kac–Rice theorem (Corollary 11.2.2 of [1]) 678 D. Beliaev et al. Eρn  N (n) crit[ℓn, ℓ]  =  B(1) E det ∇2 fn(t)  1 fn(t)∈[ℓn,ℓ]  ∇fn(t) = 0  p∇fn(t)(0) dt = π E det ∇2 fn(0)  1 fn(0)∈[ℓn,ℓ]  p∇fn(0)(0) ≤E det ∇2 fn(0)  2 1 2 P ( fn(0) ∈[ℓn, ℓ]) 1 2 1 2 1 √det Var(∇fn(0)) (18) (18) where we have used the independence of a field and its gradient at a point along with the Cauchy-Schwarz inequality. It is well known that the covariance structure of a stationary Gaussian field along with its derivatives at a point can be expressed in terms of the spectral measure (see [1, Chapter 5]). For example, Var(∂x fn(0)) =  R2 λ2 1 dρn(λ) with similar expressions for other derivatives and covariances. Since all spectral mea- sures we are considering are supported on B(1), the definition of weak-∗convergence implies that the covariance structure associated with each ρn at the origin converges to that of ρ. max{|ηℓ,R|, |γℓ,R|} ≤c f ·  R + Ntang(∂B(R))  . Specifically, if f denotes the field with spectral measure ρ, then with similar expressions for other derivatives and covariances. Since all spectral mea- sures we are considering are supported on B(1), the definition of weak-∗convergence implies that the covariance structure associated with each ρn at the origin converges to that of ρ. Specifically, if f denotes the field with spectral measure ρ, then Var( fn(0), ∇fn(0), ∇2 fn(0)) →Var( f (0), ∇f (0), ∇2 f (0)). Applying this to (18), we see that det Var(∇fn(0)) is bounded away from 0 in n. Similarly E  |det ∇2 fn(0)|2 is uniformly bounded above in n. Finally we note that Var( fn(0)) is uniformly bounded away from 0, so that choosing ℓn sufficiently close to ℓensures that P ( fn(0) ∈[ℓn, ℓ]) is arbitrarily small. An identical argument works for ℓn ≥ℓ, and so combining these observations with (16)–(18) shows that |cLS(ρn, ℓn) −cLS(ρ, ℓ)| n→∞ −−−→0 asrequired.TheproofofcontinuityofcES(ρ, ℓ)isalmostidentical.AlthoughTheorem 1.3 of [10] is stated only for level sets, the proof of this result can be adapted to apply to excursion sets with no changes of any substance. ⊓⊔ asrequired.TheproofofcontinuityofcES(ρ, ℓ)isalmostidentical.AlthoughTheorem 1.3 of [10] is stated only for level sets, the proof of this result can be adapted to apply to excursion sets with no changes of any substance. ⊓⊔ ⊓⊔ Proof (Corollary 1.19) Since f is isotropic we can use the explicitly derived critical point densities for local maxima, local minima and saddle points from [5]. We recall that these are parametrised in terms of λ ∈(0, √ 2] and η2 ∈[0, ∞) as defined by (5). If 0 < λ < √ 2 then 123 12 On the number of excursion sets of planar Gaussian fields 679 pm+(x) = pm−(−x) = 1 πη2 λ2(x2 −1)φ(x)Φ λx √ 2 −λ2 + λx √ 2 −λ2 2π e− x2 2−λ2 + √ 2  π(3 −λ2) e − 3x2 2(3−λ2) Φ λx  (3 −λ2)(2 −λ2) ps(x) = 1 πη2 √ 2  π(3 −λ2) e − 3x2 2(3−λ2) where φ and Φ denote the standard normal probability density function and cumulative density function respectively. If λ = √ 2 then where φ and Φ denote the standard normal probability density function and cumulative density function respectively. max{|ηℓ,R|, |γℓ,R|} ≤c f ·  R + Ntang(∂B(R))  . If λ = √ 2 then pm+(x) = pm−(−x) = √ 2 π3/2η2 (x2 −1)e−x2 2 + e−3x2 2 1x≥0 ps(x) = √ 2 π3/2η2 e−3x2 2 . By Theorem 1.9, cLS(ℓ+ ϵ) −cLS(ℓ) ≤  ℓ+ϵ ℓ pm−(x) + ps(x) −pm+(x) dx. We denote the integrand above by I(x). In the case λ ∈(0, √ 2), evaluating this expression using the standard Gaussian inequality 1−Φ(x) ≤(1/x)φ(x) shows that I(x) ≤ 1 πη2 √ 2 −λ2 π 1 x 2 −λ2 λ e− x2 2−λ2 − λ2 √ 2π (x2 −1)e−x2 2 which is negative for x > √ 2/λ. For λ = √ 2 and x > 0 I(x) ≤− 1 4π √ 2π (x2 −1)e−x2 2 which is negative for x > √ 2/λ. For λ = √ 2 and x > 0 I(x) ≤− 1 4π √ 2π (x2 −1)e−x2 2 I(x) ≤− 1 4π √ 2π (x2 −1)e−x2 2 I(x) ≤− 1 4π √ 2π (x2 −1)e−x2 2 which is negative for x > 1 = √ 2/λ. Similarly we have which is negative for x > 1 = √ 2/λ. Similarly we have cES(ℓ+ ϵ) −cES(ℓ) ≤  ℓ+ϵ ℓ ps(x) −pm+(x) dx, which is less than our upper bound for cLS(ℓ+ϵ)−cLS(ℓ), and the first result follows. When λ = √ 2 and ℓ≤0 we see that pm+(ℓ) = 0 and so by Theorem 1.9 which is less than our upper bound for cLS(ℓ+ϵ)−cLS(ℓ), and the first result follows. When λ = √ 2 and ℓ≤0 we see that pm+(ℓ) = 0 and so by Theorem 1.9 cES(ℓ+ ϵ) −cES(ℓ) =  ℓ+ϵ ℓ ps−(x) dx ≥0. Therefore cES is weakly increasing in this case. Therefore cES is weakly increasing in this case. ⊓⊔ Therefore cES is weakly increasing in this case. ⊓⊔ D. Beliaev et al. 680 Aℓ−ϵ Aℓ+ϵ ∞ ∞ Aℓ−ϵ Aℓ+ϵ (a) Aℓ−ϵ Aℓ+ϵ Aℓ−ϵ Aℓ+ϵ (b) Fig. 7 Subfigures a and b give two examples of the excursion sets (shaded) at a level below and above a lower connected saddle point in R2 and T respectively (the dashed rectangles in b are identified with the torus by the standard quotient relation) (b) (a) Aℓ−ϵ ∞ Aℓ−ϵ ( ) Fig. max{|ηℓ,R|, |γℓ,R|} ≤c f ·  R + Ntang(∂B(R))  . 7 Subfigures a and b give two examples of the excursion sets (shaded) at a level below and above a lower connected saddle point in R2 and T respectively (the dashed rectangles in b are identified with the torus by the standard quotient relation) 123 3 Critical point densities In this section we prove Proposition 1.8. We begin by giving the definition for lower and upper connected saddle points in full generality. Let M be a manifold and let A ⊂M. We say that A is simple if it is compact, connected and every loop in A (i.e. every continuous map h : S1 →A) is M-contractible. We will be solely interested in the case that A is a component of an excursion set of f : M →R. For such A, and in the case that M is simply connected (e.g. for M = R2 or B(R)), the condition of being simple is just the same as being bounded. Definition 3.1 Let M be a 2-dimensional Riemannian manifold without boundary and let f : M →R be a Morse function with a saddle point x ∈M such that f (x) = ℓ. For c ≤ℓ, let Ac denote the component of { f ≥c} containing x and for c > ℓlet Ac = Aℓ∩{ f ≥c}. We say that x is lower connected if either of the following conditions hold for ϵ > 0 sufficiently small: 1. Aℓ−ϵ is simple and Aℓ+ϵ consists of two simple components; or 2. Aℓ−ϵ is not simple but Aℓ+ϵ has a simple component. We say that x is upper connected if it is a lower connected saddle for −f . We say that x is upper connected if it is a lower connected saddle for −f . When f is an aperiodic Gaussian field, by Lemma 2.4 it will be Morse on R2 so we may take this as our Riemannian manifold and use the definition of lower/upper connected saddle points directly (we show in Sect. 4 that this definition coincides with that given in Sect. 1). If f is doubly periodic, recall that it is completely specified by its values on the associated parallelogram P which we identify with the torus. We then say that a saddle point of f is lower connected if it corresponds to a lower connected saddle of fT by the definition above (see Fig. 8). Similarly if f is singly periodic we say that a saddle point of f is lower connected if it corresponds to a lower connected saddle point of fC. Upper connected saddle points are defined analogously. The following lemma shows that our above definitions partition the set of saddle points in all cases of interest. 123 123 On the number of excursion sets of planar Gaussian fields 681 Fig. 8 Passing through the lower connected saddle points of a doubly periodic function increases the number of compact excursion sets g. 8 Passing through the lower connected saddle points of a doubly periodic function increases the number compact excursion sets Lemma 3.2 Let f be a stationary random field satisfying Conditions 2.3. Then with probability one all saddle points of f are either upper connected or lower connected but not both. This result is proven in Sect. 4. We are now ready to prove Proposition 1.8. Proof (Proposition 1.8) Let f satisfy Conditions 1.1. We fix a compact Ω ⊂R2 such that ∂Ω has finite Hausdorff-1 measure and consider Nh(ℓ), the number of critical points of f |Ω of type h with value greater than ℓ, where h = m−, s, m+ correspond to local minima, saddle points and local maxima respectively. 4 Topological lemmas In this section we prove the deterministic Lemma 2.5 using topological arguments. We also establish Lemma 3.2 and Proposition 1.20 using similar methods, thereby completing the proof of all results in the paper. To prove Lemmas 2.5 and 3.2 we require several results from Morse theory which we now introduce. We note that several aspects of this theory require us to work with compact manifolds, and so in the aperiodic and singly periodic cases we work with B(R) and C(n) rather than directly with R2 and C; in the doubly periodic case, by contrast, we work directly with the torus T. We also emphasise that we only ever work with the five examples of manifolds just mentioned, so it is sufficient to have them in mind. Recall that, for a Morse function f defined on a manifold M, the tangent points of f are defined as the critical points of f |∂M. Theorem 4.1 (Theorem 7 of [8]) Let M be a compact n-dimensional Riemannian manifold and let f : M →R be a Morse function. If f has no critical or tangent points with value in [a, b] then { f ≥b} is homotopy equivalent to { f ≥a}. Theorem 4.1 (Theorem 7 of [8]) Let M be a compact n-dimensional Riemannian manifold and let f : M →R be a Morse function. If f has no critical or tangent points with value in [a, b] then { f ≥b} is homotopy equivalent to { f ≥a}. We define a k-cell to be a copy of the closed unit disc in Rk and temporarily denote this by Bk. If Y is a topological space then we define the following operation to be ‘attaching a k-cell to Y’. First we find a continuous function g : ∂Bk →Y, then we take the disjoint union Y ⊔Bk and identify each point in ∂Bk with its image under g. By attaching a 0-cell, we simply mean taking the disjoint union of Y and a single point. Theorem 4.2 (Theorem 8 of [8]) Let M be a compact 2-dimensional Riemannian manifold and let f : M →R be a Morse function. If t is a critical point of f of index k with f (t) = ℓ, then for ϵ > 0 sufficiently small, { f ≥ℓ−ϵ} is homotopy equivalent to { f ≥ℓ+ ϵ} with a (2 −k)-cell attached. We say that x is upper connected if it is a lower connected saddle for −f . If ( f (0), ∇2 f (0)) has a non-degenerate distribution, then by a Kac–Rice formula (specifically, Corollary 11.2.2 of [1], which requires that ∂Ω has finite Hausdorff-1 measure) E(Nh(ℓ)) =  Ω E det ∇2 f (t)  1Index∇2 f (t)=i, f (t)>ℓ  ∇f (t) = 0  p∇f (t)(0) dt = Area(Ω)p∇f (0)(0)E det ∇2 f (0)  1Index∇2 f (0)=i, f (0)>ℓ  = Area(Ω)p∇f (0)(0)  ∞ ℓ E det ∇2 f (0)  1Index∇2 f (0)=i  f (0) = u  φ(u) du (19) (19) wherei is theindexcorrespondingto h andφ denotes thestandardGaussianprobability density. The third equality follows from the definition of conditioning on a Gaussian variable. If ( f (0), ∇2 f (0)) has a degenerate distribution then f (0) can be expressed as a linear combination of the elements of ∇2 f (0) almost surely. Substituting in this expressionfor f (0)allowsustoapplyKac–Riceandtheargumentsabovetoderive(19) in this case too. Then, by Gaussian regression, E(|det ∇2 f (0)|1Index∇2 f (0)=i | f (0) = u) is a continuous function of u (see, e.g., Proposition 1.2 of [2]). This proves the existence and continuity of the densities pm+, pm−and ps, and the fact that pm+(x) = pm−(−x) follows from the symmetry of f . ℓ Since E(Ns[0, ℓ, ]) =  ℓ 0 ps(x)dx, we know that E(Ns[0, ℓ]) is absolutely con- tinuous in ℓ. It is also clear that for any ℓ1 < ℓ2, E(Ns+[ℓ1, ℓ2]) ≤E(Ns[ℓ1, ℓ2]) so E(Ns+[0, ℓ]) is absolutely continuous in ℓ. Therefore there exists a function ps+ : R →[0, ∞) such that 12 123 3 D. Beliaev et al. 682 E(Ns+[0, ℓ]) =  ℓ 0 ps+(x) dx. Since E(Ns[0, ∞)) is finite, the monotone convergence theorem shows that ps+ ∈ L1(R). By symmetry of the Gaussian distribution, and the definition of lower con- nected saddles, this also shows the existence of ps−(x) = ps+(−x). The fact that ps−+ ps+ = ps follows from Lemma 3.2. ⊓⊔ Since E(Ns[0, ∞)) is finite, the monotone convergence theorem shows that ps+ ∈ L1(R). By symmetry of the Gaussian distribution, and the definition of lower con- nected saddles, this also shows the existence of ps−(x) = ps+(−x). The fact that ps−+ ps+ = ps follows from Lemma 3.2. ⊓⊔ ⊓⊔ 4 Topological lemmas If t is a tangent point and ℓ, ϵ are defined in the same way, then { f ≥ℓ−ϵ} is homotopy equivalent to either { f ≥ℓ+ ϵ} or { f ≥ℓ+ ϵ} with a k-cell attached for some k ∈{0, 1, 2}. These theorems are proven using methods very similar to those of the standard proofs for manifolds without boundary which can be found in almost any text on Morse theory. We now work towards a proof of Lemma 2.5. First we will need another definition which, in the case of aperiodic or singly periodic fields, identifies a subset of the upper/lower connected saddle points which have unfavourable topological properties 123 On the number of excursion sets of planar Gaussian fields 683 (for our purposes). Recall that if M is a manifold then A ⊂M is said to be simple if A is compact, connected and every loop in A is M-contractible. In the case that M = R2 or B(R) and A is an excursion set component, this is just the condition that A is bounded. (for our purposes). Recall that if M is a manifold then A ⊂M is said to be simple if A is compact, connected and every loop in A is M-contractible. In the case that M = R2 or B(R) and A is an excursion set component, this is just the condition that A is bounded. Definition 4.3 Let M∞be a Riemannian 2-manifold without boundary, with f∞∈ C2 loc(M∞) a Morse function on M∞, and let M be a compact submanifold of M∞ with boundary, with f := f∞|M a Morse function on M. Let x0 be a saddle point of f at level ℓand let Ac be defined as in Definition 3.1 for f∞. If x0 is lower connected, then we say that it is four-arm in M if, for ϵ > 0 sufficiently small, all of the simple components of Aℓ+ϵ intersect ∂M. Similarly, we say that an upper connected saddle is four-arm in M if it satisfies the previous condition for −f (and −ℓ). f p f ( ) We say that x0 is an infinite-four-arm saddle point if Aℓ+ϵ has two unbounded components for all ϵ > 0 sufficiently small. 4 Topological lemmas Remark 4.4 In the proof of Lemma 3.2, we will show that when M∞= R2 (the most important case for our analysis), the level set at the level of an infinite-four-arm saddle takes the form shown in Fig. 9b. This corresponds to the way an ‘infinite-four-arm event’ is typically defined in the percolation literature: as two disjoint paths joining a point to infinity which are separated by two ‘dual’ paths joining the same point to infinity. For other choices of M∞(such as M∞= C) the level set at the level of an infinite-four-arm saddle may look quite different, so that this terminology is less intuitive. Let us explain the importance of four-arm saddles. If f is aperiodic, then its lower connected saddle points are defined so as to correspond to an increase in the number of excursion set components in R2. However if such a saddle point is four-arm in B(R), then this increase cannot be observed from inside B(R) since the excursion sets that are created when passing through the saddle intersect ∂B(R) (see Fig. 9). The case when f is singly periodic is similar (in the case that f is doubly periodic we do not have to worry about such saddles). Infinite-four-arm saddle points will be relevant when we prove Lemma 3.2. Fortunately we can control the number of four-arm saddles which occur, in terms of the boundary behaviour of f . Lemma 4.5 Let M∞be a Riemannian 2-manifold without boundary and f∞∈ C2 loc(M∞). Let M be a compact submanifold of M∞with boundary and f := f∞|M be Morse. Let N4-arm(M) be the number of saddle points of f∞contained in M which are four-arm in M or infinite-four-arm. Then N4-arm(M) ≤3Ntang(∂M). Intuitively this bound follows because as we raise the level past a saddle which is four-arm in B(R) or an infinite-four-arm saddle we separate two components of { f ≥ℓ} ∩∂B(R) (i.e. the boundary components are no longer in the same excursion set component). The total number of such separations which may occur is bounded above by the number of boundary excursion components at different levels, which in turn is bounded by the number of tangent points. This is formalised in the proof below. 123 D. Beliaev et al. 684 (a) (b) Fig. 4 Topological lemmas 9 Different types of four-arm saddle points A− S(1) j S(1) j′ {f ≥f(x1) + ϵ} ∞ A− {f ≥f(x1) + ϵ} S(1) j Fig. 10 Since x1 is a lower connected saddle point, A−may contain either one (left) or two (right) simple component of { f ≥f (x1) + ϵ} (b) (a) (a) (b) (a) (b) Fig. 9 Different types of four-arm saddle points A− S(1) j S(1) j′ {f ≥f(x1) + ϵ} ∞ A− {f ≥f(x1) + ϵ} S(1) j Fig. 10 Since x1 is a lower connected saddle point, A−may contain either one (left) or two (right) simple component of { f ≥f (x1) + ϵ} ∞ A− {f ≥f(x1) + ϵ} S(1) j A− S(1) j S(1) j′ {f ≥f(x1) + ϵ} {f ≥f(x1) + ϵ} {f ≥f(x1) + ϵ} Fig. 10 Since x1 is a lower connected saddle point, A−may contain either one (left) or two (right) simp component of { f ≥f (x1) + ϵ} Proof We describe an algorithm which will create an injective mapping from the lower connected saddle points which are four-arm in M to the tangent points of f in ∂M. Let x1, . . . , xm denote all such saddle points arranged in order of increasing level, i.e. f (xi) < f (xi+1). We fix ϵ > 0 such that there are no tangent points with value in [ f (xi)−ϵ, f (xi)+ϵ] for any i and these intervals are disjoint for different i. For each i = 1, . . . , m, we define S(i) 1 , . . . , S(i) mi to be the simple components of { f ≥f (xi)+ϵ} which intersect ∂M. We proceed by induction. Let A−denote the component of { f ≥f (x1) −ϵ} containing x1. Since x is lower connected and four-arm in M, A−will contain some S(1) j (it may contain two such elements: see Fig. 10), and so we make a preliminary association between x1 and S(1) j . j We now assume inductively that for each x1, . . . , xn we have either a preliminary association with some S(n) j (which differs across points) or a final association to some tangent point with value less than f (xn). We consider xn+1 and let A−denote the component of { f ≥f (xn+1) −ϵ} con- taining xn+1. 4 Topological lemmas First suppose that A−is contained in some S(n) j which has a preliminary association with some xi (where i ≤n). Then A−must be simple, as it is a subset of S(n) j , and so, by the definition of a lower connected four-arm saddle point, this means that A−must contain two different components S(n+1) p and S(n+1) q . We then make a preliminary association between xn+1 and S(n+1) p and make a new preliminary 123 On the number of excursion sets of planar Gaussian fields 685 association between xi and S(n+1) q (so we remove the old association between xi and S(n) j ). If A−is contained in some S(n) j which does not have a preliminary association or if A−is not contained in any S(n) j then we choose some S(n+1) p contained in A− (which must exist since xn+1 is four-arm in M) and make a preliminary association between this and xn+1. ( ) Now suppose that xi, where i ≤n, has a preliminary association with S(n) j . If the maximum level of any point in S(n) j is less than f (xn+1) then we make a final asso- ciation between xi and the highest such point in ∂M ∩S(n) j , which is by definition a tangent point. We also remove the preliminary association between xi and S(n) j . Otherwise S(n) j must be a superset of S(n+1) k for some k, and we then make a new preliminary association between xi and S(n+1) k . We perform this process for all pre- liminary associations which remain after the step described in the previous paragraph, which completes the inductive step. This algorithm will cease after the m-th step, at which stage if a point xi has a preliminary association with some S(n+1) j we make a final association between xi and the highest tangent point of S(n+1) j . j The end result of this algorithm is an injective mapping, defined by the final associ- ations, from the set of lower connected saddles which are four-arm in M to the tangent points of f . An identical argument applied to −f creates such a map for the the upper connected saddles which are four-arm in M. A very similar algorithm can be applied to the infinite-four-arm saddles of f , except that the inductive step is slightly simpler. Combining these results proves the lemma. 4 Topological lemmas ⊓⊔ ⊓⊔ Our next result is a preliminary version of Lemma 2.5 for Morse functions on manifolds without boundary. This setting allows us to prove the desired relationship between the number of excursion set components and critical points in the simpler case that all critical points have different levels. Lemma 4.6 Let M∞be a Riemannian 2-manifold without boundary and f ∈ C2 loc(M∞) be a Morse function. Let M be a compact submanifold of M∞and f := f∞|M be Morse. For ℓ∈R, let Nsimple(ℓ) be the number of simple components of { f ≥ℓ} which do not intersect the boundary of M and let Nm+(ℓ) and Ns−(ℓ), be the number of local maxima and lower connected saddle points respectively of f with level in [ℓ, ∞]. Then Nsimple(ℓ) = Nm+(ℓ) −Ns−(ℓ) + ζ where |ζ| ≤5Ntangent. So in particular, if M has no boundary then ζ = 0. The proof involves applying the Morse theorems to each critical/tangent point to derive the change in the number of simple components at the corresponding levels and then summing these changes. Proof First suppose that f has no critical or tangent points with level in [a, b]. By Theorem 4.1, { f ≥b} is a deformation retract of { f ≥a} under some map h : { f ≥ 123 D. Beliaev et al. 686 a} × [0, 1] →{ f ≥a}. In particular, this is also true for each component of { f ≥b} and the component of { f ≥a} in which it is contained. Since h is a homotopy, the number of simple components is the same in each set. If A is a component of { f ≥a} which intersects ∂M, then we claim that A ∩{ f ≥b} also intersects ∂M. To see why, suppose A intersects ∂M but A ∩{ f ≥b} does not. Then by considering the infimum ℓ∗of ℓ∈[a, b] such that A ∩{ f ≥ℓ} does not intersect ∂M and taking a sequence of points in A ∩{ f ≥ℓ∗−1/n} ∩∂M we see that A ∩{ f ≥ℓ∗} contains a tangent point at level ℓ∗∈[a, b], which is a contradiction. Combining all these observations, we see that Nsimple is constant on intervals which contain no critical or tangent points. Now let x be a critical or tangent point of f at level c and take ϵ > 0 small enough to apply Lemma 4.2. We consider in turn the different types of critical or tangent point and calculate Nsimple(c −ϵ) −Nsimple(c + ϵ). Let Ac−ϵ denote the component of { f ≥c −ϵ} containing x and Ac+ϵ = Ac−ϵ ∩{ f ≥ℓ+ ϵ}. We note that to determine Nsimple(c −ϵ) −Nsimple(c + ϵ) it is enough to consider Ac−ϵ and Ac+ϵ, since by the arguments in the previous paragraph, the number of simple components of { f ≥ℓ} in M\Ac−ϵ which do not intersect ∂M will be constant as ℓvaries in [c −ϵ, c + ϵ]. If x is a local maximum, then by Lemma 4.2, Ac−ϵ is homotopy equivalent to Ac+ϵ with a 0-cell attached. So in particular { f ≥c −ϵ} has one more component than { f ≥c + ϵ}, and this extra component is M-contractible. where |ζ| ≤5Ntangent. So in particular, if M has no boundary then ζ = 0. Since Nsimple(a) →0 as a →∞, we see that Nsimple(ℓ) equals the sum of the finite 123 On the number of excursion sets of planar Gaussian fields 687 number of jumps Nsimple(c −ϵ)−Nsimple(c +ϵ) at each level c with a critical/tangent point. This sum equals the number of local maxima of f above level ℓminus the cor- responding number of lower connected saddles with an error ζ bounded in absolute value by N4-arm(M) + 2Ntang(M). By Lemma 4.5, |ζ| ≤5Ntang(M). g g Finally, we note that { f ≥ℓ} = ∩ϵ>0{ f ≥ℓ−ϵ} and that the intersection of a decreasing family of compact, connected sets is connected, so that { f ≥ℓ} and { f ≥ℓ−ϵ} have the same number of components for ϵ sufficiently small. Repeating the earlier argument for tangent points shows that these sets have the same number of components which do not intersect ∂M. Then, if f has a critical point x at level ℓ, applying the Morse lemma on a neighbourhood of x and Theorem 4.1 outside this neighbourhood shows that Nsimple(ℓ−ϵ) = Nsimple(ℓ). (If no such point exists, we simply apply Theorem 4.1 to M.) Therefore we can apply the above arguments to the level ℓ−ϵ such that there are no critical or tangent points with level in [ℓ−ϵ, ℓ) to prove the lemma. ⊓⊔ ⊓⊔ With the above preliminary lemma, the proof of Lemma 2.5 in the case of aperiodic functions is straightforward. Proof (Lemma 2.5 in the case of aperiodic functions) Let f be an aperiodic function satisfying the assumptions of Lemma 2.5. Applying Lemma 4.6 with M = B(R) and M∞= R2 gives exactly the stated relationship for excursion sets. To complete the proof, we prove a corresponding relationship for level sets. We fix a level ℓand let fR = f |B(R), we construct a graph on the vertex set V := {Components of { fR ≥ℓ}} ∪{Components of { fR ≤ℓ}} by declaring two vertices to be joined by an edge if they have non-empty intersection. Clearly the graph is bipartite and each edge corresponds to a component of { f = ℓ}. where |ζ| ≤5Ntangent. So in particular, if M has no boundary then ζ = 0. Since the extra component contains a local maximum, (which cannot be in ∂M) for ϵ > 0 sufficiently small, this component is disjoint from ∂M and so Nsimple(c −ϵ) −Nsimple(c + ϵ) = 1. p p If x is a local minimum, then Ac−ϵ is homotopy equivalent to Ac+ϵ with a 2-cell attached. Attaching a 2-cell does not change the number of components of { f ≥c+ϵ}, and does not affect whether the component it is attached to is simple or not. Once again since the local minimum is not in ∂M, restricting ϵ sufficiently small ensures that the attached 2-cell does not intersect ∂M and so Nsimple(c −ϵ) −Nsimple(c + ϵ) = 0. p ( ) p ( ) Next we suppose that x is a saddle point, so that by Lemma 4.2, Ac−ϵ is homotopy equivalent to Ac+ϵ with a 1-cell attached. So in particular, Ac+ϵ consists of either one or two components. First we note that if Ac−ϵ is simple and does not intersect ∂M, then any components of Ac+ϵ have both these properties, therefore Nsimple(c −ϵ) − Nsimple(c+ϵ) ≤0. If Ac−ϵ is simple but intersects ∂M, then by repeating the sequential argument in the first paragraph of this proof we see that Ac+ϵ must intersect ∂M and so cannot consist of two simple components disjoint from the boundary. Furthermore, if Ac−ϵ is not simple, then by the above homotopy, Ac+ϵ cannot consist of two simple components. Combining these two observations show that Nsimple(c−ϵ)−Nsimple(c+ ϵ) ≥−1. So the number of simple components disjoint from ∂M is either constant or increases by one on passing through the saddle point. If x is not a lower connected saddle, then by definition Nsimple(c−ϵ)−Nsimple(c+ϵ) = 0, if x is a lower connected saddle which is not four-arm in M, then Nsimple(c−ϵ)−Nsimple(c+ϵ) = −1 and if x is a lower connected saddle which is four-arm in M, then Nsimple(c−ϵ)−Nsimple(c+ϵ) = 0. Finally let x be a tangent point, so that Ac−ϵ is homotopy equivalent to Ac+ϵ with a k-cell attached for some k ∈{0, 1, 2}. In each case, Ac+ϵ has at most two simple components disjoint from ∂M, so |Nsimple(c −ϵ) −Nsimple(c + ϵ)| ≤2. p p Now suppose that there are no critical or tangent points at level ℓ. where |ζ| ≤5Ntangent. So in particular, if M has no boundary then ζ = 0. This graph is acyclic, and so by Euler’s formula #{Components of { fR = ℓ}} = #{Components of { fR ≥ℓ}} + #{Components of { fR ≤ℓ}} −1 The number of components of { fR = ℓ} which intersect ∂B(R) is bounded above by the number of tangent points of f in ∂B(R), and the same bound holds for the components of { fR ≥ℓ} and { fR ≤ℓ}. Therefore we can express the equation above as NLS,R( f , ℓ) = NES,R( f , ℓ) + NES,R(−f , −ℓ) + η(1)(ℓ) where |η(1)(ℓ)| ≤4Ntang(∂B(R)). Applying the first part of this lemma to each of the NES,R terms here then completes the proof. ⊓⊔ where |η(1)(ℓ)| ≤4Ntang(∂B(R)). Applying the first part of this lemma to each of the NES,R terms here then completes the proof. ⊓⊔ For the periodic cases, the argument is a little more technical since we cannot apply Lemma 4.6 to B(R) directly. Instead, we tile B(R) with translated parallelograms or rectangles, apply Lemma 4.6 to fT or fC(n) on each translated domain and aggregate the results. 12 123 688 D. Beliaev et al. Proof (Lemma 2.5 in the case of periodic functions) Let f be a doubly or singly periodic function satisfying the assumptions of Lemma 2.5. It suffices to prove the excursion set relationship, since the level set relationship will follow by the same argument as in the aperiodic case. Suppose f is doubly periodic with periodic vectors y, z ∈R2 and associated parallelogram P. By assumption, fT is a Morse function almost surely. Suppose that { fT ≥ℓ} has Nsimple simple components. p Let A be a component of { f ≥ℓ} and A′ be the corresponding component of { fT ≥ℓ}. If A is compact, then clearly A′ cannot contain a non-T-contractible loop, and so it is simple. Since f |∂P has a finite number of local extrema, { f ≥ℓ}∩∂P has a finite number of components. Therefore if A intersects the boundary of a translated parallelogram P + n1y + n2z where n1, n2 ∈Z then it must contain the translation of one of these boundary components. So if A is unbounded, it must contain a path joining two translated versions of the same boundary component, which implies that A′ is not simple. We can tile R2 with the translated parallelograms P + n1y + n2z where n1, n2 ∈ Z. where |ζ| ≤5Ntangent. So in particular, if M has no boundary then ζ = 0. We associate each bounded component A of { f ≥ℓ} to a particular translation P+n1y+n2z of P such that (P+n1y+n2z)∩A ̸= ∅, Nsimple components are mapped to each translated parallelogram and if A is associated with P, then A + n1y + n2z is associated with P + n1y + n2z. If A is a component of { f ≥ℓ} contained in B(R) then it must be associated with a parallelogram which intersects B(R), therefore NES,R(ℓ) ≤Nsimple · #{Translations of P in B(R + d)} (20) (20) where d = diam(P). Similarly, each compact component of { f ≥ℓ} which is associ- ated with a parallelogram inside B(R) must be in B(R) unless it intersects ∂B(R), but the number of such intersections is bounded above by the number of tangent points of f to B(R). Therefore (21) Nsimple · #{Translations of P in B(R)} −Ntang(∂B(R)) ≤NES,R(ℓ) (21) The number of translated parallelograms contained in the ball of a given radius can be approximated by a generalisation of Gauss’ circle problem. It is shown in [11] that The number of translated parallelograms contained in the ball of a given radius can be approximated by a generalisation of Gauss’ circle problem. It is shown in [11] that #{Translations of P in B(R)} = π R2 Area(P) + o(R) as R →∞. So combining this with (20) and (21) as R →∞. So combining this with (20) and (21) as R →∞. So combining this with (20) and (21) NES(R, ℓ) = Nsimple · π R2 Area(P) + O(R + Ntang(∂B(R))) as R →∞. 123 689 On the number of excursion sets of planar Gaussian fields Now suppose that { fT ≥ℓ} contains m local maxima. Reasoning as above, it is clear that m · #{Translations of P in B(R)} ≤Nm+,R[ℓ, ∞) m · #{Translations of P in B(R)} ≤Nm+,R[ℓ, ∞) ≤m · #{Translations of Pin B(R + d)} and so and so Nm+,R[ℓ, ∞) = m · π R2 Area(p) + O(R) with an analogous result holding for lower connected saddles. Applying Lemma 4.6 to fT (i.e. with the setting M∞= M = T) then shows that with an analogous result holding for lower connected saddles. Applying Lemma 4.6 to fT (i.e. with the setting M∞= M = T) then shows that NES,R(ℓ) = Nm+,R[ℓ, ∞) −Ns−,R[ℓ, ∞)) + O(R + Ntang(∂B(R))) as R →∞where the constant in the O(·) notation is independent of ℓ, as required (although it may depend on d). Now suppose that f is singly periodic with periodic vector y = (y1, 0) and define R(n) = [ny1, (n + 1)y1) × R. By assumption f almost surely has a finite number of tangent points in [0, y1] × {n1} or {n2y1} × [−R, R] for any n1, n2 ∈Z or R > 0. Repeating the argument from the doubly periodic case then shows that the compact components of { f ≥ℓ} correspond precisely to the simple components of { fC ≥ℓ}. As before, we can associate each compact component of { f ≥ℓ} with a translated rectangle R(n) for n ∈Z, in such a way that if A is a compact component of { f ≥ℓ} which is mapped to R(n) then A intersects R(n), and if A′ = A + y then A′ maps to R(n + 1). We recall our earlier definitions: if S(n1, n2) = [n1y1, (n1 + 1)y1] × [n2, n2 + 1] where n1, n2 ∈Z, then we define Bint(x, R) to be the union over n1, n2 ∈Z of all S(n1, n2) contained in B(x, R). Similarly we define Bext(x, R) to be the union over n1, n2 ∈Z of S(n1, n2) which intersect B(x, R). as R →∞. So combining this with (20) and (21) We now fix R > 0 and define mn for n ∈Z to be the largest integer such that [ny1, (n + 1)y1] × [−mn, mn] ⊂B(R) and note that under the standard quotient relationship, this rectangle can be identified with C(mn). Let Nsimple( fC(mn), ℓ) denote the number of simple components of { f |C(mn) ≥ℓ} which do not intersect ∂C(mn). By the mapping described above, there will be at least Nsimple( fC(mn), ℓ) compact components of { f ≥ℓ} which are associated with R(n) and intersect [ny1, (n+1)y1]×[−mn, mn]. If any of these components intersect ∂Bint(0, R), then they will do so at distinct components of { f |∂Bint(0,R) ≥ℓ} and so in particular the number of such components intersecting the boundary is at most the number of tangent points on ∂Bint(0, R). Therefore 123 3 D. Beliaev et al. 690 NES(R, ℓ) ≥  n Nsimple( fC(mn), ℓ) −Ntang(∂Bint(0, R)) n WecanthenapplyLemma4.6to fC(mn) (i.e.withthesetting M∞= C and M = C(mn)) for each n to get this inequality in terms of local maxima and lower connected saddles points. Let r = max{ √ 2, y1}. Then since Bint(0, R) covers B(R −r) we see that NES(R, ℓ) ≥Nm+(R, ℓ) −Ns−(R, ℓ) −6Ntang(∂Bint(R)) −Ncrit(B(R)\B(R −r)). NES(R, ℓ) ≥Nm+(R, ℓ) −Ns−(R, ℓ) −6Ntang(∂Bint(R)) −Ncrit(B(R)\B(R −r)). NES(R, ℓ) ≥Nm+(R, ℓ) −Ns−(R, ℓ) −6Ntang(∂Bint(R)) −Ncrit(B(R)\B(R −r) This gives the required lower bound for NES(R, ℓ). The upper bound follows from a very similar argument. First we define Mn to be the largest integer such that This gives the required lower bound for NES(R, ℓ). The upper bound follows from a very similar argument. First we define Mn to be the largest integer such that [ny1, (n + 1)y1] × [Mn −1, Mn] ⊂Bext(0, R) so that B(R) is covered by the finite union of rectangles [ny1, (n+1)y1]×[−Mn, Mn]. Then NES(R, ℓ) ≤  n Nsimple( fC(Mn), ℓ) and by applying Lemma 4.6 to each cylinder we see that and by applying Lemma 4.6 to each cylinder we see that NES(R, ℓ) ≤Nm+(R, ℓ) −Ns−(R, ℓ) + 5Ntang(∂Bext(R)) + Ncrit(B(R + r)\B(R)) NES(R, ℓ) ≤Nm+(R, ℓ) −Ns−(R, ℓ) + 5Ntang(∂Bext(R)) + Ncrit(B(R + r)\B(R)) ⊓⊔ 123 as required. We next complete the proof of Lemma 3.2. Again we shall separate the argument into the aperiodic case and the periodic cases, so that the reader interested only in the aperiodic case can access the simplest version of the argument. Since in this case we work only with the manifolds R2 and B(R), we replace the condition that an excursion set component A is simple with the equivalent condition that it is bounded. Proof (Lemma 3.2 in the case of aperiodic fields) Let f be an aperiodic stationary field satisfying Conditions 2.3. Let g : R2 →R be a Morse realisation of f and assume that g|B(n) is Morse for all n ∈N. Let x be a lower connected saddle of g at level ℓ. Proof (Lemma 3.2 in the case of aperiodic fields) Let f be an aperiodic stationary field satisfying Conditions 2.3. Let g : R2 →R be a Morse realisation of f and assume that g|B(n) is Morse for all n ∈N. Let x be a lower connected saddle of g at level ℓ. The first step is to show that, with probability one, x is not also an upper connected saddle of g. For ϵ > 0 sufficiently small, let Aℓ−ϵ be the component of {g ≥ℓ−ϵ} containing x and Aℓ+ϵ = Aℓ−ϵ ∩{g ≥ℓ+ϵ}. From the definition of a lower connected saddle point, we have three cases to consider. In the first two cases we use the fact that g is Morse to (deterministically) rule out the possibility of x being upper connected; we show that the third case occurs with zero probability and can therefore be neglected. p y g 1)Aℓ−ϵ is unbounded and Aℓ+ϵ has one bounded and one unbounded compo- nent. Let S denote the bounded component of Aℓ+ϵ. We start by choosing n sufficiently large that B(n) contains a neighbourhood of S and a neighbourhood of x. We let A′ ℓ−ϵ be the component of {g|B(n) ≥ℓ−ϵ} containing x and A′ ℓ+ϵ denote A′ ℓ−ϵ∩{g ≥ℓ+ϵ}. We can apply Theorem 4.2 to −g to deduce that B(n) ∩{g ≤ℓ+ ϵ} is homotopy 123 12 On the number of excursion sets of planar Gaussian fields 691 equivalent to B(n) ∩{g ≤ℓ−ϵ} with a 1-cell, denoted γ , attached. as required. By reducing ϵ we can ensure that A′ ℓ+ϵ has two components, one of which is bounded, and so both end-points of γ must be contained in the same component of B(n) ∩{g ≤ℓ−ϵ}. This will in turn be contained in a component of {g ≤ℓ−ϵ} which we denote by B. Clearly B ∪γ is bounded if and only if B is, therefore B(n) ∩{g ≤ℓ−ϵ} and B(n)∩{g ≤ℓ+ϵ} have the same number of bounded components. This holds for any n sufficiently large (possibly after reducing ϵ) but if x were upper connected we could find m large enough that B(m) ∩{g ≤ℓ−ϵ} has one more bounded component than B(m)∩{g ≤ℓ+ϵ} (this argument is stated more formally in the proof of Lemma 4.6). Therefore x is not an upper connected saddle point. 2) Aℓ−ϵ is bounded and Aℓ+ϵ has two bounded components. The arguments in the previous case are also valid in this case where S is chosen to be either of the two components of Aℓ+ϵ. 2) Aℓ−ϵ is bounded and Aℓ+ϵ has two bounded components. The arguments in the previous case are also valid in this case where S is chosen to be either of the two components of Aℓ+ϵ. 3)Aℓ−ϵ is unbounded and Aℓ+ϵ is bounded. We will show that when f is ape- riodic, it almost surely has no saddle points of this type. We fix R > 0 and let gR = g|B(R) which we assume is Morse. For y ∈R2 we define Ay,−ϵ to be the component of {g ≥g(y) −ϵ} containing y and Ay,ϵ = Ay,−ϵ ∩{g ≥g(y) + ϵ}. Let x1, . . . , xn be the saddle points of gR for which Axi,−ϵ is unbounded but Axi,ϵ is bounded for all ϵ > 0 sufficiently small. We then choose a fixed ϵ sufficiently small that this condition holds for each i and such that gR has no other critical points and no tangent points in {gR(xi) −3ϵ ≤gR ≤gR(xi) + 3ϵ} for any i. This ensures the intervals [g(xi) −ϵ, g(xi) + ϵ] are non-overlapping for i = 1, . . . , n. Finally we assume that the xi are ordered so that g(xi) < g(x j) for i < j. j Since Axi,−ϵ is unbounded for each i, we see that B− i := Axi,−ϵ ∩∂B(R) is non-empty for each i. as required. Since there are no tangent points with level in [gR(xi) − ϵ, gR(xi) + ϵ], we can repeat the arguments in the proof of Lemma 4.6 to show that is non-empty for each i. Since there are no tangent points with level in [gR(xi) − ϵ, gR(xi) + ϵ], we can repeat the arguments in the proof of Lemma 4.6 to show that B+ i := Axi,ϵ ∩∂B(R) is non-empty and has the same number of components as B− i for each i = 1, . . . , n (see Fig. 11). Suppose there exists a point y ∈B− i ∩B− j for some i < j. Since Ax j,−ϵ is unbounded and path connected, there exists an unbounded path started at y and contained in {g ≥g(x j) −ϵ)}. Since y ∈Axi,ϵ which is path connected, this path must also be contained in Axi,ϵ which is bounded. This contradiction implies that B− 1 , . . . , B− n are disjoint. Since each of these sets must have a local maximum, we see that n ≤Ntang(∂B(R)). Since f is stationary, we know that the expected number of lower connected saddle points x contained in B(R) for which Ax,−ϵ is unbounded and Ax,ϵ is bounded for ϵ > 0 sufficiently small is c0R2 for some c0 ≥0. However by the argument just given, this number is bounded above by the expected number of tangent points of f to ∂B(R) which is O(R) by Conditions 2.3. Hence c0 = 0 so f almost surely has no saddle points of this type. 123 123 D. Beliaev et al. 692 Fig. 11 We show that almost surely an unbounded excursion set component of f cannot shrink into a bounded excursion set by passing through a saddle point Fig. 11 We show that almost surely an unbounded excursion set component of f cannot shrink into a bounded excursion set by passing through a saddle point Fig. 11 We show that almost surely an unbounded excursion set component of f cannot shrink into a bounded excursion set by passing through a saddle point Fig. 12 The existence of the path γ implies that x is a lower connected saddle point Fig. as required. 12 The existence of the path γ implies that x is a lower connected saddle point We have shown that the set of lower connected saddles of f is almost surely disjoint from the set of upper connected saddles. Using the same notation as above, we will now show that any saddle point of g must be lower connected or upper connected, completing the proof of the lemma. For a small neighbourhood N of x, {g > ℓ} ∩N and {g < ℓ} ∩N each have two components. Since g is Morse, it has no other critical points at level ℓand so the level set {g = ℓ} consists of non-intersecting curves except for the component which contains x. Since g has no infinite-four-arm saddles there are two mutually exclusive possibilities to consider; either there exists a path in {g < ℓ} joining the two components of {g < ℓ} ∩N or there exists a path in {g > ℓ} joining the two components of {g > ℓ} ∩N. We now show that in the first case x will be lower connected. By symmetry, this will imply that in the second case, x is upper connected and so complete the proof for aperiodic fields. We fix a path γ in {g < ℓ} connecting the components of {g < ℓ}∩N. From the existence of γ it is clear that Aℓ\{x} has two components B1, B2 and that without loss of generality, B1 is bounded (see Fig. 12). For ϵ > 0 sufficiently small, Aℓ+ϵ therefore has at least one bounded component B1∩{g ≥ℓ+ϵ}. If Aℓ−ϵ is compact for some ϵ > 0 then clearly B2 is also bounded and so Aℓ+ϵ has two bounded components so x is lower connected. If Aℓ−ϵ is unbounded for arbitrarily small ϵ we can assume that Aℓ+ϵ has an unbounded component, by the argument given above, and so x is again lower connected. We note that this also proves the equivalence of Definition 3.1 in the aperiodic case and Definition 1.7. ⊓⊔ ⊓⊔ Proof (Lemma 3.2 in the case of periodic fields) The arguments here are very similar to those in the aperiodic case (i.e. replacing the condition of boundedness with the more general condition of simplicity). Let f be a stationary field satisfying Conditions 2.3. as required. Let g : M →R be a Morse realisation of f restricted to M, where M is one of T or 123 12 On the number of excursion sets of planar Gaussian fields 693 Fig. 13 An example of the excursion sets at a level below (left) and above (right) a saddle point at level ℓ for which Aℓ−ϵ is not simple and Aℓ+ϵ has one simple and one non-simple component when M = T Fig. 13 An example of the excursion sets at a level below (left) and above (right) a saddle point at level for which Aℓ−ϵ is not simple and Aℓ+ϵ has one simple and one non-simple component when M = T C depending on whether f is doubly periodic or singly periodic. We also assume that g|C(n) is Morse for all n ∈N in the singly periodic case. Let x be a lower connected saddle of g at level ℓ. Again the first step is to show that, with probability one, x is not an upper connected saddle of g. For ϵ > 0 sufficiently small, let Aℓ−ϵ be the component of {g ≥ℓ−ϵ} containing x and Aℓ+ϵ = Aℓ−ϵ ∩{g ≥ℓ+ ϵ}. Again we have three cases to consider, the first two of which are proven deterministically: 1)Aℓ−ϵ is not simple and Aℓ+ϵ has one simple and one non-simple component. Let S denote the simple component of Aℓ+ϵ. If f is singly periodic, we set Mc = C(n) where n is sufficiently large that Mc contains a neighbourhood of S and a neighbour- hood of x. If f is doubly periodic we take Mc = M = T. We define A′ ℓ−ϵ to be the component of {g|Mc ≥ℓ−ϵ} containing x and A′ ℓ+ϵ := A′ ℓ−ϵ ∩{g|Mc ≥ℓ+ ϵ}. By Theorem 4.2 applied to −g we deduce that Mc ∩{g ≤ℓ+ ϵ} is homotopy equivalent to Mc ∩{g ≤ℓ−ϵ} with a 1-cell, denoted γ , attached. By reducing ϵ we can ensure that A′ ℓ+ϵ has two components, one of which is simple, and so both end-points of γ must be contained in the same component of Mc ∩{g ≤ℓ−ϵ}. This will then be contained in a component of {g ≤ℓ−ϵ} which we denote by B. If B is not simple, then clearly B ∪γ is not simple. as required. Suppose that B is simple but B ∪γ is not simple, then B ∪γ must contain a loop, denoted η, which is not M-contractible and in particular η must intersect γ . However B is path connected and B ∪γ ‘surrounds’ a region which is homotopy equivalent to S and so must be simple (see Fig. 13 for an example of these sets in the doubly periodic case). Therefore η is homotopy equivalent to a loop which is contained in B and so B is not simple, which is a contradiction. We have shown that B ∪γ is simple if and only if B is, therefore Mc ∩{g ≤ℓ−ϵ} and Mc ∩{g ≤ℓ+ ϵ} have the same number of simple components. This holds for any Mc sufficiently large (possibly after reducing ϵ) but if x were upper connected we could find Mc such that Mc ∩{g ≤ℓ−ϵ} has one more simple component than Mc ∩{g ≤ℓ+ ϵ} (again, we note that this argument is stated more formally in the proof of Lemma 4.6). We conclude that x is not an upper connected saddle point. 2) Aℓ−ϵ is simple and Aℓ+ϵ has two simple components. Once again, the argu- ments in the previous case are also valid in this case where S is chosen to be either of the two components of Aℓ+ϵ. 2) Aℓ−ϵ is simple and Aℓ+ϵ has two simple components. Once again, the argu- ments in the previous case are also valid in this case where S is chosen to be either of the two components of Aℓ+ϵ. 12 123 D. Beliaev et al. 694 Fig. 14 The paths γ1 and γ2 cut through x but γ3 does not Fig. 14 The paths γ1 and γ2 cut through x but γ3 does not 3)Aℓ−ϵ is not simple and Aℓ+ϵ is simple (in particular, connected). We can repeat the argument given in this section of the proof for aperiodic fields to show that if f is singly periodic then Aℓ−ϵ must be bounded. Clearly in the doubly periodic case, Aℓ−ϵ ⊂T must also be bounded. We therefore choose a compact domain Mc ⊂M of the form T or C(n) for some n which contains a neighbourhood of Aℓ−ϵ. Let B1, . . . , Bn denote the components of {g ≤ℓ+ ϵ} ∩Mc which intersect Aℓ+ϵ. as required. Since Aℓ+ϵ is simple, we know that at most one of these sets is not simple and the remainder are. (One of the sets will ‘surround’ Aℓ+ϵ which in turn will ‘surround’ the remaining sets.) Without loss of generality, we assume B1 is the ‘surrounding’ set, which may not be simple. By Theorem 4.2, Aℓ−ϵ is homotopy equivalent to Aℓ+ϵ with a 1-cell, denoted γ attached. Clearly γ is contained in B1 and B1 is not simple, otherwise Aℓ+ϵ ∪γ would be simple. If M = C then B1\γ has two components and each of these components contains a loop which is not C-contractible (since Aℓ+ϵ ∪γ is compact and so separated from ∞and −∞by B1\γ ). If M = T then B1\γ may have one or two components, in either case each component will contain a non-T- contractible loop (to see this, consider a path on either side of γ which then traverses the boundary of Aℓ+ϵ). This means that passing through the saddle point x can only create non-simple components of {g ≤ℓ−ϵ}, so x is not upper connected. This completes the proof that the sets of upper and lower connected saddle points of f are almost surely disjoint. We continue to use the notation defined above, and we now show that any saddle point of g must be lower connected or upper connected. As in the aperiodic case, Lemma 4.5 and Conditions 2.3 allow us to conclude that f almost surely has no infinite four-arm saddles. Suppose that f is doubly periodic so that g is defined on the torus T. We fix a small neighbourhood N of x such that {g > ℓ} ∩N has precisely two components denoted N1, N2. If γ : [0, 1] →T is a path contained in {g > ℓ} ∪{x}, we say that γ cuts through x if x ∈γ [0, 1] and for every t ∈(0, 1) such that γ (t) = x, for ϵ > 0 sufficiently small γ ((t −ϵ, t)) ⊂N1 and γ ((t, t + ϵ)) ⊂N2 (intuitively, this means that the image of γ just before hitting x is always on the same side of the saddle point; see Fig. 14). We make an analogous definition for paths contained in {g < ℓ} ∪{x}. First we suppose that there exists a T-contractible loop γ in {g < ℓ}∪{x} which cuts through x. as required. Since x is a saddle point, it is clear that Aℓ+ϵ must have two components, one of which is surrounded by γ and so is simple. If Aℓ−ϵ is simple, then both components of Aℓ+ϵ must be simple. If Aℓ−ϵ is not simple, we know from Lemma 4.6 that Aℓ+ϵ can contain at most one simple component. In either case, we see that x is lower connected. By symmetry, x is upper connected if there exists a T-contractible loop in {g > ℓ} ∪{x} cutting through x. 123 123 12 695 On the number of excursion sets of planar Gaussian fields Now we suppose that all loops cutting through x are non-T-contractible. We define C+ to be the union of {x} and the components of {g > ℓ} which x is in the closure of. We define C−to be the analogous set for {g < ℓ}. At least one of C+ or C−must contain a non-contractible loop cutting through x (in order to stop any paths in C+ cutting through x from joining to form a loop, there must be a loop in T\C+ cutting through x which blocks them, and since x is the only critical point at level ℓ, such a ‘blocking’ loop can be found in C−). We also note that both C+ and C−must contain non-T-contractible loops (if C+ was simple, then we could find a T-contractible loop in C−cutting through x, and this argument is symmetric). Suppose that γ1 is a loop contained in C+ which cuts through x. If every non-T- contractible loop in C+ intersects x, then C+\{x} is simple, so Aℓ+ϵ is simple and hence x is lower connected. Therefore we may assume that C+ contains a non-T- contractible loop γ2 which does not intersect x. Let η1 and η2 be two loops which generate the fundamental group of T, we denote the concatenation of n copies of η1 and m copies of η2 by nη1 + mη2. We suppose γ1 ≃n1η1 + m1η2 and γ2 ≃n2η1 + m2η2 where ≃denotes being T-homotopic. It is known (see [7, Section 1.2.3]) that any loop homotopic to n1η1 + m1η2 must intersect any loop homotopic to n2η1 + m2η2 unless n1m2 = n2m1. If n1m2 ̸= n2m1 then we know that any non-contractible loop in C−must intersect either γ1 or γ2. Since C−∩C+ = {x} clearly such a loop must intersect γ1 at x. By the previous paragraph this means that C−\{x} is simple and so x is upper connected. If n1m2 = n2m1 then by choosing a path ξ in C+\{x} which joins γ1 to γ2 we consider the concatenated loop n2γ1 + ξ + n1(−γ2) + (−ξ) where −denotes inverting the direction of the path. This path is contained in C+ and cuts through x (since γ1 does but γ2 and ξ do not hit x) and since n1m2 = n2m1 this loop is T-contractible. where α + β + γ = 1 so that the covariance function of f is κ(x) = α + β cos(2π K · x) + γ cos(2π L · x). Then f has the representation Then f has the representation f (x) = X0 + Y1 cos(2π K · x + θ1) + Y2 cos(2π L · x + θ2) (22) (22) where X0 ∼N(0, α), Y1 ∼Ray(√β), Y2 ∼Ray(√γ ), the θi are uniformly dis- tributed on [0, 2π] and all of these random variables are independent. (By well known properties of the Rayleigh distribution, the field defined by the right hand side of (22) will have Gaussian finite dimensional distributions and simple calculations show that the covariance function of this field is κ.) u = 1 K1L2 −K2L1 L2 −L1 v = 1 K1L2 −K2L1 K2 −K1 and P = {tu + sv : t, s ∈[0, 1]}. Then P is the parallelogram associated with f and we consider fT to be the restriction of f to P when P is identified with the two dimensional torus. By rotating the axes, we may assume that K1 > 0 (since this does not affect the definition of cLS). Some basic calculations show that, on the event {Y1 ̸= 0} ∩{Y2 ̸= 0} ∩{Y1 ± Y2 ̸= 0} , fT has four critical points which occur at different levels and are all non-degenerate. Therefore fT is almost surelyaMorsefunction. Moreover, thecritical points of fT−X0 occur at levels −Y1 −Y2, −|Y1 −Y2|, |Y1 −Y2|, Y1 + Y2. Clearly the critical points at levels Y1 + Y2 and −Y1 −Y2 are a local maximum and local minimum respectively. We can use Lemma 4.6 to characterise the other two critical points and the number of simple components of { fT −X0 ≥ℓ}, denoted Nsimple(ℓ), at different levels. Specifically, since { fT −X0 ≥Y1 + Y2} = ∅and { fT −X0 ≥−Y1 −Y2} = T, we see that Nsimple(ℓ) = 0 whenever |ℓ| > Y1 + Y2. Then, since the critical points at level Y1 + Y2 and −Y1 −Y2 must be a local maximum and local minimum respectively, applying Lemma 4.6 shows that Nsimple(ℓ) does not change as ℓpasses through −Y1 −Y2 and decreases by one as ℓpasses through Y1 + Y2. Therefore fT has four critical points which occur at different levels and are all non-degenerate. 123 However this contradicts the above supposition, so this case is not possible. This completes the proof in the doubly periodic case. The proof in the singly periodic case is simply a repetition of parts of the proof for aperiodic and doubly periodic fields, so we omit it. ⊓⊔ ⊓⊔ All that remains is to complete the calculations for the special class of degenerate fields. Proof (Proposition 1.20) We recall that a random variable Y is Rayleigh distributed with parameter σ > 0 if P(Y ≤x) = 1 −e−x2/(2σ 2) for all x ≥0, and we denote this as Y ∼Ray(σ). If Y ∼Ray(σ) and θ is an independent uniform-[0, 2π] random variable then it is well known that Y cos(θ) ∼ N(0, σ 2). for all x ≥0, and we denote this as Y ∼Ray(σ). If Y ∼Ray(σ) and θ is an independent uniform-[0, 2π] random variable then it is well known that Y cos(θ) ∼ N(0, σ 2). Let f be the Gaussian field with spectral measure ρ = αδ0 + β 2 (δK + δ−K ) + γ 2 (δL + δ−L) 123 123 D. Beliaev et al. 696 where α + β + γ = 1 so that the covariance function of f is where α + β + γ = 1 so that the covariance function of f is Th f f i l t l M f ti M th iti l i t f f X Nsimple(ℓ) = ⎧ ⎪⎪⎪⎨ ⎪⎪⎪⎩ 0, if ℓ> Y1 + Y2, 1, if ℓ∈(|Y1 −Y2|, Y1 + Y2), 0, if ℓ∈(−Y1 −Y2, −|Y1 −Y2|), 0, if ℓ< −Y1 −Y2. Nsimple(ℓ) = ⎧ ⎪⎪⎪⎨ ⎪⎪⎪⎩ 0, if ℓ> Y1 + Y2, 1, if ℓ∈(|Y1 −Y2|, Y1 + Y2), 0, if ℓ∈(−Y1 −Y2, −|Y1 −Y2|), 0, if ℓ< −Y1 −Y2. Now assume that θ1 = θ2 = 0. Then by traversing the parallelogram P across one of its edges (depending on which of Y1, Y2 is bigger) we can find a closed path which is not T-contractible, on which fT −X0 is bounded below by |Y1 −Y2|, so in particular 123 On the number of excursion sets of planar Gaussian fields 697 is positive. This shows that { fT −X0 ≥0} has a non-simple component (for general values of θ1, θ2, such a path will also exist, but it will be translated). Since { fT −X0 ≥ |Y1 −Y2|} consists of a single simple component, this implies that the critical point at level |Y1 −Y2| must be a lower connected saddle point. Then by Lemma 4.6 we see that Nsimple(ℓ) =  1, if ℓ∈(|Y1 −Y2|, Y1 + Y2], 0, otherwise. It is then clear that It is then clear that pm+(x) = 1 Area(P) pX0+Y1+Y2(x) and ps−(x) = 1 Area(P) pX0+|Y1−Y2|(x). Arguments given in the proof of Lemma 2.5 for periodic functions show that NES,R(ℓ) = 1ℓ−X0∈(|Y1−Y2|,Y1+Y2] · π R2 Area(P) + O(R + Ntang(∂B(R))). Arguing as in the proof of Lemma 2.4 it can be shown that E(Ntang(∂B(R)) = O(R) as R →∞. Then for ℓ∈R Arguing as in the proof of Lemma 2.4 it can be shown that E(Ntang(∂B(R)) = O(R) as R →∞. Then for ℓ∈R E  NES,R(ℓ) π R2 − 1 Area(P)1ℓ−X0∈(|Y1−Y2|,Y1+Y2]  = O 1 R , so that NES,R(ℓ)/(π R2) converges in L1 to the random variable 1 Area(P)1ℓ−X0∈(|Y1−Y2|,Y1+Y2] which is non-deterministic provided α > 0 or ℓ> 0. In particular, this convergence shows that cES(ℓ) := lim R→∞ 1 π R2 E(NES,R(ℓ)) = 1 Area(P)P(ℓ−X0 ∈(|Y1 −Y2|, Y1 + Y2]). We can repeat these arguments for the sum of upper and lower excursion sets to derive the corresponding expression for cLS(ℓ). where α + β + γ = 1 so that the covariance function of f is ⊓⊔ We can repeat these arguments for the sum of upper and lower excursion sets to derive the corresponding expression for cLS(ℓ). ⊓⊔ ⊓⊔ Acknowledgements The authors would like to thank Manjunath Krishnapur for pointing out [16] and also Igor Wigman and Mikhail Sodin for useful comments and suggestions. OpenAccess ThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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’) B. IV 125-129 118851. 460. R. WeiSgerber: Ober dae Iaoobinolin im Stsinkohlenteer. gMitteilnng rns dem wisseoschaftlichen Laboratorium der Geeellschaft fir Teerverwertung m. b. H. in Duisbnrg-Meiderich.] (Eingegangen am 25. November 1914.) S. Hoogewerff und W.A. v a n Dorpl) haben schon im Jahre 1885 in einer wertvollen Experimentalarbeit gezeigt, daO dss aus dem Steinkohlenteer erhiiltliche Rohchinolin in geringer Menge eine Base enthat, welche mit dem Chinolin isomer ist und mit diesem eine 80 weitgehende Ahnlichkeit hinsichtlich ibres gesamten chemischen Ver- baltens aufweist, daB sich die Trennung beider KZirper voneinnnder nicbt auf eiufacbe Weise bewerkstelligen Iffit. Die genannteo Formher fandeo, daD ein geringer Unterschied in der Liislichkeit der sauren Sulfate der Basen in Alkohol eine Handhabe bietet, um die Trennung dennoch auszufiihren. Ibr Weg Fiihrte sie zur Isolierung und Rein- darstellung des neuen Kiirpers. welcher von h e n auf Grund seines Verhaltens und seiner Oxydationsprodukte als ’\ /‘ Isochinolin, 1 \,LJN 1) Die Zusammensetzurig wurde auf Grund der Emtarrungspunkte kiinst- licher Gemische mit anntihernder Genauiglteit ermittelt. erkannt und charakterisiert wurde. Das Verfabren, velcbes die hollandischen Chemiker zur Trennung der beiden Basen anwandten, liiDt zwar hinsichtlicb der Sicherheit, mit welcher es Zuni Ziele fubrt, nichts zu wunscheu ubrig, leidet aber an dem Ubelstand, daB, da das Rohchinolin nur sebr geringe Mengen Isochinolin entblilt (sie belaufeo sicb meist nur auf Bruchteile eines Prozentea vom Ausgnngsmaterial) unverb5ltnismliBig groBe Mengen des Ausgrngsmaterials in Arbeit genommen werden miissen und dsO erst aus zahlreicben, mit Verlusten verkniipften Krystallisationen dee Rohsulfates dss reine Salz des Isocbinolins hervorgeht. Das Bedurtnis oach einer technischen Vereinfachung dieses, groSe Massen eines an sich ganz gleichgeltigen Begleitkiirpers (des Cbinolins) mitverarbeitenden Trennungsverhhrens, gab die Veranlassung zur erneuten Besrbeitung der Aufgabe, Chinolio und Isochinolin von einander zu trennen. Her- bei gelang es, ausgehend von theoretischen Erwiigungen, einen Weg zu finden, welcher durch Ausschaltung der Hauptmenge des Chinoline, die Gewinnung der Isobase ungemein vereinfacht : Bus den Formelbildern der beiden Basen ergibt sich, daf3 im Isochinolin die basischen Funktionen des Stickstoffatoms von dem ’) B. IV 125-129 118851. 1) Das Verfahren ist Gegenstand der P. A. 40941, 12p der Gesellschaft fur Teervemertung m. I. H., Duisburg-Meiderich. 3176 Benzolkern nicht in dem MaBe beeinllul3t werden kannen wie im Chinolin, bei welchem der Stickstoff unmittelbar mit dem, als scbwach .negative. Gruppe wirkenden Benzolkern in Verbindung stebt. Es wird daher, iihnlich wie das Dimetbylanilip zweifellos weniger basisch ist als das Dimethylbenzylamin, zu ernarten seio, daS der basiscbe Charakter des Cbinolins hinter dem des Isochinolins zuriicktritt. Dies ist nun in der Tat der Fall; das Isocbinolin besitzt eine groBere Basizitat als das Cbinolin und dieser Umstand ermoglicht, in einer das praktrische Rediirfnis befriedigenden Weire die Treunung auszu- liihren. Diese, aus Theorie uud Praxis hervorgegaugene Erkeniitnis wird iibrigens auch durch folgendcn einfachen Versuch bestatigt, der zugleich einen Anbalt uber die quantitativen Verhiiltnisse bietet: Wenn man ein Gemiscb aus gleichen Teilen Cbioolin und Isochinolin (vom Erstarrungspunkt 20°) in etwn der sechsfaclien Menge Toluol lost und zuniichst die Hiilfte der Basen mit der berechneten hfenge n-Scbsefei- saure, hierauf den Rest durch die gleiche Menge Siiure ausziebt, so ergibt sicb, nachdem die Basen aus beiden Aurziigen wieder iu Freiheit gesetzt sind, folgende Diflereozierung: Erstarrungspunkt des Anagrngsmaterinla 1 O enlhLlt 5Ou,o Taochiriolin '1 u 1. Atlsxugs . . . 8.5' 68'10 B n n 11. 4 . . -7' P 33",0 n Bei der Anwendung dieses Verhnltens :id die Prnxis ergibt sich nun folgender einfacbe Weg zur Gewinnung von Isochinolin: Geht man z. B. von der iiblichen Handelsmarke des Steinkohleochiaolins aus, einem Baseagemiscb, welches etws von 230-2459 siedet, so liist man dieses ziir Gewinnung der in ihm enthaltenen - iibrigens sebr geringen - hlengen Ieocbinolin in etwa der doppelten Menge Beozol und schiittelt aus dieser Liisung beispielsweise den sechsten Teil der Bssen niit der berechneten Menge Scbwefelsaure voo 20°/0 aus. Die nus der Sullatliisung in Freiheit gesetzten Basen zeigen eiuen atiffallend weit auseinander gezogenen Siedepunkt, denn sie beginnen bereits gegen 2200 uberzugehen und ihre letzten Anteile sieden bis gegen 260°. IXese, scbeinbar eine QualitLtsverschlecbterung vortirischende Erscheinung berubt, wie die Erfahrung gelehrt hat, offenbar darsuf, daS irn roben Teerchinolin neben Chinolin und Isochinolin, basische Anteile unbekannter Zusammensetzung enthalten sind, welcbe, bochst- wahrscheinlicb vom Cbarakter der mehrfach methylierten Pyridine, mit dem Isochinolin die Eigenschaft der gr6Beren Basizitat gemein haben und daher mit diesem in dem ersten Saureauszug enthnlten 31 77 I) Hoogcweiff uod van Dorp, It. 5, 308 [1887]. *) CIaur; und Svelemann, J. pr. p2] 52, 1 [1895]. 31 77 sind. Fiir die weitere Verarbeitung hat diese Erscheinung indessen keine Bedeutung, da durch Fraktionieren dieses stiirker basischen An- teils leicht eine brauchbare etwa von 235-245 O siedende Fraktion herausgeschnitten werden kann, welche nunmehr bereits ansehnliche Mengen Isochinolin enthiilt. Da als Begleiter des Letzteren auch in dieser Fraktion in erster Linie Chinolin in Frage kommt, so liegt es nahe, das Verfahren der partiellen Bindung an Siiure mit dem bereits angereicherten Material zii wiederholen, urn auf solche Weise schlie6lich zu reinem oder nahezu reinem Isochinolin zu gelangen. Dieser Weg liil3t sich in der Tat mit Erfolg beechreiten, allein aus praktiechen’ Griinden mu0 es ale zweckmiifliger bezeichnet werden, hier das alte Reinigungsverfahren der Entdecker des Isochinolins mit dem neuen zu Yerbinden und die angereicherte, in oben beschriebener Weise dar- gestellte Fraktion iiber das Sulfat weiter zu reinigeu. I n wenigen (etwa 3-4) Krystallisationen des Sulfates dieser Fraktion gelingt es, Isochinolin-sulfat in einer fur die meisten Zwecke geniigenden Reinheit zu erhalten. Die Ausbeute ist selbstverstiindlich abhangig van dem wechselnden, wie erwiibnt meist nur Bruchteile eines Prozentee be- tragenden Gehalt des angewandten Rohchinolins an Isochinolin, wird aber hier, wie schon aus dem beschriebenen Verlauf der Aufarbeitung hervorgeht, in ungleich schneller verlaufenden, an Zahl wesentlich verminderten Operationen unter Ausschaltung der Hauptmenge der Begleitkorper erzielt. Trotzdem eignet sich, wie man sieht, das rohe Teerchinolin -’ eben wcgen seiues geringen Gehaltes an lsobase - nur wenig zur Herstellung der Letzteren, vielmehr empfiehlt es sich, hierzn auf das chinolinhaltige Teerol selbst zuruckzugehen. Der groDe, pralrtische Vorteil in der Anwendung des neuen Verfabrens auf diesea Material, besteht vor Allem darin, dal3 man auf das Ausziehen der groBten Menge, des in diesem Falle liistigen Begleitkorpers, des Chinolina verzichten kann, d. h., da8 man durch beschriinkte Bindung der ba- sischen Bestandteile dieser 6le aberhaupt nur die isochinolinhaltigen Anteile zu gewinnen braucbt und demnach von vornherein nur mit einer kleineren, leicht zu handhabenden Menge eines Ausgangsmateriale von wesentlich erhohtem Isochinolingehalt zn rechnen hat I). Das partielle Ausziehen der Basen aus den Teerolen, welches zweckmiiBig nur bei griifleren, im Fabrikbetriebe zur Verfiigung ste- henden olmengen erfolgt, laBt sich in iihnlicher Weise, wie oben be- schrieben, leicht bewerkstelligen. Die erhaltenen Basen, deren Menge z. B. ’ 25 O/O der in den 6len iiberhaupt vorhnndenen basischen Bestand- 3178 3178 teile betragen kann, zeigen gleichfalls die oben erwahnte Erscheinnng der weit auseinander gezogenen Siedegrenzen, konnen aber ebenfalls leicht durch einmaliges, vorsicbtiges Fraktiooieren auf ein zur nberfiihrung in das Sulfat geeipetes Rohmaterial eingeeogt werden. Allem An- schein nach werden bei der Darstellung des Sulfates in alkoholischer Losung iiberhaupt nur Chinolin und Isochinolin als Sulfate gefiillt, so daB schon hierdurch alle, dern Teerol entstammenden, aber nicht dieser Gruppe angehbrenden Rmen entfernt werden und die weitere Reinigung bis zum einheitlichen Isochinolin-sulfat, wie erwHhnt, rnit leichter Miihe bewerkstelligt werden kann. Trenniing der Basen durch d i e Sulfosiiuren. Bus der Konstitution der beiden Basen liilit sich, zunkhst rein theoretisch, noch ein andrer Weg zu ihrer Trennuhg ableiten. Wahrendr im Chinolin der Benzolkern sowobl an Kohlenstoff wie an tertiaren Stickstoff gebunden ist, sein Verhalten demnach in zweifacher Hiosicht beeinflufit werden muB, ist im Isochinolin der Benzolkern nur an Kohlenstoff gebunden, so daB von ihm ein Verbalten erwartet werden dad, welches in gewisser Beziebung von den] des Chinolins abweicht. g g Der mit tertiiirein Stickstoff verbundene Benzolkern zeigt bei einigen Substitutionsvorbane;en, zii denen z. B. die Sulfurierung ge- bort, eine wesentlich geringere Angreifbarkeit als das nicht substituierre Benzol oder die nur Kohlenstoffbindungen enthaltenden Homologen und wie z. B. das Dimethylanilin wesentlich schwerer in seine Sulfo- siiure uberzufuhren ist, als etwa das Toluol, so kiinnte man erwarten, da13 auch das Cbinolin weit schwieriger zu sulfurieren wire als das Isochinolin. Auch bier entspricht das tatsiichliche Verhalten der theoretischen Forderung. Isochinolin ist in der Tat weit leichter in seine Sulfouiiure uberfuhrbar, als Chinolin, allein d r sich herrtusge- stellt hat, da(3 aus der erhaltenen Sulfosiiure nach den gebrauchlichen Methoden die Sulfogruppe sich nur scbwierig oder gar uicht wieder abspalten lafit. so hat dieser Weg wohl fiir die Darstellung der Iso- chinolin-sulfosiiure, nicbt aber fur die Gewinnung des Isochinolins selber eine praktische Bedeutung. Wahrend beim Chinolin die Sulfurierung sehr eingehend unter- sucht worden ist, liegen beziiglich das Verhaltens des Ieochinolins bei dern gleichen Vorgang nur die kurzen Angaben der Entdecker dieser Base’) sowie die Arbeiten von Claus und seinen Schulern2) vor, welche Letztere die zuerst von Hoogewerff und van D o r p gemachten Beobachtungen nicbt wesentlich erweitert haben. 1 ) Claus iind (:etzcit, ,I. pr. [2] 52, TI-22 [lSttjJ. '?) Der Schmelrpunkt sird YOU CI:r us, .J. pr. [2] 59, 1 I zu 1300 dagogai, J. pr. p2] 47, 137 zu 2300 angegeben. Nur die letzterc Angabe ist ricbtig (ich fand 2980), und es ist zu bedauern. d 3 der falsche Schmelzpunkt von 130" in den ))Boilstein nnd ltichtert, iibergegangen id. 3180 mag diese aber auf ublichem Wege nicht wieder in Isochinolin iiberzu- fiihren. Weder Erhitzen mit verdunnter Scbwelelsiinre auf Tempe- raturen bis iiber 20O0, noch trockne Destillation des Ammonsalzes, noch Reduktion vermogen aus der genannten Siiure nennenswerte hfengen Isochinolin abzuspalten, so' daB diese Base auf dem be- schriebenen Wege uber ihre Sulfosailren vorliiufig nicht dargestellt werden kann. Zur Feststellung der Einheitlichkeit und des Charakters der Isochinolin-sulfoshure, konnte, da die Bereitung des Chlorids und Amids Schwierigkeiten machte, nur das Verhalten dieser Siiure in der Kaliscbmelze herangezogen werden, wobei, wie schon C1 aus und seine Scbuler feststellten l), aus der a-SPure das a-Oxy-isochinolin vom Schnip. ?30° gewoonen wird 3. Bei den xahlreichen, im Laufe der Untersuchung ausgefuhrten Kalischmelzen wurde nun eine Beobach- tung gemaclit, welche bei der Auslegung der Versuchsergebnisse leicht zu Irrtumern fiihren krnn. Schmilzt man die a-Isochinolin-sulfosiiure mit Kali bei Temperaturen von 200-250° so wird sie leicht in das erwiihnte Oxyderivat ubergeftihrt; erhtiht man dagegen die Schmelz- temperatur a d 30O0 und dariiber und setzt die Schmelze fort bis zum deutlichen Auftreten von Ammoniak, so erfjihrt das entstandene Oxg- isocbinolin eine IJmwandlung, die sich u. a. auch durch erneutes Auf- schiiumen der Schmelze kundgibt. Beim Losen einer solchen Schmelze in Wasser fiillt nach dem Ansluern sogleich ein schon krystallisierter Korper in gelblich weiBen Nadeln aus, welche nach dem Umkrystalli- sieren aus Wasser bei 2730 unter Briiunung und Zersetzung schmelzen. Diese T'erbinduog besitzt im Gegensatz zum Oxy-isochinolin .keinerlei basische Eigenschaften mehr, lost sich dagegen in Soda und kombi- aiert mit Diaeoniumverbindungen zu orangeroten Farbstoffen. Von den meisten Losungsmitteln, mit Ausnahme von heil3em Wasser, wird sie nur schwer aufgenommen. Sie ist ihrem Verhalten und ibrer Analyse nach als ein Dioxy-isochinolin anzusprechen. mag diese aber auf ublichem Wege nicht wieder in Isochinolin iiberzu- fiihren. Weder Erhitzen mit verdunnter Scbwelelsiinre auf Tempe- raturen bis iiber 20O0, noch trockne Destillation des Ammonsalzes, noch Reduktion vermogen aus der genannten Siiure nennenswerte hfengen Isochinolin abzuspalten, so' daB diese Base auf dem be- schriebenen Wege uber ihre Sulfosailren vorliiufig nicht dargestellt werden kann. Zur Feststellung der Einheitlichkeit und des Charakters der Isochinolin-sulfoshure, konnte, da die Bereitung des Chlorids und Amids Schwierigkeiten machte, nur das Verhalten dieser Siiure in der Kaliscbmelze herangezogen werden, wobei, wie schon C1 aus und seine Scbuler feststellten l), aus der a-SPure das a-Oxy-isochinolin vom Schnip. ?30° gewoonen wird 3. 3179 Aus diesen Arbeiten geht zuniichst hervor, dalJ augepscheinlich auch das Isochinolin nur schwer sulfurierbar ist, sowie, daB unter den verschiedensten Bedingungen stets zwei Sulfosiiuren entstehen, von denen nur die eine sich einigermaoen leicht in reiner Form er- halten IPBt. Beide Umstiinde 'erscheinen fur den vorliegenden Zweck recht hinderlich, doch ergab die neue Bearbeitung dieses Vorganges, daB die Sulfurierung des Isochinoline, welche nach Clsus unter l l O o iiberhaupt nicht rnebr erfolgen sollte, sich bei geeigneter Wahl der Siiurekonzentrrtion schon bei mPRiger Temperatur mit grol3er Leichtig- keit vollzieht, sowie, da13 bei Anwendung tiefer Temperaturen fast nusschliefilich die a-Siiure in leicht zu reinigender Form erhalten wird. Unter derartigen, gemiisigten Bedingungen liiRt sich auch der Unterscbied in dem Verhalten der beiden Basen leicht beobachten und in gewisser Weise quantitativ bestimmen: 50 g Chinolin- bezw. Isochinolinsulfat werden bei 30-40° unter Ruhren und Kuhlen in 110 g Oleum von 50°/0 allmiihlich eingetragen, wobei nur anfangs eine betriichtliche Temperatursteigerung zu bemerken ist. Nach 24- sttindigern Stehen bei Zimmertemperatur erfolgt die Aufarbeitung der Sulfurierung in der iiblicben Weise iiber Barium- und Natriumsalz, nachdem man zuvor mittels dmmoniaks die unsulfurierten Basen aus- gefiillt hat. Irn Falle des Isochinolins versetzt inan zweckrniiBig die eingeengte Liisung des Natriumsalzes rnit Salzsiiure, worauf sogleich die schon von den obeu genannten Forschern beschriebene n-Isochino- lin-sulfosiiure in schiinen, einheitlichen Nadeln auskrystallisiert. Man erhiilt ; 1. aus Chinolin 10.5 g Base xurtick, demnach Ausboute an Solfos. 63 */a '2. )) Tsochinolin 1.0 )) I) >> n B n n 96.5 a Von dem sulfurierten Isochinolin konnten nahezu 85 O/O der theo- retischen Menge an reiner, wasserfreier or-Sulfosiiure erhalten werden. Auch in den letzten Mutterlaugen konnte diese Siiure noch beob- achtet, jedoch nicht mit Sicherheit als viillig rein identifiziert werden. Auf Grund ihrer verschiedenen Sulfurierbarkeit ist es nun maglich, Chinolin von Isochinolin innerhalb gewisser, durch die Praxis ge- gebenen Grenzen zu trennen; allein auch hier ist es angezeigt, mit Hilfe der Sulfate und des zuerst beschriebenen Verfahrens das Basen- gemisch vor Uberfuhrung in die SulFosiiuren an Isochinolin anzu- reichern. Man geht daher zweckmiiBig von einem isochinolinhaltigen Rohsulfat, dessen Schmelzpunkt miiglichst nicht unter 180° liegen sollte, aus, fiihrte dieses in oben beechriebener Weise in seine Sulfo- siiure Cber und reinigt sie gegebenenfalls noch durch Umkrystallirlieren. Man gelangt so, wie schon erwiihnt, leicht zur reinen or-Siiure, ver- 3180 1) Der Berliner Aksdemie der Wissenscbaften vorgelegt am 25. Jani 1914. 2) A. 409, 364 [1914]. Siehe Sitznngsberichte 1914, 714 und C. 1914. 11, 981. Berlchte d. D. Chem. Oesellschaft. Jahrg. XXXXVII. 207 3180 Bei den xahlreichen, im Laufe der Untersuchung ausgefuhrten Kalischmelzen wurde nun eine Beobach- tung gemaclit, welche bei der Auslegung der Versuchsergebnisse leicht zu Irrtumern fiihren krnn. Schmilzt man die a-Isochinolin-sulfosiiure mit Kali bei Temperaturen von 200-250° so wird sie leicht in das erwiihnte Oxyderivat ubergeftihrt; erhtiht man dagegen die Schmelz- temperatur a d 30O0 und dariiber und setzt die Schmelze fort bis zum deutlichen Auftreten von Ammoniak, so erfjihrt das entstandene Oxg- isocbinolin eine IJmwandlung, die sich u. a. auch durch erneutes Auf- schiiumen der Schmelze kundgibt. Beim Losen einer solchen Schmelze in Wasser fiillt nach dem Ansluern sogleich ein schon krystallisierter Korper in gelblich weiBen Nadeln aus, welche nach dem Umkrystalli- sieren aus Wasser bei 2730 unter Briiunung und Zersetzung schmelzen. Diese T'erbinduog besitzt im Gegensatz zum Oxy-isochinolin .keinerlei basische Eigenschaften mehr, lost sich dagegen in Soda und kombi- aiert mit Diaeoniumverbindungen zu orangeroten Farbstoffen. Von den meisten Losungsmitteln, mit Ausnahme von heil3em Wasser, wird sie nur schwer aufgenommen. Sie ist ihrem Verhalten und ibrer Analyse nach als ein Dioxy-isochinolin anzusprechen. COZ, 0.0646 g H?O. - 0.066:: g Sbst.: 5.6 ccm N (300, 758 mm). - 0.1.338 g Sbst.: 11.1 ccm N (3flo, 763mm). O.llS8 g Sbst.: 0.2!133 g COs, 0.0446 g HsO. - 0.1582 g Sbst.: 0.3903 g Ber. C 67.05, H 4.34, N 8.6!1. Gel. I) 67.33, 67.29, D 420, 4.57, D !).09, 8.S5. 1) Der Berliner Aksdemie der Wissenscbaften vorgelegt am 25. Jani 1914. 2) A. 409, 364 [1914]. Siehe Sitznngsberichte 1914, 714 und C. 1914. 11, 981. pp (Eingegangen am 16. November 1914.) pp (Eingegangen am 16. November 1914.) Bei der Vertauschung zweier Substituenten am asymrnetrischen Kohlenstoffatom rnuI3 nach der Theorie eine Umkehrung des optischen DrehungsvermBgens erfolgen. Wir haben friiher ’) veraucht, diese Folgerung zu priifen bei der optisch-aktiven Athyl-isopropyl-malon- rrninsiiure, die in den Methylester und dann durch sslpetrige SPure in den aktiven ~thyl-isopropyl-malonsiiure-monomethylester verwandelt wurde. Leider mi0lang die Riickverwandlung dieses Esters in die Malonaminsiiure, wail Ammoniak bei gewohnlicher Temperatur nicht amidiert und bei hoherer Temperatur komplizierte Vorgiinge statt- finden. Wir haben aber jetzt das Ziel auf etwas andrem Wege er- reicht bei der Isopropyl-malonaminsiiure (Formel I), die aus dem Isopmpyl-cyan-essigester durch Verseifung mit starker Schwefelsiiure gewonnen und durch das Chininsalz in die optisch-aktive Form iiber- gefiihrt wurde. d-Isopropyl-malonaminsiiure d-Isopropyl-malonami~~ure-methyle d-Isopropyl-malonaminsiiure d-Isopropyl-malonaminsiiure d-Isopropyl-malonaminsiiure d-Isopropyl-malonami~~ure-methyleste 1) Der Berliner Aksdemie der Wissenscbaften vorgelegt am 25. Jani 1914. Siehe Sitznngsberichte 1914, 714 und C. 1914. 11, 981. Berlchte d. D. Chem. Oesellschaft. Jahrg. XXXXVII. d-Isopropyl-malonami~~ure-methyleste Die Siiure liil3t sich durch Diazomethan leicht in den Methyl- ester (11.) und dieser dnrch salpetrige Siiure in den optisch-aktiven Monomethylester der Isopropyl-malonsiiure (111.) verwandeln. CIsopropyl-malonhydidsaure. 461. Elmil Fisoher und Frits Braune: Perwandlung der d-18opropyl-malonaminellure in den optischen Antipoden durch Vertauschung von Carboxyl- und S&ureamid-Gruppel). (Eingegangen am 16. November 1914.) Perwandlung der d-18opropyl-malonaminellure in den optischen Antipoden durch Vertauschung von Carboxyl- und S&ureamid-Gruppel). 3181 Aulfallend ist die leichte Entstehung dieses Dioxyderivates in der Sie erfolgt ohne nachweisbare Nebenprodukte und in Kalischmelze. einer Menge von etwas iiber 5Ooh der Theorie. Herr Dr. Vielitz hatte die Freundlichkeit, mich bei der vor- g liegenden Arbeit durch seine analytische Tiitigkeit zu unterstutzen. 461. Elmil Fisoher und Frits Braune: Perwandlung der d-18opropyl-malonaminellure in den optischen Antipoden durch Vertauschung von Carboxyl- und S&ureamid-Gruppel). (Eingegangen am 16. November 1914.) CIsopropyl-malonhydidsaure. CIsopropyl-malonhydidsaure. I-Isopropyl-malonmethyleRtersiiure 207
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General solution of the chemical master equation and modality of marginal distributions for hierarchic first-order reaction networks
Journal of mathematical biology
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18,952
Mathematical Biology Mathematical Biology Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00285- 018-1205-2) contains supplementary material, which is available to authorized users. J. Math. Biol. (2018) 77:377–419 https://doi.org/10.1007/s00285-018-1205-2 J. Math. Biol. (2018) 77:377–419 https://doi.org/10.1007/s00285-018-1205-2 B Edda Klipp edda.klipp@hu-berlin.de Matthias Reis matthias.reis@hu-berlin.de Justus A. Kromer justus.kromer@tu-dresden.de 1 Institute of Biology, Theoretical Biophysics, Humboldt-Universität zu Berlin, 10117 Berlin, Germany 2 Center for Advancing Electronics Dresden, Technische Universität Dresden, 01062 Dresden, Germany 1 Introduction The development of single-molecule methods such as Fluorescence in situ Hybridiza- tion (FISH) has resulted in considerable advances in fundamental research in biology (Trcek et al. 2011, 2012). Those methods provide researchers with discrete, single- molecule data on (bio-)chemical reaction networks. The measured molecule numbers are often small, requiring a modeling approach that accounts for the discreteness of the molecule numbers (Klipp et al. 2009). Networks of stochastically reacting molecule species are often described by the Chemical Master Equation (CME) (Gardiner 2009; Kampen 2011). The CME is a difference–differential equation. By using Gillespie’s Stochastic Simulation Algo- rithm (Gillespie 1977), an exact realization of the CME’s underlying Markov jump process is sampled and an indirect solution is obtained (Gillespie 1992). The CME may also be studied by approximation methods such as finite state projection (Munsky and Khammash 2006), or exactly using analytical approaches. Stationary solutions to the CME are known for a wide range of chemical reaction networks (Kampen 1976; Anderson et al. 2010). However, time-dependent, analytical solutions only exist for monomolecular reaction networks (Gans 1960; Jahnke and Huisinga 2007), consist- ing exclusively of conversion Si ↔Sj, degradation Si →∅and production ∅→Sj reactions, where Si defines a species of molecules. In contrast to numerical solution methods, analytical solutions allow for an abstract study of reaction networks, inde- pendently of the concrete value of reaction rate constants and network graphs. Here, we present an extension of the class of analytically solvable CME’s to a subset of first-order reactions, namely hierarchic first-order networks. In addition to monomolecular reactions, first-order reactions include (auto-)catalytic and splitting reactions, and therefore allow for more real-world applications. As a subset of those networks, we define hierarchic networks (Def. 3), characterized by a division into independent and dependent subnetworks. Common examples of hierarchic networks are the transcription–translation model in molecular biology (Friedman et al. 2006; Shahrezaei and Swain 2008), or nuclear decay chains in physics (Bateman 1910; Pressyanov 2002), both of which we discuss here. In contrast to monomolecular networks, first-order networks result in marginal distributions of individual species that are not unimodal in general, as we show in Theorems 3 and 4. These distributions are poorly characterized by mean and vari- ance, two essential measures used to describe statistical data in natural sciences. On one hand, characterizing multimodal distributions by mean and variance is unintu- itive, since the mean is often a very unlikely outcome. Mathematics Subject Classification 92C45 · 60J27 · 05A15 Mathematics Subject Classification 92C45 · 60J27 · 05A15 General solution of the chemical master equation and modality of marginal distributions for hierarchic first-order reaction networks Matthias Reis1 · Justus A. Kromer2 · Edda Klipp1 Received: 7 April 2017 / Revised: 10 October 2017 / Published online: 20 January 2018 © The Author(s) 2018. This article is an open access publication Abstract Multimodality is a phenomenon which complicates the analysis of sta- tistical data based exclusively on mean and variance. Here, we present criteria for multimodality in hierarchic first-order reaction networks, consisting of catalytic and splitting reactions. Those networks are characterized by independent and dependent subnetworks. First, we prove the general solvability of the Chemical Master Equa- tion (CME) for this type of reaction network and thereby extend the class of solvable CME’s. Our general solution is analytical in the sense that it allows for a detailed analysis of its statistical properties. Given Poisson/deterministic initial conditions, we then prove the independent species to be Poisson/binomially distributed, while the dependent species exhibit generalized Poisson/Khatri Type B distributions. General- ized Poisson/Khatri Type B distributions are multimodal for an appropriate choice of parameters. We illustrate our criteria for multimodality by several basic models, as well as the well-known two-stage transcription–translation network and Bateman’s model from nuclear physics. For both examples, multimodality was previously not reported. B Edda Klipp edda.klipp@hu-berlin.de Matthias Reis matthias.reis@hu-berlin.de Justus A. Kromer justus.kromer@tu-dresden.de 1 Institute of Biology, Theoretical Biophysics, Humboldt-Universität zu Berlin, 10117 Berlin, Germany 2 Center for Advancing Electronics Dresden, Technische Universität Dresden, 01062 Dresden, Germany 1 Institute of Biology, Theoretical Biophysics, Humboldt-Universität zu Berlin, 10117 Berlin, Germany 1 Institute of Biology, Theoretical Biophysics, Humboldt-Universität zu Berlin, 10117 Berlin, Germany 2 Center for Advancing Electronics Dresden, Technische Universität Dresden, 01062 Dresden, Germany 3 378 M. Reis et al. Keywords Hierarchically linear ODE system · Hierarchic reaction network · Chemical master equation · Conditional multimodality · Compound process · Bursting process · Generalized poisson distribution · Generalized binomial distribution · Nuclear decay chain · Transcription–translation model 123 1 Introduction On the other hand, many unimodal distributions, such as the Gaussian distribution, are fully specified by the 123 General solution of the chemical master equation… 379 first two cumulants, i.e. mean and variance, or just the mean in case of the Poisson distribution. Contrastingly, an infinite number of cumulants can be necessary to fully specify the multimodal distributions derived in this article. We therefore argue that CME approximation methods based on the first two cumulants or moments, such as the moment-closure method (Milner et al. 2012), yield insufficient results when reaction networks lead multimodal molecule distributions. Albeit to a lesser degree, this hold true even when more than the first two moments are used to approximate the CME’s solution in approaches such as Constantino et al. (2016). Such distribu- tions have already been reported for networks that include two-state or three-state random variables such as promoter switches studied in the context of gene-regulation (Shahrezaei and Swain 2008; Thomas et al. 2014). In the same context, a maximum- entropy approach has been used to approximate multimodal molecule distributions successfully using the first seven moments (Andreychenko et al. 2017). In the present paper, we develop an exact theoretical description for hierarchic first-order networks, based on probability generating functions. Hierarchic first-order networks are introduced in Sect. 2, followed by the main results in Sect. 3. In Sect. 3.1, we establish the existence of analytical solutions (Theorem 1) and derive the joint prob- ability generating functions (Proposition 2), which are complete characterizations of the underlying statistics. Then, the analytical form of the marginal distributions of indi- vidual species is derived in Sect. 3.2. We show that the independent part of the network exhibits Poissonian and Binomial marginal distributions, while the dependent part is described by Discrete Compound Poisson (DCP) and Kathri Type B (KTB) marginal distributions. Next, we present criteria for the marginal distributions to be multi- modal in Sect. 3.3. Since Poissonian/Binomial distributions are generally unimodal, theindependentpartexhibitsunimodalmarginaldistributions(Theorem2).Incontrast, DCP/KTB marginal distributions from the dependent part can lead to multimodality under quite general conditions (Theorems 3 and 4). We illustrate these general results by several basic models (Sect. 4.1) and two real-world models (Sect. 4.2). Among these are the transcription–translation model (Example 5) and Bateman’s model for nuclear decay chains (Example 6). 1 Introduction For the former, multimodality was previously only discussed in an extended three-stage model variant (Shahrezaei and Swain 2008), whereas Theorem 3 proves the protein numbers to be multimodally distributed even in the simple two-species version. To the best of our knowledge, our exact solution to the CME for Bateman’s model of nuclear decay chains is a novel result, applying the techniques developed in the preceding sections. We apply Theorem 4 to show multimodality for Bateman’s model. y The derivation of probability mass functions from generating functions is reviewed in Appendix A.1. 2.1 Chemical master equation and characteristic ODEs First of all, we define a network of n species with m chemical reactions by the n × m stoichiometric matrices Q = (Qi j) and R = (Ri j), where Qi j, Ri j ∈N0. These matrices enable us to write 12 3 3 380 M. Reis et al. QTS k→RTS, with S = (S1, . . . , Sn)T being the vector of species and k = (k1, . . . , km)T the vector of rate constants. with S = (S1, . . . , Sn)T being the vector of species and k = (k1, . . . , km)T the vector of rate constants. In the present paper, we consider discrete and stochastic models of biochemical reaction networks. Let P(x, t|x0, t0) be the probability distribution for the number of molecules x of each species at time t, given the initial condition x0 at t0. This distribution is described by the Chemical Master Equation, a difference–differential equation defined by1 ∂t P(x, t) = m  i=1  αi(x −ni)P(x −ni, t) −αi(x)P(x, t)  , (1) (1) where where αi(x) :=ki n  j=1 x j! (x j −Q ji)! and ni := coli(R −Q). (2) (2) In the last equation coli denotes the ith column of a matrix. The CME (1) is based on the Markovian assumption and is derived in detail in Gardiner (2009) or Kampen (2011). Applicable for (multivariate) distributions of discrete random variables, we introduce the concept of generating functions: In the last equation coli denotes the ith column of a matrix. The CME (1) is based on the Markovian assumption and is derived in detail in Gardiner (2009) or Kampen (2011). Applicable for (multivariate) distributions of discrete random variables, we introduce the concept of generating functions: Definition 1 (Generating function) A probability generating function (PGF) is a power series2 g(s, t) := ∞  x=0 sxP(x, t) (3) (3) defined for s ∈Cn within some radius of convergence ≤1 in each coordinate respec- tively. defined for s ∈Cn within some radius of convergence ≤1 in each coordinate respec- tively. In the univariate case, a large list of correspondences between PGFs and distributions is provided by Johnson et al. (2005). Next, we represent the Master Eq. (1) as a partial differential equation (PDE) in terms of a generating function: Lemma 1 The Master Eq. 3 See Evans (2010) for an introduction. 2.1 Chemical master equation and characteristic ODEs (1) can equivalently be expressed as the partial differential equation ∂tg(s, t) = m  i=1 ki ⎛ ⎝ n  j=1 s R ji j − n  j=1 s Q ji j ⎞ ⎠ ⎛ ⎝ n  j=1 ∂ Q ji s j ⎞ ⎠g(s, t). (4) (4) 1 We suppress the dependence on the initial states x0 in the following. 2 We suppress the generating function’s dependence on x0 and t0 in the following. 123 123 381 General solution of the chemical master equation… This result can be found in textbooks such as Gardiner (2009), Sect. 11.6.4, p. 297. We provide a proof in “Appendix A.2” to the convenience of the reader. This result can be found in textbooks such as Gardiner (2009), Sect. 11.6.4, p. 297. We provide a proof in “Appendix A.2” to the convenience of the reader. We define first-order networks as those obeying a first-order PDE for the gen- erating function ( n j=1 Q ji ≤1), or equivalently a linear ordinary differential equation (ODE) system for the moments. In terms of chemical reactions, first-order networks consist exclusively of reactions of the type Sj → l Rli Sl, i.e. monomolec- ular plus splitting and (auto-)catalytic reactions. First-order PDEs are solvable by the method of characteristics,3 a method to convert the PDE solution problem into ODEs. The basic idea is that the solution of the PDE is given by a set of integral curves s(τ) called characteristic curves, parametrized by some τ ∈R. We obtain the ODE system, describing these curves s(τ), by interpreting (4) as a total derivative, i.e. dg(s(τ), t(τ)) dτ = ∂g(s(τ), τ) ∂t dt dτ + n  i=1 ∂g(s(τ), τ) ∂si dsi(τ) dτ != f (s(τ), τ)g(s(τ), τ). (5) (5) (5) Then, the characteristic system of ODEs, obtained by comparing the coefficients of (5) and (4), reads Then, the characteristic system of ODEs, obtained by comparing the coefficients of (5) and (4), reads dt dτ = 1 m n  dt dτ = 1 dt dτ = 1 ds j(τ) dτ = − m  i=1 ki Q ji n  l=1 sl(τ) Rli −s j(τ)  ( j ∈{1, . . . , n}) (6) dg(s(τ), t(τ)) dτ = m  i=1 n  j=1 ki(1 −Q ji) n  l=1 sl(τ) Rli −1     := f (s(τ),τ) g(s(τ), τ). 4 The same result may be obtained without the use of generating functions (Jahnke and Huisinga 2007). 2.1 Chemical master equation and characteristic ODEs (7) (6) (7) := f (s(τ),τ) In case we not only require n j=1 Q ji ≤1, but also n j=1 R ji ≤1, the equations for dsi(τ) dτ and dg(s(τ),t(τ)) dτ are linear and we are restricted to monomolecular reactions. In consequence, the characteristic ODE system is solvable and we obtain a general solution of the CME, as shown in Gans (1960).4 For the complete definition of the ODE system, we specify the initial conditions s0 : = s(0) g(s0, 0) = d(s0). (8) (8) The same result may be obtained without the use of generating functions (Jahnke and Huisinga 2007). 12 3 3 382 M. Reis et al. The generating function d(s0) specifies the initial distribution. Here, we study deter- ministic initial conditions d(s0) = s0 x0 and product Poissonian initial conditions The generating function d(s0) specifies the initial distribution. Here, we study deter- ministic initial conditions d(s0) = s0 x0 and product Poissonian initial conditions d(s0) = exp  ⟨x⟩0 · (s0 −1)  , where  x  0 is the expected number of molecules at t = 0. where  x  0 is the expected number of molecules at t = 0. 5 The temporal dependence of the functions is suppressed as usual. Furthermore, xi : R+ 0 →Cmi , fi : Cmi+1 ×Cmi+2 ×· · ·×Cmn ×R+ 0 →Cmi and hi : Cmi+1 ×Cmi+2 ×· · ·×Cmn ×R+ 0 →Cmi ×Cmi , where n, mi ∈N. 2.2 Hierarchically linear ODE systems and reaction networks (11) (11) If the matrix-valued function h1 does not commute for two different instants of time, the matrix exponent is expressed in terms of Magnus series (see Magnus 1954). Even in that case [h1(x2(t1)), h1(x2(t2))] ̸= 0, it is still possible to write down an analytical expression in terms of matrix exponentials. ⊓⊔ ⊓⊔ A hierarchically linear ODE system translates to the study of chemical reaction networks modeled by the CME as follows: Definition 3 (Hierarchic first-order reaction network) A first-order reaction network, described by a hierarchically linear characteristic ODE system is called hierarchic first-order reaction network. 2.2 Hierarchically linear ODE systems and reaction networks Compared to monomolecular reaction networks, a more general subset of first-order networks are hierarchically linear networks, which we characterize in this article. Definition 2 (Hierarchically linear ODE system) Let xi(t), fi(·, t) and hi(·, t) be con- tinuously differentiable, vector- and matrix-valued functions respectively.5 An ODE system exhibiting the structure dx1 dt = f1(x2, x3, . . . , xn) + h1(x2, x3, . . . , xn)x1 dx2 dt = f2(x3, x4, . . . , xn) + h2(x3, x4, . . . , xn)x2 ... dxn dt = fn + hnxn dx1 dt = f1(x2, x3, . . . , xn) + h1(x2, x3, . . . , xn)x1 dx2 dt = f2(x3, x4, . . . , xn) + h2(x3, x4, . . . , xn)x2 dxn dt = fn + hnxn (9) (9) is called a hierarchically linear ODE system. is called a hierarchically linear ODE system. The next proposition gives a reason for our interest in hierarchically linear ODE systems: Proposition 1 All hierarchically linear ODE systems are solvable by means of matrix exponentials. Proof We inspect a system of two levels only, since an extension to the n-dimensional case follows by mathematical induction. Then, the system for n = 2 ˙x1 = f1(x2) + h1(x2)x1 ˙x2 = f2 + h2x2 has a solution in terms of simple matrix exponentials if h1(x2(t1)) and h1(x2(t2)) commute, i.e. has a solution in terms of simple matrix exponentials if h1(x2(t1)) and h1(x2(t2)) commute, i.e. h1(x2(t1))h1(x2(t2)) = h1(x2(t2))h1(x2(t1)) . h1(x2(t1))h1(x2(t2)) = h1(x2(t2))h1(x2(t1)) . h1(x2(t1))h1(x2(t2)) = h1(x2(t2))h1(x2(t1)) . 5 The temporal dependence of the functions is suppressed as usual. Furthermore, xi : R+ 0 →Cmi , fi : Cmi+1 ×Cmi+2 ×· · ·×Cmn ×R+ 0 →Cmi and hi : Cmi+1 ×Cmi+2 ×· · ·×Cmn ×R+ 0 →Cmi ×Cmi , where n, mi ∈N. 123 General solution of the chemical master equation… General solution of the chemical master equation… 383 In this case, the solution can be written6 as In this case, the solution can be written6 as In this case, the solution can be written6 as x1(t) = exp  t 0 h1(x2(t′))dt′  x0 1 + exp  t 0 h1(x2(t′))dt′   t 0 exp  −  t′ 0 h1(x2(t′′))dt′′  f1(x2(t′))dt′ (10) x2(t) = exp{h2t}x0 2 + exp{h2t}  t 0 exp{−h2t′}f2dt′ . 6 For a review of the solution of a non-homogeneous system see Brannan (2010). 2.3 Two-level hierarchic networks In contrast to the general form of the preceding definition, we focus on two-level hierarchic networks, composed of two subsystems. That is, we have nind indepen- dent species Sind = (S1, . . . , Snind, 0, . . . , 0)T and ndep dependent species Sdep = (0, . . . , 0, Snind+1, . . . , Sn)T, where nind + ndep = n. We express a two-level hierar- chic network in terms of chemical reactions as QTSind k→RTSind + RTSdep (first order reactions, system I), (12) QTSdep k→RTSdep (monomolecular reactions, system II). (13) (12) (13) The first-order reactions’ products are split into two parts: Sind appears only once per reaction ( nind j=1 R ji ≤1 for i ∈{1, . . . , m}), while an arbitrary number of molecules Sdep is allowed. The monomolecular reactions are defined by n j=nind+1 R ji ≤1 for i ∈{1, . . . , m}. Note that by ignoring Sdep, we might study QTSind →RTSind independently of the monomolecular reactions (13), and obtain another monomolecular network, since nind j=1 R ji ≤1 for i ∈{1, . . . , m}. We therefore refer to (12) as independent part and (13) as dependent part of the hierarchic system, since the latter cannot be modeled 12 3 3 384 M. Reis et al. Fig. 1 Scheme depicting a hierarchic first-order network. In system I, all non-monomolecular reactions take place, such as splitting or catalytic reactions, while in system II only monomolecular reactions take place Fig. 1 Scheme depicting a hierarchic first-order network. In system I, all non-monomolecular reactions take place, such as splitting or catalytic reactions, while in system II only monomolecular reactions take place Fig. 1 Scheme depicting a hierarchic first-order network. In system I, all non-monomolecular reactions take place, such as splitting or catalytic reactions, while in system II only monomolecular reactions take place Fig. 1 Scheme depicting a hierarchic first-order network. In system I, all non-monomolecular reactions take place, such as splitting or catalytic reactions, while in system II only monomolecular reactions take place independently of system I. Figure 1 depicts an example of such a hierarchic first-order two-level system. independently of system I. Figure 1 depicts an example of such a hierarchic first-orde two-level system. independently of system I. Figure 1 depicts an example of such a hierarchic first-order two-level system. Mathematically, (12) and (13) translate to a hierarchically linear characteristic ODE system. That is, the general form (6) is constrained to ds j dt = − m  i=1 Q jiki ⎧ ⎨ ⎩ ⎡ ⎣ n  l=nind+1 sl Rli ⎤ ⎦ nind  l=1 Rlisl + δ nind l=1 Rli,0  −Q jis j ⎫ ⎬ ⎭ ( j ∈{1, . . . nind}), (14) ds j dt = − m  i=1 Q jiki ⎛ ⎝ n  l=nind+1 Rlisl + δ n l=nind+1 Rli,0 −Q jis j ⎞ ⎠ ( j ∈{nind + 1, . . . n}). (15) ds j dt = − m  i=1 Q jiki ⎧ ⎨ ⎩ ⎡ ⎣ n  l=nind+1 sl Rli ⎤ ⎦ nind  l=1 Rlisl + δ nind l=1 Rli,0  −Q jis j ⎫ ⎬ ⎭ ( j ∈{1, . . . nind}), (14) ds j dt = − m  i=1 Q jiki ⎛ ⎝ n  l=nind+1 Rlisl + δ n l=nind+1 Rli,0 −Q jis j ⎞ ⎠ ( j ∈{nind + 1, . . . n}). (15) (14) (15) Note that the algebraic hierarchy is inverse to the reaction network hierarchy. The dependent part of the reaction network (system II) is described by an autonomous ODE system, while the independent part (system I) is non-autonomous due to the term &n l=nind+1 sl Rli . & l nind+1 The monomolecular system II can be expressed more compactly as Sj α jk →Sk Sj α j0 →∅, where j, k ∈{nind + 1, . . . , n} and A := (αi j) is the matrix holding the conversion rates. Using that matrix, we rewrite (15) as where j, k ∈{nind + 1, . . . , n} and A := (αi j) is the matrix holding the conversion rates. Using that matrix, we rewrite (15) as dsdep dt = −AT(sdep −1). independently of system I. Figure 1 depicts an example of such a hierarchic first-orde two-level system. (16) (16) 123 General solution of the chemical master equation… 385 The inversion of the hierarchic and algebraic structure, mentioned in the previous paragraph, also becomes clear by the traditional rate equations for the concentration vector Cdep of system II The inversion of the hierarchic and algebraic structure, mentioned in the previous paragraph, also becomes clear by the traditional rate equations for the concentration vector Cdep of system II dCdep dt = ACdep + influx from system I. (17) (17) It was shown by Jahnke and Huisinga (2007) using a statistical argument that the solutiontothemonomolecularCMEisaproductPoissondistribution,givenPoissonian initialconditions,andamultinomialdistribution,givendeterministicinitialconditions. Thereby, the parameters of the distributions are given by the traditional rate Eq. (17). This parametrization is also evident by the characteristic ODEs (16) because these can be expressed using the transposed Jacobian matrix of (17). It was shown by Jahnke and Huisinga (2007) using a statistical argument that the solutiontothemonomolecularCMEisaproductPoissondistribution,givenPoissonian initialconditions,andamultinomialdistribution,givendeterministicinitialconditions. Thereby, the parameters of the distributions are given by the traditional rate Eq. (17). This parametrization is also evident by the characteristic ODEs (16) because these can be expressed using the transposed Jacobian matrix of (17). However, it also becomes clear that the exact solution to the CME for a general first- order network, is not fully parametrized by the traditional rate equations: The ODE system (13) is structurally different from the traditional rate equations for system I due to the time-dependent coefficients &n l=nind+1 sl Rli . In contrast to the characteristic Eq. (14), the traditional rate equation system is generally autonomous if the rate constants ki are time-independent as we assume. In order to highlight the relationship between traditional rate equations and char- acteristic ODEs for system II, we use the convention QTSind k→RTSind + RTSdep , Sj α jk →Sk, Sj α j0 →∅, ∅ b→S, (18) (18) instead of (12) and (13) to fully express the two-level hierarchic reaction network. Note that we summarized influx reactions to both system I and II in (18), which yields the characteristic equations ds j dt = − m  i=1 Q jiki ⎧ ⎨ ⎩ ⎡ ⎣ n  l=nind+1 sl Rli ⎤ ⎦ nind  l=1 Rlisl + δ nind l=1 Rli,0  −Q jis j ⎫ ⎬ ⎭ ( j ∈{1, . . . nind}) dsdep dt = −AT(sdep −1) dg(s, t) dt = b · (s −1)g(s, t) instead of (14) and (15). instead of (14) and (15). 3.1 Solvability of hierarchic first-order networks First, we show that the CME is solvable for any hierarchically linear reaction network Theorem 1 (Existence of analytical solutions for hierarchic first-order networks) The Master equation corresponding to a hierarchic first-order reaction network is analyt- ically solvable. Proof Applying Proposition 1, we find that the characteristic system (6) is solvable as a linear equation system as s = eCs0 + v, where C is a square matrix and v a column vector. For the initial condition of (7), we need s0. Since eC is generally invertible, the characteristic system s = eCs0 + v can be solved for s0. This in turn yields the generating function and the distribution, defined as the coefficients of the former. ⊓⊔ Remark 1 The term “analytical solution”, which is often used in a more intuitive sense, requires some explanation. One may argue that the Master equation is a system of linear ODEs and can always be formally solved by a matrix exponential as such. This remains true even for bimolecular networks such as the Lotka–Volterra model (Lotka 1925). However, a formal solution of the Master equation does not tell us anything about the structure of the distribution, if the matrix exponential cannot be calculated explicitly. Contrary to this non-analytical case, the generating function stated in the next proposition earns the word “analytical”, even if it also contains a matrix exponential. As shown in Sect. 3.2, the matrix exponential from the next proposition enables us to analyze properties of the distribution and may be used to find the stationary distribution, to compute moments and cumulants, etc. Proposition 2 Let b = 0, i.e. no reactions7 ∅ b→S. Given that the molecules are initially product Poisson distributed, i.e. g(s, 0) = exp ' x  0 · (s −1) ( , the generating function is given by g(s, t) = exp ) ⟨x1⟩0 · ' e− * t 0 J(sdep,t′,t)dt′sind −  t 0 e− * t′ 0 J(sdep,t′′,t)dt′′f(sdep, t′, t)dt′ −1 ( + exp(At)⟨x2⟩0 · (sdep −1) + . (19) g(s, t) = exp ) ⟨x1⟩0 · ' e− * t 0 J(sdep,t′,t)dt′sind −  t 0 e− * t′ 0 J(sdep,t′′,t)dt′′f(sdep, t′, t)dt′ −1 (19) + exp(At)⟨x2⟩0 · (sdep −1) + . independently of system I. Figure 1 depicts an example of such a hierarchic first-orde two-level system. 12 3 M. Reis et al. 386 Without this requirement, we obtain an additional factor e * t 0 b·(sind(t′′′)−1)dt′′′, where sind(t′′′) is defined (55). For more compact expressions, this factor was left out without loss of generality. 3.1 Solvability of hierarchic first-order networks In the last expression, the Jacobian J is defined by In the last expression, the Jacobian J is defined by Jpq(sdep, t′, t) := m  i=1 Q piki 1 −ci(sdep, t′, t)Rqi , p, q ∈{1, . . . , nind} (20) 7 Without this requirement, we obtain an additional factor e * t 0 b·(sind(t′′′)−1)dt′′′, where sind(t′′′) is defined in (55). For more compact expressions, this factor was left out without loss of generality. 123 General solution of the chemical master equation… 387 General solution of the chemical master equation… 387 with8 [J(sdep, t1, t), J(sdep, t2, t)] = 0 and f is given by with8 [J(sdep, t1, t), J(sdep, t2, t)] = 0 and f is given by with8 [J(sdep, t1, t), J(sdep, t2, t)] = 0 and f is given by f j(sdep, t′, t) := − m  i=1 Q jikici(sdep, t′, t) , j ∈{1, . . . , nind} (21) (21) where ci(sdep, t′, t) := n  l=nind+1  exp , −A(t′ −t) - ϵl · (sdep −1) + 1 Rli . (22) (22) For deterministic initial conditions, i.e. P(x, 0) = δx,x0, where x0 = (x0 1, . . . , x0 n)T, we obtain For deterministic initial conditions, i.e. P(x, 0) = δx,x0, where x0 = (x0 1, . . . , x0 n)T we obtain g(s, t) = ) n  i=1 ' e− * t 0 J(sdep,t′,t)dt′sind −  t 0 e− * t′ 0 J(sdep,t′′,t)dt′′f(sdep, t′, t)dt′( ϵ(1) i + exp(At)ϵ(2) i · (sdep −1) + 1 x0 i + , (23) g(s, t) = ) n  i=1 ' e− * t 0 J(sdep,t′,t)dt′sind −  t 0 e− * t′ 0 J(sdep,t′′,t)dt′′f(sdep, t′, t)dt′( ϵ(1) i + exp(At)ϵ(2) i · (sdep −1) + 1 x0 i + , (23) + exp(At)ϵ(2) i · (sdep −1) + 1 x0 i + , (23) with ε(1) i := coliInind×nind and ε(2) i := coliIndep×ndep. with ε(1) i := coliInind×nind and ε(2) i := coliIndep×ndep. We postpone the proof to “Appendix A.3” and interpret this result first. Remember that the deterministic or product Poisson distributions, assumed as an initial distributions in the preceding proposition, imply that all random variables are uncorrelated for a fixed time t. 8 Without this requirement, Magnus series are needed to express the solution. For more compact expressions we restrict ourselves to [J(sdep, t1, t), J(sdep, t2, t)] = 0 without loss of generality. 3.1 Solvability of hierarchic first-order networks This can be shown by using the cumulant generating function κ(ξ) = log(g(eξ)), that is given for a product Poisson distribution by We postpone the proof to “Appendix A.3” and interpret this result first. Remember that the deterministic or product Poisson distributions, assumed as an initial distributions in the preceding proposition, imply that all random variables are uncorrelated for a fixed time t. This can be shown by using the cumulant generating function κ(ξ) = log(g(eξ)), that is given for a product Poisson distribution by κ(ξ) = log(e  x  1(eξ1−1)+···+  x  n(eξn −1)) =  x  1(eξ1 −1) + · · · +  x  n(eξn −1) . Here, “uncorrelated” means that the covariances and higher order mixed cumulants, defined as the coefficients of κ(ξ), are zero: Here, “uncorrelated” means that the covariances and higher order mixed cumulants, defined as the coefficients of κ(ξ), are zero: ∂ξ1,...,ξn  x  1(eξ1 −1) + · · · +  x  n(eξn −1) ... ξ=0 = 0. For monomolecular reaction networks, the variables remain uncorrelated after some time t > 0, since a monomolecular system given Poissonian initial conditions stays Poissonian, as shown in Jahnke and Huisinga (2007), Proposition 2. Contrastingly, for first-order processes, covariances between the variables appear for t > 0, because the exponent of Eq. (19) is in general not a first-order polynomial. In other words, monomolecular systems stay uncorrelated for uncorrelated initial conditions, while first-order systems do not. 12 3 3 388 M. Reis et al. We understand the correlations in terms of chemical reactions by the difference between the first-order splitting reaction Sj →Sk + Sl and the two monomolecular reactions Sj →Sk and Sj →Sl: The molecules Sk and Sl appear simultaneously in the splitting reaction, while the two monomolecular conversions are statistically independent events. 3.2 Analytical form of marginal distributions As we show in the following, the hierarchic network structure yields marginal distri- butions that are best interpreted as generalized distributions. We follow Johnson et al. (2005) for the next definition. Definition 4 (Generalizing distribution) The distribution P1 is generalized by the generalizing distribution P2: P ∼P1 / P2 :⇔g(s) = g1(g2(s)), where g1(s) and g2(s) are the corresponding generating functions. where g1(s) and g2(s) are the corresponding generating functions. Generalized distributions are related to mixing distributions by Gurland’s theorem (Gurland 1957), if certain requirements are met. Next, we define two classes of gen- eralized distributions that arise as marginal distributions for hierarchic networks. Definition 5 (Discrete compound Poisson distribution (DCP)) A discrete compound Poisson distribution PDCP is a univariate distribution (Zhang et al. 2014), generalizing the Poisson distribution with another distribution P2: PDCP ∼PPoisson / P2. The generating function is defined accordingly: gDCP(s) := gPoisson(g2(s)) = eλ(g2(s)−1) = e ∞ i=1 λαi(si−1) , where λ > 0 and αi ∈[0, 1] with ∞ i=1 αi = 1 are parameters. We introduce the notation ∞ where λ > 0 and αi ∈[0, 1] with ∞ i=1 αi = 1 are parameters. We introduce the notation ∞ DCPN :⇔degs ∞  i=1 λαi(si −1) = N. (24) (24) The next type of distribution was previously defined by Khatri and Patel (1961) and will be important for our study of reaction networks given deterministic initial conditions: The next type of distribution was previously defined by Khatri and Patel (1961) and will be important for our study of reaction networks given deterministic initial conditions: Definition 6 (Khatri’s Type B distribution) Khatri’s Type B distribution PKTB is a univariate distribution, generalizing the deterministic distribution with another distri- bution P2: PKTB ∼PDeterministic / P2. PKTB ∼PDeterministic / P2. 123 123 12 General solution of the chemical master equation… 389 The generating function is defined accordingly: The generating function is defined accordingly: The generating function is defined accordingly: gKTB(s) := g2(s) ν = 0 ∞  i=0 αisi 1ν , gKTB(s) := g2(s) ν = 0 ∞  i=0 αisi 1ν , αi ∈[0, 1] are parameters and ν ∈N0 is the number of trials. Furthermore, g2(s) must be a generating function, i.e. ∞ i=1 αi = 1. We introduce the notation KTBN :⇔degs ∞  i=0 αisi = N. 9 The matrix exponential and the following expression, obtained by the Cayley–Hamilton theorem, is reviewed in Rowell (2004). 3.2 Analytical form of marginal distributions (25) (25) These distributions specify the marginal distributions of the dependent part (system II) of hierarchic first-order reaction networks. We will show in Sect. 3.3, that the DCPN and KTBN distributions are conditionally multimodal for N > 1. Proposition 3 (Marginal distributions given ∂sX λi(sX) = 0) Let λi(sX) be the ith eigenvalue of J(sX, t′, t). Furthermore, let ∂sX λi(sX) = 0, f = 0 and b = 0. Given Poissonian initial conditions, the marginal distribution of any species X from the dependent part of the network is DCPN, where N < ∞. Given deterministic initial conditions, this distribution is KTBN. To prove this statement, we need an auxiliary result: Lemma 2 Let H(x) be an n×n matrix, whose entries depend polynomially on x ∈C. Let λi(x) be the ith eigenvalue of H(x). Then, ∂xλi(x) = 0 ⇒degx eH(x) i j < ∞. Proof By the Cayley–Hamilton theorem, H(x) fulfills its own characteristic polyno- mial Δ(λ), i.e. Δ(H(x)) = 0. Furthermore, any polynomial p(λ) might be expressed as p(λ) = q(λ)Δ(λ) + r(λ), where q is found by long division p/q with remainder r of degree ≤n −1. Since Δ(H(x)) = 0, we might express9 eH(x) as eH(x)    p(H(x)) = n−1  k=0 αk(x)Hk(x)    r(H(x)) (26) (26) for some coefficients αk(x). for some coefficients αk(x). for some coefficients αk(x). Furthermore, the eigenvalues λi(x) fulfill Furthermore, the eigenvalues λi(x) fulfill eλi(x) = ∞  k=0 λk i (x) k! = n−1  k=0 αk(x)λk i (x)    := Lα i (i ∈{1, . . . , n}). (27) (27) 9 The matrix exponential and the following expression, obtained by the Cayley–Hamilton theorem, is reviewed in Rowell (2004). 12 390 M. Reis et al. Here, the second relation follows again from long division by the characteristic poly- nomial of H(x). For ∂xλi(x) = 0, the coefficients of the characteristic polynomial do not depend on x and thus ∂xαk(x) = 0. Here, the second relation follows again from long division by the characteristic poly- nomial of H(x). For ∂xλi(x) = 0, the coefficients of the characteristic polynomial do not depend on x and thus ∂xαk(x) = 0. Therefore, the expression (26) introduces only a finite-order dependence on x. 10 We suppress the time-dependence of J in the following. 3.2 Analytical form of marginal distributions ⊓⊔ Using this Lemma, we prove Proposition 3: Proof We obtain the marginal distribution of species X by setting all si = 1 except sX in Eqs. (19) and (23) respectively. First of all, the integrals in Eq. (19) do not change the order of the polynomial in the exponent with respect to sX, that is degsX e−J(sX,t,t′) i j = degsX e− * t 0 J(sX,t′,t)dt′ i j. Therefore, we have a DCPN dis- tribution, where N is given by (24). We find10 N = degsX  s0 ind(sX) ·  x1  0  = degsX  e−J(sX)1 ·  x1  0  = degsX  i, j  e−J(sX) i j (28) ≤ max i, j∈{1,...,nind} degsX  e−J(sX) i j . (29) (29) Because J(sX) depends polynomially on sX, we apply Lemma 2 to obtain N ≤degsX  e−J(sX) i j < ∞. In case [J(sX, t1, t), J(sX, t2, t)] ̸= 0, the same argument can be made for the Magnus series. By replacing  x1  0 in Eq. (28) with ε(1) i := coliInind×nind for deterministic initial conditions, we have the result KTBN, where N < ∞. ⊓⊔ In case [J(sX, t1, t), J(sX, t2, t)] ̸= 0, the same argument can be made for the Magnus series. By replacing  x1  0 in Eq. (28) with ε(1) i := coliInind×nind for deterministic initial conditions, we have the result KTBN, where N < ∞. ⊓⊔ ⊓⊔ For simple reaction networks such as those from the next proposition, we show the order of the marginal distributions to be larger than one. We first investigate networks whose independent part is mass-conservative, i.e. nind l=1 Rli ̸= 0 ⇒f = 0 and nind l=1 Qli ̸= 0 ⇒b = 0. Proposition 4 Let ∂sX λi(sX) = 0, f = 0, b = 0 and [J(sX, t1, t), J(sX, t2, t)] = 0. The marginal distribution of any dependent species X is DCPN (KTBN for determin- istic initial conditions), with N > 1 for the following minimal reaction networks: 1. Sind 1 →Sind 2 + RX, (Type I) where R > 1 and nind = 2. where R > 1 and nind = 2. 2. 2. 11 Here, “∝” means that lower order terms and constant factors in sX are dropped. 3.2 Analytical form of marginal distributions Sind 1 →Sind 2 + R1X Sind 2 →Sind 3 + R2X, (Type II) Sind 1 →Sind 2 + R1X Sind 2 →Sind 3 + R2X, (Type II) where R1 ≥1, R2 ≥1 and nind = 3. where R1 ≥1, R2 ≥1 and nind = 3. where R1 ≥1, R2 ≥1 and nind = 3. 10 We suppress the time-dependence of J in the following. 10 We suppress the time-dependence of J in the following. 123 General solution of the chemical master equation… 39 General solution of the chemical master equation… 391 General solution of the chemical master equation… 391 Proof We expand the matrix powers in Proof We expand the matrix powers in degsX  p,q (e−J(sX))pq (26) = degsX  p,q 0 nind−1  k=0 (−J(sX))kαk 1 pq , (30) (30) starting with J1: Jpq(sX) (20) = m  i=1 ki Q pi(1 −ci(sX)Rqi), p, q ∈{1, . . . , nind} For reaction Type I, it suffices to consider the first order J1 of (30). Since there are no reactions within the dependent part A = 0, we then have For reaction Type I, it suffices to consider the first order J1 of (30). Since there are no reactions within the dependent part A = 0, we then have ci(sX, t′, t) (22) =  1 · (sX −1) + 1 Ri = s Ri X (31) (31) In consequence of (31), we get degsX p,q(eJ(sX))pq > 1 whenever Ri > 1. This translates to a splitting reaction of Type I. To see ∂sX λi(sX) = 0, consider ds1 dt = −kS1→S2+X    :=1 (s R Xs2 −s1) ds2 dt = 0 ds1 dt = −kS1→S2+X    :=1 (s R Xs2 −s1) and det(J −λ1) = det((∂s j dsi dt )i j −λ1) = det 0 1 −λ −s R X 0 −λ 1 = λ2 −λ. For reaction Type II, nind = 3 and we need to calculate the second order J2 in (30). We have11 J 2 pq(sX) ∝ nind  r=1 m  i=1 ki Q pi(1 −s Ri X Rri)  ⎛ ⎝ m  j=1 k j Qr j(1 −s R j X Rqj) ⎞ ⎠ ∝ nind  r=1 m  i, j=1 kik j Q pi Rri Qr j Rqjs Ri+R j X . 3.2 Analytical form of marginal distributions Note that only i ̸= j terms are non-zero due to nind l=1 Rli ≤1. This implies that the products from the independent part must appear in different reactions. In terms of chemical reaction networks, we have 12 3 3 392 M. Reis et al. Q p1Sind p →Rr1Sind r + Rξ1X Qr2Sind r →Rq2Sind q + Rξ2X. Q p1Sind p →Rr1Sind r + Rξ1X Qr2Sind r →Rq2Sind q + Rξ2X. Q p1Sind p →Rr1Sind r + Rξ1X Qr2Sind r →Rq2Sind q + Rξ2X. Q p1Sind p →Rr1Sind r + Rξ1X Qr2Sind r →Rq2Sind q + Rξ2X. We arbitrarily chose p = 1, r = 2, q = 3 to obtain the result. To see ∂sX λi(sX) = 0, consider We arbitrarily chose p = 1, r = 2, q = 3 to obtain the result. To see ∂sX λi(sX) = 0 consider We arbitrarily chose p = 1, r = 2, q = 3 to obtain the result. To see ∂sX λi(sX) = 0, consider ds1 dt = −kS1→S2+R1X    :=1 (s R1 X s2 −s1) ds2 dt = −kS2→S3+R2X    :=1 (s R2 X s3 −s2) ds3 dt = 0 ds1 dt = −kS1→S2+R1X    :=1 (s R1 X s2 −s1) ds2 dt = −kS2→S3+R2X    :=1 (s R2 X s3 −s2) ds3 dt = 0 and and det J = det(∂s j dsi dt )i j = det ⎛ ⎝ 1 −λ −s R1 X 0 0 1 −λ −s R2 X 0 0 −λ ⎞ ⎠= −λ  λ2 −2λ + 1  . ⊓⊔ Proposition 5 Let J(t′, t) be independent of sX, b = 0 and ∂sX f(sX, t′, t) ̸= 0, then the marginal distribution of any species X from the dependent part is DCPN (KTBN for deterministic initial conditions), with N > 1 for the following minimal reaction network: Sind 1 →RX, where R > 1 and nind = 1. where R > 1 and nind = 1. Proof We express the reaction Sind 1 →RX by Proof We express the reaction Sind 1 →RX by f1(sX) (21) = −k1  1 · (sX −1) + 1 R = −k1s R X and plug the same expression into (19) to obtain log g(sX, t) ∝s R X. In consequence, we get a DCPN distribution with N > 1 whenever R > 1. 3.2 Analytical form of marginal distributions The analogous statement for deterministic initial conditions follows by plugging f1(sX) into (23). ⊓⊔ and plug the same expression into (19) to obtain log g(sX, t) ∝s R X. In consequence, we get a DCPN distribution with N > 1 whenever R > 1. The analogous statement for deterministic initial conditions follows by plugging f1(sX) into (23). ⊓⊔ The next proposition holds for the transcription–translation model, as examined in detail in Example 5. Proposition 6 (Marginal distributions given ∂sX λi(sX) ̸= 0) Let λi(sX) be the ith eigenvalue of J(sX, t′, t) and let ∂sX λ(sX) ̸= 0. Given Poissonian initial conditions, the marginal distribution of any species X from the dependent part of the network is DCP∞(KTB∞for deterministic initial conditions). 123 123 ion of the chemical master equation… 393 General solution of the chemical master equation… 3 General solution of the chemical master equation… 393 Proof In case all eigenvalues are distinct, we solve the system (27) by inverting the matrix12 L(sX), so Proof In case all eigenvalues are distinct, we solve the system (27) by inverting the matrix12 L(sX), so eJ(sX) = nind−1  k=0 nind−1  i=0 (L−1(sX))kieλi(sX)Jk(sX). (32) (32) Since (L−1)ki is a rational function of the eigenvalues, we have Since (L−1)ki is a rational function of the eigenvalues, we have max i, j∈{1,...,nind} degsX eJ(sX) i j = ∞. (33) (33) In case there is an eigenvalue λ j of multiplicity μ, the matrix L is not invertible. Since In case there is an eigenvalue λ j of multiplicity μ, the matrix L is not invertible. Since dμ−1Δ(λ) dλμ−1 .... λ j = dμ−1 dλμ−1 (λ −λ j)μ nind−μ  i=1 (λ −λi) .... λ j = 0 , we derive (27) for λ and obtain μ −1 additional equations: we derive (27) for λ and obtain μ −1 additional equations: dieλ j(sX) dλi = di dλi nind−1  k=0 αk(sX)λk (i ∈{1, . . . , μ −1}). We solve this system together with (27) for αk(sX) by inverting a matrix. The entries of this matrix are rational functions of λ, so the terms eλi(sX) in (32) do not cancel, i.e. Equation (33) holds. In the last step, we plug Eq. (32) into (19) for Poissonian and into (23) for deterministic initial conditions to obtain the generating function for the marginal distribution of X. 3.2 Analytical form of marginal distributions Since the exponent of (19) is of infinite degree, we obtain a DCP∞class distribution. For deterministic initial conditions, we obtain a KTB∞ class distribution. ⊓⊔ ⊓⊔ 12 The time-dependence of L and J is suppressed in the following. 3.3 Modality of marginal distributions In this section, we investigate under which conditions the generalized distributions from Proposition 2 have infinitely many modes. We need several definitions for this end: Definition 7 (Unimodality)Adistribution pn issaidtobeunimodal withmodea ∈N0 efinition 7 (Unimodality)Adistribution pn issaidtobeunimodal withmodea ∈N0 pn≥pn−1, ∀n ≤a and pn+1 ≤pn, ∀n ≥a. If this property does not depend on the parameters of the distribution, the latter is said to be unconditionally unimodal. A unimodal distribution that results in a unimodal 12 3 394 M. Reis et al. distribution upon convolution with another unimodal distribution is called strongly unimodal.13 distribution upon convolution with another unimodal distribution is called strongly unimodal.13 The classes DCP1 and KTB1 are unconditionally unimodal, since the only members of these sets are the Poissonian and Binomial distribution respectively. For the indepen- dent species (system I) of a hierarchic first-order reaction network, we obtain unimodal marginal distributions: Theorem 2 (Unconditional unimodality of marginal distributions of independent species (system I)) Given deterministic or Poissonian initial conditions, the species from the independent part of the hierarchic network exhibit unconditionally unimodal marginal distributions at all times t > 0. Proof Upon setting all si = 1, except one sX from the independent part, we obtain a Poissonian distribution for Eq. (19). For deterministic initial conditions, Eq. (23) yields a convolution of binomial and Poisson distributions. Since the binomial and Poisson distribution are strongly unimodal, the resulting distribution is unimodal as well, as shown by Keilson and Gerber (1971). ⊓⊔ ⊓⊔ For the second part of a hierarchic first-order network, we need the following definition: Definition 8 (Conditional multimodality) A distribution that is not unconditionally unimodal is called conditionally multimodal. As an example for the preceding definition, we have Neyman’s Type A distribution (Neyman 1939), defined by the generating function exp(λ(eφ(s−1) −1)), where λ > 0 and φ > 0. Because of the double exponential generation function, it belongs to the class of DCP∞distributions that we prove to be conditionally multimodal in the next proposition. Proposition 7 (Three classes of conditionally multimodal distributions) The classes of distributions DCP∞, KTB∞, DCP2, KTB2 and DCP3, KTB3 are conditionally multimodal. Proposition 7 (Three classes of conditionally multimodal distributions) The classes of distributions DCP∞, KTB∞, DCP2, KTB2 and DCP3, KTB3 are conditionally multimodal. Proof Since conditional multimodality is defined to be the contrary of uncondi- tional unimodality, a counterexample is enough to prove conditional multimodality. Respective counterexamples are shown in Fig.2. 14 Note that the coefficients of eφ(s−1) are defined as the parameters of DCP∞, not φ. 13 Defined by Keilson and Gerber (1971). 13 Defined by Keilson and Gerber (1971). 3.3 Modality of marginal distributions Figure 2a shows Neyman’s Type A distribution, defined by the DCP∞generating function g(s) = exp(λ(eφ(s−1) −1)), can be multimodal.14 The same is done for the Hermite distribution by the gener- ating function g(s) = ea1(s−1)+a2(s2−1). As a DCP2 distribution, it is conditionally multimodal, as demonstrated in Fig. 2a. A counterexample for DCP3 is given by 12 123 General solution of the chemical master equation… 395 (a) (b) (c) (d) (e) (f) Fig. 2 Some conditionally multimodal distributions. The thick gray lines depict the expectation value, while the dashed lines stand for the standard deviation. The arrow marks the probability for a zero outcome. a Hermite (DCP2) distribution, a1 = 1, a2 = 5.1. b Triple stuttering-Poisson (DCP3) distribution, a1 = 0.1, a2 = 1.1, a3 = 2. c Neyman’s type A (DCP∞) distribution, λ = 2, φ = 18.1. d Binomial–Binomial (KTB2) distribution, p1 = 0.5, p2 = 0.8. e Binomial–Binomial (KTB3) distribution, p1 = 0.5, p2 = 0.8. f Binomial–Binomial (KTBinf inity) distribution, p1 = 0.5, p2 = 0.8 (a) (b) (c) (c) (d) (e) (f) (d) (f) (e) Fig. 2 Some conditionally multimodal distributions. The thick gray lines depict the expectation value, while the dashed lines stand for the standard deviation. The arrow marks the probability for a zero outcome. a Hermite (DCP2) distribution, a1 = 1, a2 = 5.1. b Triple stuttering-Poisson (DCP3) distribution, a1 = 0.1, a2 = 1.1, a3 = 2. c Neyman’s type A (DCP∞) distribution, λ = 2, φ = 18.1. d Binomial–Binomial (KTB2) distribution, p1 = 0.5, p2 = 0.8. e Binomial–Binomial (KTB3) distribution, p1 = 0.5, p2 = 0.8. f Binomial–Binomial (KTBinf inity) distribution, p1 = 0.5, p2 = 0.8 g(s) = ea1(s−1)+a2(s2−1)+a3(s3−1) from Fig. 2b. Figure 2d–f shows counterexamples for the generalized binomial distributions KTB2, KTB3 and KTB∞: gKTB2(s) = 1 −p1 + p1(1 −p2 + p2s)2 4 Binomial(4, p1) / Binomial(2, p2) gKTB3(s) = 1 −p1 + p1(1 −p2 + p2s)3 4 Binomial(4, p1) / Binomial(3, p2) gKTB∞(s) = 1 −p1 + p1eλ(s−1) 4 Binomial(4, p1) / Poisson(λ) . ⊓⊔ Since we cannot provide an infinite number of counterexamples, one for each DCPN or KTBN class, where N > 1, we extend the result of the last proposition as a conjecture. Conjecture 1 The classes DCPN and KTBN are conditionally multimodal for N > 3. 3.3 Modality of marginal distributions The intuition behind this conjecture is that the DCPN and KTBN classes are modeling events that can occur in bursts. In terms of Definition 4, the generalizing distribu- tion P2 is modeling the (stochastic) number of events per burst or burst size, whereas the generalized distribution P1 models the (stochastic) number of bursts, yielding the total number of events as P ∼P1 2 P2. In consequence, the distances between the modes seen in Fig. 2 approximately correspond to multiples of the burst sizes. For the Binomial–Binomial distribution from Fig. 2d the burst size is 2, for Fig. 2e it is 3, and for the Binomial–Poisson distribution from Fig. 2f it is λ = 10.8. The same applies for the generalized Poisson distributions. For Neyman’s Type A (Poisson–Poisson) 12 396 M. Reis et al. distribution shown in Fig. 2c, the modes are clearly visible at multiples of 18.1. Thus, the bursting behavior is linked to conditional multimodality. In consequence, what we are conjecturing is that this link stays the same as we increase the number of possible bursts beyond 3. distribution shown in Fig. 2c, the modes are clearly visible at multiples of 18.1. Thus, the bursting behavior is linked to conditional multimodality. In consequence, what we are conjecturing is that this link stays the same as we increase the number of possible bursts beyond 3. In the next Theorems, we summarize the cases in which conditional multimodality appears for the species of the dependent system II. Theorem 3 (Sufficient criterion for conditional multimodality, given deterministic or Poissonian initial conditions and ∂sX λ(sX) ̸= 0) Let ∂sX λ(sX) ̸= 0. Then the depen- dent species X in a hierarchic first-order network obeys a conditionally multimodal marginal distribution at all times t > 0. Proof ∂sX λ(sX) ̸= 0 implies DCP∞and KTB∞class distributions (Proposition 6) that are conditionally multimodal by Proposition 7. ⊓⊔ ⊓⊔ Theorem 4 (Minimal reaction networks exposing conditional multimodality, given deterministic or Poissonian initial conditions and ∂sX λ(sX) = 0) Let ∂sX λ(sX) = 0, b = 0 and assume Conjecture 1 is true. 3.3 Modality of marginal distributions Then, given Poissonian or deterministic initial conditions, the following minimal hierarchic first-order networks exhibit a condition- ally multimodal marginal distribution at all times t > 0 for the dependent species Theorem 4 (Minimal reaction networks exposing conditional multimodality, given deterministic or Poissonian initial conditions and ∂sX λ(sX) = 0) Let ∂sX λ(sX) = 0, Theorem 4 (Minimal reaction networks exposing conditional multimodality, given deterministic or Poissonian initial conditions and ∂sX λ(sX) = 0) Let ∂sX λ(sX) = 0, b = 0 and assume Conjecture 1 is true. Then, given Poissonian or deterministic initial conditions, the following minimal hierarchic first-order networks exhibit a condition- ally multimodal marginal distribution at all times t > 0 for the dependent species X: b = 0 and assume Conjecture 1 is true. Then, given Poissonian or deterministic initial conditions, the following minimal hierarchic first-order networks exhibit a condition- ally multimodal marginal distribution at all times t > 0 for the dependent species X: 1. For mass-conservative independent networks, i.e. f = 0: ( ) (a) Sind 1 →Sind 2 + RX, (Type I) where R > 1 and nind = 2. (b) where R > 1 and nind = 2. ) (b) Sind 1 →Sind 2 + R1X Sind 2 →Sind 3 + R2X, (Type II) where R1 ≥1, R2 ≥1 and nind = 3. where R1 ≥1, R2 ≥1 and nind = 3. 2. For open independent networks and nind l=1 Rli = 0 for all reactions, i.e. ∂sX J = 0 and ∂sX f ̸= 0, the minimal network is: 2. For open independent networks and nind l=1 Rli = 0 for all reactions, i.e. ∂sX J = 0 and ∂sX f ̸= 0, the minimal network is: Sind 1 →RX where R > 1 and nind = 1. Proof By Proposition 4 we know that the distribution class is DCPNProp4 whenever f = 0, where the degree NProp4 > 1 for both types of minimal reaction networks. Proposition 5 applies if ∂sX f ̸= 0, and yields the minimal reaction network Sind 1 →RX for which NProp5 > 1. 123 397 General solution of the chemical master equation… These distribution classes are conditionally multimodal if NProp4 and NProp5 are larger than one according to Proposition 7 and Conjecture 1. 4 Examples In the following two sections, we illustrate how our results can be used to predict conditional multimodality by several examples. The calculations can be verified using the supplemented Wolfram Mathematica files. 3.3 Modality of marginal distributions ⊓⊔ In general conditional multimodality arises, if the degree of the exponent of (19) is larger than one upon setting all si = 1 except sX. 4.1 Basic models 3 Marginal distributions for catalysis product py := P(y, t|y0 = 0, 0), given the parameters kcat = 20 s−1, deterministic (y0 = 0) and Poissonian initial conditions for  x  0 = 2. The thick gray lines mark the expectation value, and the dashed lines the range of one standard deviation from the mean. The arrow marks the probability for a zero outcome and sY = s0 Y we obtain the generating function and sY = s0 Y we obtain the generating function and sY = s0 Y we obtain the generating function and sY = s0 Y we obtain the generating function gGenPoiss(sX, sY , t) = exp  x  0(ekcatt(sY −1)sX −1) +  y  0(sY −1)  . (37) (37) The marginal distribution of Y can be represented as a convolution of Neyman’s type A distribution and a Poisson distribution: The marginal distribution of Y can be represented as a convolution of Neyman’s type A distribution and a Poisson distribution: P ∼ ' Poisson(  x  0) / Poisson(kcatt)    Neyman Type A ( ∗Poisson(  y  0). (38) (38)  Neyman Type A In case we set y0 = 0 as a deterministic initial condition, the distribution is identical to Neyman’s Type A distribution. In Fig. 3, we depict a numerical evaluation of this multimodal distribution. Conditional multimodality is expected by Theorem 3, since the Jacobian exhibits eigenvalues dependent on sY . Note that the most likely outcome is zero, is far from the mean (solid gray line in Fig. 3), even outside the standard deviation (dashed gray line). This clearly demonstrates the misleading character of mean and standard deviation for multimodal distributions. The high probability for a zero outcome, depicted by the black arrow in Fig. 3, can be explained by the high likelihood of an “extinction scenario”: If there are initially no molecules of species X, the number of molecules of type Y must always be zero. This phenomenon can be found in many hierarchic networks, as we show in the following figures. Using deterministic initial conditions, we obtain a contrasting picture, since the resulting marginal distributions are Poissonian: gdet(sX, sY , t) = (sX)x0(sY )y0 = (ekcatt(sY −1)sX)x0(sY )y0 ⇒gdet(1, sY , t) = (ekcatt(sY −1))x0    :=gcat(sY ) . (39) (39) In consequence, no multimodality is observed. 4.1 Basic models Example 1 (Catalysis) The model of the simple catalytic reaction X kcat →X+Y is a triv- ial example of a hierarchic system, since system II is governed by a time-independent characteristic ODE. By Eqs. (6–7) we have dsX dt = −kcat(sXsY −sX) (34) dsY dt = 0 (35) dg dt = 0 ⇔g = g(s0 X, s0 Y , 0). (36) (34) (35) (36) The solution of (35) simply reads sY (t) = s0 Y . We reorder the terms in (34) The solution of (35) simply reads sY (t) = s0 Y . We reorder the terms in (34) dsX dt = sX(−kcat(s0 Y −1)), dsX dt = sX(−kcat(s0 Y −1)), and solve the same equation as sX(t) = e−kcatt(s0 Y −1)s0 X. The generating function, given by (8) for Poissonian initial conditions, reads The generating function, given by (8) for Poissonian initial conditions, reads gPoiss(s0 X, s0 Y , 0) = exp  x  0(s0 X −1) +  y  0(s0 Y −1)  . By replacing s0 X = ekcatt(s0 Y −1)sX, 12 3 398 M. Reis et al. Fig. 3 Marginal distributions for catalysis product py := P(y, t|y0 = 0, 0), given the parameters kcat = 20 s−1, deterministic (y0 = 0) and Poissonian initial conditions for  x  0 = 2. The thick gray lines mark the expectation value, and the dashed lines the range of one standard deviation from the mean. The arrow marks the probability for a zero outcome Fig. 3 Marginal distributions for catalysis product py := P(y, t|y0 = 0, 0), given the parameters kcat = 20 s−1, deterministic (y0 = 0) and Poissonian initial conditions for  x  0 = 2. The thick gray lines mark h i l d h d h d li h f d d d i i f h Th Fig. 3 Marginal distributions for catalysis product py := P(y, t|y0 = 0, 0), given the parameters kcat = 20 s−1, deterministic (y0 = 0) and Poissonian initial conditions for  x  0 = 2. The thick gray lines mark the expectation value, and the dashed lines the range of one standard deviation from the mean. The arrow marks the probability for a zero outcome Fig. 4.1 Basic models However, since Pcat ∈KTB∞, Propo- sition 7 predicts conditionally multimodality. This is not a contradiction because conditional multimodality is a notion defined by the order of the generalizing dis- tribution g2(s) (see Def. 6), not by the model parameters (here kcat). For a concrete 123 General solution of the chemical master equation… General solution of the chemical master equation… 399 Fig. 4 Plot of the joint distribution for the educt X and product Y for the same parameters and time points as Fig. 3 model, only subsets of the parameter space, defined by the set of coefficients of g2(s), are reachable by appropriate choices of the model parameters (here kcat). In our model this subset does not contain to multimodal distributions. The joint distribution is plotted in Fig. 4. To obtain a multimodal distribution for deterministic initial conditions, we add a Fig. 4 Plot of the joint distribution for the educt X and product Y for the same parameters and time points as Fig. 3 model, only subsets of the parameter space, defined by the set of coefficients of g2(s), are reachable by appropriate choices of the model parameters (here kcat). In our model this subset does not contain to multimodal distributions. The joint distribution is plotted in Fig. 4. model, only subsets of the parameter space, defined by the set of coefficients of g2(s), are reachable by appropriate choices of the model parameters (here kcat). In our model this subset does not contain to multimodal distributions. The joint distribution is plotted in Fig. 4. To obtain a multimodal distribution for deterministic initial conditions, we add a degradation reaction, as shown in the next example. Example 2 (Catalysis with degradation) We see a different picture for deterministic initial conditions once we add a degradation mechanism: X kcat →X + Y X kdeg →∅. These reactions result in the characteristic system These reactions result in the characteristic system dsX dt = −kcat(sXsY −sX) −kdeg(1 −sX) dsY dt = 0, solved by solved by sX(t) = sX(kcat(1 −sY ) + kdeg) −kdeg et(kcat(1−sY )+kdeg) + kdeg kcat(1 −sY ) −kdeg . 3 3 M. Reis et al. 400 (a) (b) Fig. 5 Marginal distributions py := P(y, t|y0 = 0, 0) at t = 1.4 s. Parameters: kcat = 10.1 s−1, kdeg = 0.5 s−1. The thick gray lines depict the expectation value, while the dashed lines stand for the standard deviation. The arrow marks the probability for a zero outcome. a Poissonian initial conditions:  x  0 = 1,  y  0 = 0.001. b Deterministic initial conditions: x0 = 1, y0 = 0 (a) (a) (b) (b) (b) (a) Fig. 5 Marginal distributions py := P(y, t|y0 = 0, 0) at t = 1.4 s. General solution of the chemical master equation… Parameters: kcat = 10.1 s−1, kdeg = 0.5 s−1. The thick gray lines depict the expectation value, while the dashed lines stand for the standard deviation. The arrow marks the probability for a zero outcome. a Poissonian initial conditions:  x  0 = 1,  y  0 = 0.001. b Deterministic initial conditions: x0 = 1, y0 = 0 Deterministic initial conditions result in the following generating function: Deterministic initial conditions result in the following generating function: Deterministic initial conditions result in the following generating function: g(sX, sY , t) = 0e−t(kcat(1−sY )+kdeg) −sX(kcat(1 −sY ) + kdeg) + kdeg −et(kcat(1−sY )+kdeg) + kdeg kcat(sY −1) −kdeg 1x0 (sY )y0. g(sX, sY , t) g( ) = 0e−t(kcat(1−sY )+kdeg) −sX(kcat(1 −sY ) + kdeg) + kdeg −et(kcat(1−sY )+kdeg) + kdeg kcat(sY −1) −kdeg 1x0 (sY )y0. Figure 5 shows the comparison between Poissonian and deterministic initial condi- tions. For Poissonian initial conditions, the scenario of starting without any molecules of species X is possible. This “extinction scenario” does not lead to the production of any Y molecules at any time and is seen by the large peak at y = 0 in Fig. 5a. For deterministic initial conditions, we set x0 = 1 and Fig. 5b therefore does not exhibit a peak of the same size at y = 0. However, after some time, the species X may well become extinct and therefore the mode at y = 0 still appears, albeit with a lower size compared to Fig. 5a. Example 3 (Simple splitting) The simple splitting reaction X k1 →Y + Z, obeys the characteristic system dsX dt = −k1(sY sZ −sX) dsY dt = 0 dsZ dt = 0. It is solved by sX(t) = s0 Xek1t + sY sZ(1 −ek1t) ⇔s0 X = e−k1tsX(t) −sY sZ(e−k1t −1). 123 General solution of the chemical master equation… 401 Poissonian initial conditions yield the generating function Poissonian initial conditions yield the generating function y g g g(sX, sY , sZ, t) g(sX, sY , sZ, t) = exp ) x1  0(e−k1tsX(t) −sY sZ(e−k1t −1) −1)+  x2  0(sY −1)+  x3  0(sZ −1) + . (40) = exp ) x1  0(e−k1tsX(t) −sY sZ(e−k1t −1) −1)+  x2  0(sY −1)+  x3  0(sZ −1) + . General solution of the chemical master equation… (40) + (40) Therefore, the marginal distributions are Poissonian and the joint distribution is a multivariate Poisson distribution, studied in detail by Kawamura (1979). The product Poisson distribution differs from the multivariate Poisson distribution by the fact that the variables Y and Z correlate, yet the marginal distributions stay unimodal for both. Without the explicit solution (40), we prove unconditional unimodality by not- ing that either Y or Z may be assigned to the independent part of the network. In consequence, Theorem 2 can be applied to both Y and Z. Example 4 (Additional conversion) Things get more interesting as we add a conver- sion reaction to the same network: X k1 →Y + Z Y k2 →X. The characteristic system is given by The characteristic system is given by The characteristic system is given by The characteristic system is given by dsX dt = −k1(sY sZ −sX) dsY dt = −k2(sX −sY ) dsZ dt = 0, and the Jacobian by J(sZ) = 0 k1 −k1sZ −k2 k2 1 . The characteristic polynomial The characteristic polynomial det(λI − 0 k1 −k1sZ −k2 k2 1 ) = −k1λ −k2λ −k1k2sZ + k1k2 + λ2 depends on sZ, which implies ∂sZ λ(sZ) ̸= 0. Proposition 6 therefore applies and the resulting distributions are conditionally multimodal. Multiple modes, obtained by assuming Poisson distributed initial numbers, are visible in the marginal distribution Fig. 6 as well as the joint distribution Fig. 7. k By adding a degradation reaction X k3 →∅to this model and setting k2 ≫k1, we obtain a model that behaves like Example 2. In consequence, multimodal distributions 12 3 402 M. Reis et al. Fig. 6 Marginal distribution pz := P(z, t|z0 = 0, 0), given Poissonian initial conditions  x  0 = 1.875,  y  0 = 1.875, as well as the deterministic initial condition ξZ = 0. Parameters are k1 = 7.0 s−1, k2 = 1.875 s−1. The thick black line represents the mean while the dashed lines depict the standard deviation. The arrow marks the probability for a zero outcome Fig. 6 Marginal distribution pz := P(z, t|z0 = 0, 0), given Poissonian initial conditions  x  0 = 1.875,  y  0 = 1.875, as well as the deterministic initial condition ξZ = 0. Parameters are k1 = 7.0 s−1, k2 = 1.875 s−1. General solution of the chemical master equation… The thick black line represents the mean while the dashed lines depict the standard deviation. The arrow marks the probability for a zero outcome Fig. 6 Marginal distribution pz := P(z, t|z0 = 0, 0), given Poissonian initial conditions  x  0 = 1.875,   1 Fig. 6 Marginal distribution pz := P(z, t|z0 = 0, 0), given Poissonian initial conditions  x  0 = 1.875,  y  0 = 1.875, as well as the deterministic initial condition ξZ = 0. Parameters are k1 = 7.0 s−1, k2 = 1.875 s−1. The thick black line represents the mean while the dashed lines depict the standard deviation. The arrow marks the probability for a zero outcome Fig. 7 Plot of the joint distribution for the educt X and product Z for the same parameters as Fig. 6 Fig. 7 Plot of the joint distribution for the educt X and product Z for the same parameters as Fig. 6 Fig. 7 Plot of the joint distribution for the educt X and product Z for the same parameters as Fig. 6 123 General solution of the chemical master equation… 403 Fig. 8 Marginal distribution of protein numbers py := P(y, t|y0 = 0, 0) created from 105 simulated trajectories. Parameters: t = 2.7 s and t = 15.0 s, kmRNA deg = 1.2 s−1, kmRNA syn = 1.2 s−1, kprot deg = 0.3 s−1, ktransl = 10.0 s−1, deterministic initial conditions with x0 = y0 = 0. The thick gray lines depict the expectation value, while the dashed lines stand for the standard deviation. The arrow marks the probability for a zero outcome in the left hand side plot Fig. 8 Marginal distribution of protein numbers py := P(y, t|y0 = 0, 0) created from 105 simulated trajectories. Parameters: t = 2.7 s and t = 15.0 s, kmRNA deg = 1.2 s−1, kmRNA syn = 1.2 s−1, kprot deg = 0.3 s−1, ktransl = 10.0 s−1, deterministic initial conditions with x0 = y0 = 0. The thick gray lines depict the expectation value, while the dashed lines stand for the standard deviation. The arrow marks the probability for a zero outcome in the left hand side plot appear for deterministic and Poissonian initial conditions for k1 = kcat and k3 = kdeg (data not shown). 4.2 Real world models Example 5 (Transcription–translation model) The two-stage model, which excludes any effects of promoter activity, consists of the reactions ∅ kmRNA → X X dmRNA →∅ X ktl →X + Y Y dprot →∅, where X are mRNA molecules and Y proteins. We use the implementation of Gille- spie’s algorithm by Maarleveld et al. (2013) to simulate this model. The appearance of multimodal distributions is due to the reaction X →X + Y, studied in Example 2. As mentioned there, the Jacobian’s eigenvalues depend on sY , conditional multimodality is expected. Figures 8 and 9 show that the protein distribution can be multimodal for deterministic and Poissonian initial conditions respectively. Previously, the two-stage transcription–translation model was not shown to be conditionally multimodal (Shahrezaei and Swain 2008). Given small degradation rates, the apparent difference between the shape of Neyman’s Type A distribution (see Figs. 2c and 3), solving the CME for X →X + Y, and the simulation results is rather small. Furthermore, the previously described “extinction scenario” is visible by the large peak at y = 0 in Figs. 8 and 9. This highlights the usefulness of basic models for the study of more complex models. 12 3 404 M. Reis et al. Fig. 9 Marginal distribution of proteins py := P(y, t|y0 = 0, 0), simulated using Poissonian initial conditions for the mRNA,  x  0 = 2. Parameters: kmRNA deg = 0.2 s−1, kmRNA syn = 0.4 s−1, kprot deg = 0.3 s−1, ktransl = 20.0 s−1. The number of simulated trajectories was 106. The thick gray lines depict the expectation value, while the dashed lines stand for the standard deviation. The arrow marks the probability for a zero outcome Fig. 9 Marginal distribution of proteins py := P(y, t|y0 = 0, 0), simulated using Poissonian initial conditions for the mRNA,  x  0 = 2. Parameters: kmRNA deg = 0.2 s−1, kmRNA syn = 0.4 s−1, kprot deg = 0.3 s−1, ktransl = 20.0 s−1. The number of simulated trajectories was 106. The thick gray lines depict the expectation value, while the dashed lines stand for the standard deviation. The arrow marks the probability for a zero outcome Fig. 9 Marginal distribution of proteins py := P(y, t|y0 = 0, 0), simulated using Poissonian initial conditions for the mRNA,  x  0 = 2. 4.2 Real world models Parameters: kmRNA deg = 0.2 s−1, kmRNA syn = 0.4 s−1, kprot deg = 0.3 s−1, ktransl = 20.0 s−1. The number of simulated trajectories was 106. The thick gray lines depict the expectation value, while the dashed lines stand for the standard deviation. The arrow marks the probability for a zero outcome The right panel in Fig. 8 shows that the probability of still having zero proteins after 15 s is much smaller than at 2.6 s. Therefore, the corresponding mode disap- pears. However, the distribution class is still conditionally multimodal as predicted by Theorem 3. Remember that conditional multimodality is a notion defined by the order of the generalizing distribution g2(s) (see Def. 6), not by the model parameters. Example 6 (Natural decay series including decay particles) In an extension of the classical model by Bateman (1910), we now formulate the reaction network for nuclear decay chains including decay particles α, β and γ . The stoichiometry of the model is Xi ki→Xi+1 + Ri,αα + Ri,ββ + Ri,γ γ , where Ri,α, Ri,β and Ri,γ are the numbers of decay particles involved in each reaction. The generating function PDE corresponding to the Master equation is where Ri,α, Ri,β and Ri,γ are the numbers of decay particles involved in each reaction. The generating function PDE corresponding to the Master equation is ∂tg = n−1  i=1 ki(s Rα,i α s Rβ,i β s Rγ,i γ si+1 −si)∂si g. (41) (41) 123 General solution of the chemical master equation… 40 General solution of the chemical master equation… 405 Asbefore,wesolvethePDE(41)usingthemethodofcharacteristics.Thecharacteristic ODEs corresponding to Eq. (41) are: Asbefore,wesolvethePDE(41)usingthemethodofcharacteristics.Thecharacteristic ODEs corresponding to Eq. (41) are: dsi dt = −ki(s Rα,i α s Rβ,i β s Rγ,i γ si+1 −si) (i ∈1, . . . , n −1) (42) dsn dt = 0 (43) dsα dt = dsβ dt = dsγ dt = 0 dg(s(t), t) dt = 0 ⇒g(s(t), t) = f (s0). (44) (42) (43) (44) These equations represent a hierarchically linear ODE system. It can be interpreted as being composed of two subsystems, just as in Theorem 2. System I consists of all isotopes, while system II represents the α, β and γ particles. System II is a trivial example of a monomolecular reaction network, since no reactions take place within the system, we just have to consider the influx of particles from system I. 15 This identity can be seen by writing out the components of the scalar and matrix product: 4.2 Real world models We note that the Jacobian matrix of the characteristic system is identical to the rate equation’s transposed, as discussed in Sect. 2.3. The ODE system is homogeneous and we have s = eA(sα,β,γ )ts0 ⇔s0 = e−A(sα,β,γ )ts, where A(sα,β,γ ) = (∂s j dsi dt ) is the Jacobian. We express the generating function for Poissonian initial conditions by this matrix as where A(sα,β,γ ) = (∂s j dsi dt ) is the Jacobian. We express the generating function for Poissonian initial conditions by this matrix as g(s, t) = exp ) x  0 · (s0 −1) + = exp ) xα,β,γ  0 · (sα,β,γ −1) +  x1,...,n  0 · (exp(−A(sα,β,γ )t)s −1) + = exp ) xα,β,γ  0 · (sα,β,γ −1) + exp(−A(sα,β,γ )Tt)  x1,...,n  0 · s −  x1,...,n  0 · 1 + . In the last line, we have used the identity Ab · c = b · ATc = ATc · b, which holds for any square matrix A and two vectors b, c of the same dimension.15 The insight of the equivalence of the characteristic system and the transposed rate equation system, as discussed in Sect. 2.3, greatly simplifies the following computations. In consequence, the solution of the Master equation is not much more difficult than the solution of the rate equations, as provided by Bateman (1910) originally. n  i=1  Ab  ici = n  i=1 bi  ATc  i n  i=1 n  j=1 Ai jb jci = n  i=1 bi n  j=1 AT i jc j = n  i=1 bi n  j=1 A jic j. 123 123 M. Reis et al. 406 The difference to Bateman’s system is that the negative Jacobian matrix AT depends on the constants sα, sβ and sγ : −AT(sα,β,γ ) = ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎝ −k1 0 . . . 0 k1s Rα,1 α s Rβ,1 β s Rγ,1 γ 0 ... −ki 0 kis Rα,i α s Rβ,i β s Rγ,i γ ... −kn−1 0 0 kn−1s Rα,n−1 α s Rβ,n−1 β s Rγ,n−1 γ 0 ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎠ . Since this matrix is triangular, the eigenvalues are the diagonal entries. Because the eigenvalues do not depend on sα,β,γ , the marginal distribution of the corresponding particles obeys Proposition 3. 16 The cumulants are defined as the coefficients of the cumulant generating function κ(ξ) := log(g(eξ )) at ξ = 0. 17 This can be seen by looking at the number of factors &i−1 l=m. 4.2 Real world models Furthermore, Theorem 4 predicts that conditional mul- timodality depending on the choice of Rα,i, Rβ,i and Rγ,i. Note that Theorem 4 gives us this information without the exact solution of the system presented here. We now proceed along the lines of Pressyanov (2002) and solve the rate equation system, transposed to the characteristic system, to obtain exp(−AT(sα,β,γ )t)  x1,...,n  0. The computations can be found in “Appendix A.4” and yield  exp −AT(sα,β,γ )t  x1,...,n  0  i =  xi  0e−kit + i−1  m=1  xm  0  i−1  l=m kls Rα,l α s Rβ,l β s Rγ,l γ  i l=m e−klt &i j=m, j̸=l(k j −kl) . In consequence, we use the matrix exponential to express the generating function as In consequence, we use the matrix exponential to express the generating function as g(s, t) = exp ) xα,β,γ  0 · (sα,β,γ −1) + n  i=1 50 xi  0e−kit + i−1  m=1  xm  0  i−1  l=m kls Rα,l α s Rβ,l β s Rγ,l γ  i l=m e−klt &i j=m, j̸=l(k j −kl) 1 si −  xi  0 6+ . i 1 + i−1  m=1  xm  0  i−1  l=m kls Rα,l α s Rβ,l β s Rγ,l γ  i l=m e−klt &i j=m, j̸=l(k j −kl) 1 si −  xi  0 6+ . 123 General solution of the chemical master equation… General solution of the chemical master equation… 407 Fig. 10 Marginal distribution py := P(y, t|y0 = 0, 0) of decay particles Y. Parameters:  x1  0 = 2,  x2  0 = 0.1,  x3  0 = 0.001, k1 = 3.1 s−1, k2 = 1 s−1. The thick black line represents the mean while the dashed lines depict the standard deviation Fig. 10 Marginal distribution py := P(y, t|y0 = 0, 0) of decay particles Y. Parameters:  x1  0 = 2,  x2  0 = 0.1,  x3  0 = 0.001, k1 = 3.1 s−1, k2 = 1 s−1. The thick black line represents the mean while the dashed lines depict the standard deviation Interestingly, the further the isotopes are downstream of the decay chain, the more they contribute to the higher order cumulants16 of the α, β and γ particles.17 An insight like that is not easily gained by doing stochastic simulations, because the simulation results alone would not directly point to this fact. We also see that the distribution is not simply characterized by mean and covariance. Next, we consider multimodality of the marginal distributions. Since the eigenval- ues of AT(sα,β,γ ) do not depend on sα,β,γ , Theorem 4 applies and predicts conditional multimodality for several minimal networks. First, we consider networks correspond- ing to case 1. We evaluate the solution for the decay chain 12 3 3 408 M. Reis et al. X1 k1 →X2 + Y (45) X2 k2 →X3 + Y (46) (45) (46) and plot the marginal distributions for Y in Fig. 10. The plots, depicting the temporal evolution of the marginal distribution, clearly demonstrate multimodality. Here, (45– 46)representtheminimalreactionnetworkTypeIfromTheorem4.Aminimalreaction network of Type II is obtained by reducing the network to and plot the marginal distributions for Y in Fig. 10. The plots, depicting the temporal evolution of the marginal distribution, clearly demonstrate multimodality. Here, (45– 46)representtheminimalreactionnetworkTypeIfromTheorem4.Aminimalreaction network of Type II is obtained by reducing the network to X1 k1 →X2 + 2Y. (47) (47) Finally, by assigning X2 to the dependent part of the network, we obtain an even smaller network as X1 k1 →(X2) + 2Y. Now the independent part of the network is open. If we ignore X2, this represents case 2 in Theorem 4. Finally, by assigning X2 to the dependent part of the network, we obtain an even smaller network as X1 k1 →(X2) + 2Y. General solution of the chemical master equation… Now the independent part of the network is open. If we ignore X2, this represents case 2 in Theorem 4. Note that X2 can either be assigned to the independent or the dependent part of the reaction network. However, in either case Theorem 4 applies and predicts conditional multimodality. 123 5 Conclusions In this article, we analyzed the CME for hierarchic first-order reaction networks, based on a general solution method. We showed in Theorem 1 that hierarchic reaction networks are generally treatable in an analytic manner. We derived the analytical solu- tion for the joint probability generating function for Poisson and deterministic initial conditions. Next we analyzed the multimodality of resulting marginal distributions of individual species. The analysis revealed that the marginal distributions of species from the independent part of the network are unimodal (Theorem 2), while the dependent part yields conditionally multimodal distributions (Theorems 3 and 4). Furthermore, we presented several basic models of hierarchic reaction networks, which we consider insightful for the understanding of larger reaction networks. We illustrated this point by showing the similarity between the catalysis basic model and the transcription– translation model. As a proof of principle, we showed that even more complex models such as the nuclear decay chain presented here, are amenable to an exact analytical treatment. Underlining the prevalence of multimodality, we saw that even trivial networks like the one from Example 1, consisting of the reaction X →X + Y, are conditionally multimodal given Poissonian initial conditions. We also showed that deterministic initial conditions give rise to multimodal distributions in Examples 2 and 5. For the two-stage transcription–translation model, multimodality was previously not reported (Shahrezaei and Swain 2008). Since the main Theorems 3 and 4 only require knowledge about the dependence of the eigenvalues of the Jacobian matrix on the variable of interest, it is sufficient to compute the characteristic polynomial of the Jacobian matrix. However, the char- acteristic polynomial of any square matrix is algorithmically computable and so our Theorems are algorithmically applicable in practice. We furthermore demonstrated 12 123 General solution of the chemical master equation… 409 how insight about a network may be gained by simply dividing the network into dependent and independent parts (see Example 3), without doing any calculations or simulations. Therefore, we believe that our abstract treatment of hierarchic reaction networks may inspire other researchers to analyze and design networks with respect to their multimodality properties. 18 Write the author for a sketch of the calculations. 6 Further developments As a future project, we suggest the development of a numerical software to determine the parameter regions of multimodality of our basic models. In that study, the collection of basic models maybe extended, since the ones presented here are far from the only ones whose generating function may be written in a compact form. It might also be interesting to study n-fold hierarchies instead of the two-level hierarchies presented here. Furthermore, we note that the Luria–Delbrück model (Zheng 1999), describing the division of wild-type cells X into mutants Y may be investigated in a similar way we proposed. One of the many variants of the model is defined by X kwt →2X X kmut wt →X + Y X dwt →∅ Y kmut →2Y Y dmut →∅. X kwt →2X X kmut wt →X + Y X dwt →∅ Y kmut →2Y Y dmut →∅. X kwt →2X X kmut wt →X + Y X dwt →∅ Y kmut →2Y Y dmut →∅. X kwt →2X X kmut wt →X + Y X dwt →∅ Y kmut →2Y Y dmut →∅. Even though the reaction equations show the hierarchic character of the model, they do not fit our definition of hierarchic first-order networks. This fact can by seen by the characteristic ODEs: dsX dt = −kwt(s2 X −sX) −kmut wt (sXsY −sX) −dwt(1 −sX) (48) dsY dt = −kmut(s2 Y −sY ) −dmut(1 −sY ) dg dt = 0. (49) dsX dt = −kwt(s2 X −sX) −kmut wt (sXsY −sX) −dwt(1 −sX) (48) dsY dt = −kmut(s2 Y −sY ) −dmut(1 −sY ) (48) dg dt = 0. (49) (49) The system is not hierarchically linear due to the s2 X and s2 Y terms, which is why we excluded autocatalytic reactions X →2X from our study. However, (48) and (49) are examples of Riccati differential equations, and may be linearized as such by a coor- dinate transformation. In this way, an exact solution of the system can be obtained,18 even though it involves large expressions involving hypergeometric functions. 12 3 3 410 M. Reis et al. Acknowledgements The authors thank Jakob Löber for fruitful discussions. Matthias Reis was funded b the Leibniz Graduate School of Molecular Biophysics. 6 Further developments Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 Interna- tional License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 123 A.1 How to obtain distributions from generating functions Before finally coming to the proof of Proposition 2, we discuss how the probability mass function (PMF), defined as the coefficients of the generating function, can be found. In the first-order case, this is not as easy as in the monomolecular case, where the product Poisson and multinomial distributions are obtained (Jahnke and Huisinga 2007). Even the PMF of the relatively simple Neyman Type A distribution given by g(s) = eλ(exp(φ(s−1))−1), almost identical to Eq. (38) from Example 1, is defined only in terms of a multinomial expansion (Zhang et al. 2014). In general, the distribution corresponding to the generating function of Eq. (19) is not expressible in terms of elementary functions, except for special cases. Generally, the coefficients of a generating function g(s, t) are given by the multi- variate Cauchy integral formula P(x, t) = 1 (2πi)n 7 C1×···×Cn g(s, t) sx+1 ds. (50) (50) This identity is due to the definition of the generating function as a power series. Here, Ci are discs encircling the origin on the complex (hyper)plane. See Pemantle and Wilson (2013), Appendix A, for the technicalities of the definition of multivariate Cauchy integrals. Asymptotic joint distributions |x| →∞can be obtained by applying a saddle point integration approach to the Cauchy integral (50). Pemantle and Wilson (2013) present these techniques, developed over the course of the last 20years, in an excellent textbook. The resulting asymptotic distributions are often in very good numerical agreement with the exact ones, even for low |x|, and are a convenient tool to analyze the distributions. If only a numerical evaluation of the distributions is needed, the Discrete Fourier Transform (DFT) is obtained by defining s := e−2πiω with ω ∈[0, 2π] and the application of an inverse Fast-Fourier Transform (FFT) leads to a fast computation of the distributions. Recently, Xu and Minin (2015) have made efforts to increase the efficiency of these computations. Numerical integration of (50) is of course also a possibility. Lastly, it shall be mentioned that there are a number of algorithms based on recur- rences (Zheng 1999; Zhang et al. 2014), applicable for one dimensional distributions. The most important of these is the Lévy-Adelson-Panjer recursion for the DCPN, given 123 General solution of the chemical master equation… 411 by pk+1 = λ k + 1 k  j=0 (k + 1 −j)αk+1−j p j. A.1 How to obtain distributions from generating functions (51) (51) In practice, the sum k j=0 often converges after a constant number of terms (i.e. the convergence is linear,19) so the computational cost of evaluating n probabilities is O(n). Khatri and Patel (1961) provide recurrences for KTBN distributions, needed for the deterministic initial condition setting. The generating function can also be used directly for parameter estimation, with- out calculating the distribution first. Marcheselli et al. (2008) propose a method for parameter estimation that is based exclusively of generating functions. 19 Deuflhard and Hohmann (2003), p. 85, Definition 7. A.2 Proof of Lemma 1 Proof The proof is adapted from Gardiner (2009), Sect. 11.6.4, p. 297. We first mul- tiply with sx and sum over all x to obtain ∂tg(s, t) = ∞  x=0 m  i=1 sx (αi(x −ni)P(x −ni, t) −αi(x)P(x, t)) = m  i=1 ∞  x′=0 sx′+ni αi(x′)P(x′, t) − ∞  x=0 sxαi(x)P(x, t)  . (52) (52) Using the definition of the stochastic propensities αi(x) = ki &n j=1 x j! (x j−Q ji)!, we find the identities Using the definition of the stochastic propensities αi(x) = ki &n j=1 x j! (x j−Q ji)!, we find the identities sxαi(x) = ki n  j=1 s x j j x j! (x j −Q ji)! = ki n  j=1 s x j j (x j(x j −1)(x j −2) · · · (x j −Q ji + 1) = ki n  j=1 s Q ji j  ∂ Q ji s j s x j j  and sx+ni αi(x) = ki n  j=1 s x j+n ji j x j! (x j −Q ji)! = ki n  j=1 s R ji j  ∂ Q ji s j s x j j  . Plugging these into Eq. (52), the desired Eq. (4) is obtained. Plugging these into Eq. (52), the desired Eq. (4) is obtained. ⊓⊔ Plugging these into Eq. (52), the desired Eq. (4) is obtained. ⊓⊔ Plugging these into Eq. (52), the desired Eq. (4) is obtained. ⊓⊔ 123 123 412 M. Reis et al. A.3 Proof of solution formula for twofold hierarchy A.3 Proof of solution formula for twofold hierarchy Proof We start by writing out the PDE for a system of two parts, corresponding to the variables sind := (s1, . . . , snind)T and sdep := (snind+1, . . . , sn)T, as depicted in an exemplary manner in Fig. 1. The PDE for the generating function, derived from Lemma 1, then reads ∂g(s, t) ∂t = m  i=1 ki 0 ci(sdep)  nind  l=1 Rlisl + δ nind l=1 Rli,0  − nind  l=1 Qlisl 1 nind  l=1 Qli∂sl  g + N  i=nind+1 N  j=nind+1 αi j s j −si ∂si g(s, t) N + N  i=nind+1 αi0(1 −si)∂si g(s, t) + b · (s −1)g(s, t). ci(sdep(t′, t)) := &n l=nind+1 s Rli l (t′, t) is defined to be the function representing the produced molecules that do not belong to part I. The characteristic ODEs are obtained in the same manner as for monomolecular systems and read for part I as: ds j dt = − m  i=1 Q jiki ci(sdep)  nind  l=1 Rlisl + δ nind l=1 Rli,0  −Q jis j  (ki ∈R+). Part II is monomolecular and the characteristic equations are: Part II is monomolecular and the characteristic equations are: dsdep dt = −AT(sdep −1). The total derivative of g(s(t), t) reads The total derivative of g(s(t), t) reads The total derivative of g(s(t), t) reads dg(s(t), t) dt = b · (s −1)g(s, t). (53) (53) The Jacobian matrix for part I is given by The Jacobian matrix for part I is given by Jjk = −∂sk m  i=1 Q jiki ci(sdep)  nind  l=1 Rlisl(t) + δ nind l=1 Rli,0  −Q jis j(t)  (k ∈1, . . . nind) = − m  i=1 Q jiki ci(sdep) nind  l=1 Rliδkl −1  = m  i=1 Q jiki 1 −ci(sdep)Rki . 123 General solution of the chemical master equation… 413 If the Jacobian commutes for two different times, i.e. J(t1)J(t2) = J(t2)J(t1), the homogeneous part of this system is solved by exp ) * t 0 J(sdep(t′))dt′+ . If it does not commute, exp ) * t 0 J(sdep(t′))dt′+ is the first term of the Magnus expansion If the Jacobian commutes for two different times, i.e. A.3 Proof of solution formula for twofold hierarchy monomolecular networks can be fully parametrized by the solution of its traditional reaction rate equations translates to hierarchic first-order networks. monomolecular networks can be fully parametrized by the solution of its traditional reaction rate equations translates to hierarchic first-order networks. q Using Poissonian initial conditions, we find d(s0): Using Poissonian initial conditions, we find d(s0): g(s0, t = 0) = exp(⟨x⟩0 · (s0 −1)) = d(s0). (58) (58) We solve Eq. (55) for s0 ind: We solve Eq. (55) for s0 ind: s0 ind = exp ) −  t 0 J(sdep(t′))dt′+ sind −  t 0 exp ) −  t′ 0 J(sdep(t′′))dt′′+ f(s2(t′))dt′. The last integral is over a double exponential function and a simple exponential, hidden in f j(sdep, t′, t) := − m  i=1 Q jiki n  l=nind+1 0 exp ) −A(t′ −t) + ϵl · (sdep −1) + 1 1Rli and is not easily expressed in terms of elementary functions. A series expansion can be found, but is not very simple, which is why we keep the integral as it is. For s0 dep, we obtain the same result as in the monomolecular case, namely s0 dep = exp(ATt)(sdep −1) + 1 . The complete generating function is The complete generating function is g(s, t) = exp ) ⟨x1⟩0 · (e− * t 0 J(sdep,t′,t)dt′sind −  t 0 e− * t′ 0 J(sdep,t′′,t)dt′′f(sdep, t′, t)dt′    −1) + ⟨x2⟩0 · exp(ATt)(sdep −1) +  t 0 b · (sind(t′′′) −1)dt′′′+ = exp ) (e− * t 0 JT(sdep,t′,t)dt′ ⟨x1⟩0 · sind −  t 0 e− * t′ 0 JT(sdep,t′′,t)dt′′ ⟨x1⟩0 · f(sdep, t′, t)dt′ −⟨x1⟩0) + exp(At) ⟨x2⟩0 · (sdep −1) +  t 0 b · (sind(t′′′) −1)dt′′′+ . −  t 0 e− * t′ 0 JT(sdep,t′′,t)dt′′ ⟨x1⟩0 · f(sdep, t′, t)dt′ −⟨x1⟩0) + exp(At) ⟨x2⟩0 · (sdep −1) +  t 0 b · (sind(t′′′) −1)dt′′′+ . (59)  0 + exp(At) ⟨x2⟩0 · (sdep −1) +  t 0 b · (sind(t′′′) −1)dt′′′+ . (59) (59) If the initial condition is deterministic, that is P(x, 0) = δx,x0, where x0 = (x0 1, . . . , x0 n)T, we have If the initial condition is deterministic, that is P(x, 0) = δx,x0, where x0 = (x0 1, . . . A.3 Proof of solution formula for twofold hierarchy J(t1)J(t2) = J(t2)J(t1), the homogeneous part of this system is solved by exp ) * t 0 J(sdep(t′))dt′+ . If it does not commute, exp ) * t 0 J(sdep(t′))dt′+ is the first term of the Magnus expansion e(J(t)) = exp )  t 0 J(sdep(t′))dt′ +  t 0  t1 0 [J(sdep(t1)), J(sdep(t2))]dt2dt1 + · · · + . The nonhomogeneous part is The nonhomogeneous part is The nonhomogeneous part is f j(t) := − m  i=1 Q jikici(sdep(t)) (54) (54) for the lth component of system I, so the complete solution reads for the lth component of system I, so the complete solution reads sind(t) = exp )  t 0 J(sdep(t′))dt′+ s0 ind+ exp )  t 0 J(sdep(t′))dt′+  t 0 exp ) −  t′ 0 J(sdep(t′′))dt′′+ f(sdep(t′))dt′ (55) (55) (56) sdep(t) = exp(−ATt)(s0 dep −1) + 1. sdep(t) = exp(−ATt)(s0 dep −1) + 1. (56) (56) From Eq. (53), we know that From Eq. (53), we know that g(s(t), t) = d(s0) exp 0 t 0 b · (s(t′) −1)dt′ 1 where d(s0) is an arbitrary function to be determined by the initial conditions. The integral over ci(sdep(t′, t)) = &n l=nind+1(sl(t′, t))Rli appears both in the homogeneous and in the inhomogeneous part of the equations for sind(t′, t): where d(s0) is an arbitrary function to be determined by the initial conditions. The integral over ci(sdep(t′, t)) = &n l=nind+1(sl(t′, t))Rli appears both in the homogeneous and in the inhomogeneous part of the equations for sind(t′, t):  t 0 n  l=nind+1 s Rli l (t′, t)dt′ =  t 0 n  l=nind+1 0 exp ) −ATt′+ (s0 dep −1) · ϵl + 1 1Rli dt′ =  t 0 n  l=nind+1 0 exp ) −ATt′+ exp(ATt)(sdep −1) · ϵl + 1 1Rli dt′ =  t 0 n  l=nind+1 0 exp ) −A(t′ −t) + ϵl · (sdep −1) + 1 1Rli dt′. (57) (57) In the last line, we made use of the identity Ab · c = b · ATc = ATc · b. We note that the integral can be expressed completely in terms of the fundamental matrix of the second, monomolecular reaction network. Thus, the property that the statistics of 12 414 M. Reis et al. A.3 Proof of solution formula for twofold hierarchy , x0 n)T, we have g(s(0), 0) = ∞  x=0 δx,x0(s0)x0 = n  i=1 (s0 i )x0 i = d(s0) . (60) (60) 123 123 415 General solution of the chemical master equation… This yields the result, using ϵi := coliIn×n and ϵ(1) i := (ϵi,1, . . . , ϵi,nind)T, ϵ(2) i := (ϵi,nind+1, . . . , ϵi,n)T: This yields the result, using ϵi := coliIn×n and ϵ(1) i := (ϵi,1, . . . , ϵi,nind)T, ϵ(2) i := (ϵi,nind+1, . . . , ϵi,n)T: d(s0) = n  i=1 (s0 i )x0 i = n  i=1  s0 · ϵi x0 i = n  i=1 0' e− * t 0 J(sdep,t′,t)dt′sind −  t 0 e− * t′ 0 J(sdep,t′′,t)dt′′f(sdep, t′, t)dt′( · ϵ(1) i +  eATt(sdep −1) + 1  · ϵ(2) i 1x0 i (61) = n  i=1 0 e− * t 0 JT(sdep(t′))dt′ϵ(1) i · sind −  t 0 e− * t′ 0 JT(sdep(t′′))dt′′ϵ(1) i · f(s2(t′))dt′ +  exp(At)ϵ(2) i · (sdep −1) + 1 1x0 i . (62) +  eATt(sdep −1) + 1  · ϵ(2) i 1x0 i (61) (61) = n  i=1 0 e− * t 0 JT(sdep(t′))dt′ϵ(1) i · sind −  t 0 e− * t′ 0 JT(sdep(t′′))dt′′ϵ(1) i · f(s2(t′))dt′ +  exp(At)ϵ(2) i · (sdep −1) + 1 1x0 i . (62) = n  i=1 0 e− * t 0 JT(sdep(t′))dt′ϵ(1) i · sind −  t 0 e− * t′ 0 JT(sdep(t′′))dt′′ϵ(1) i · f(s2(t′))dt′ +  exp(At)ϵ(2) i · (sdep −1) + 1 1xi . (62) (62) ⊓⊔ 21 See Brannan (2010) for an introduction. 20 In the following, we suppress the dependence of x on s Rα,i α s Rβ,i β s Rγ,i γ for better readability. 21 See Brannan (2010) for an introduction. 20 In the following, we suppress the dependence of x on s Rα,i α s Rβ,i β s Rγ,i γ f 22 We will shortly investigate general initial conditions. A.4 Solution of characteristic ODE system for decay chains The ODE system, describing the temporal evolution of the concentration of isotope xi, is transposed to the characteristic system (43–44) of the PDE (41). More precisely, the system for xi is identical to the original one from Bateman (1910), except for the factors20 representing the decay particles, s Rα,i α s Rβ,i β s Rγ,i γ : dx1 dt = −k1x1 (63) dx2 dt = k1s Rα,1 α s Rβ,1 β s Rγ,1 γ x1 −k2x2 ... dxn dt = kn−1s Rα,n−1 α s Rβ,n−1 β s Rγ,n−1 γ xn−1 −knxn. (64) k1x1 (63) (63) dxn dt = kn−1s Rα,n−1 α s Rβ,n−1 β s Rγ,n−1 γ xn−1 −knxn. (64) (64) Due to this similarity, we are able to adapt the calculations from Pressyanov (2002) almost one-to-one. We take the Laplace transform21 on both sides of Eqs. (63) and (64), thereby introducing the complex variable σ, setting x0 i = 0 for i ≥2, and obtain Due to this similarity, we are able to adapt the calculations from Pressyanov (2002) almost one-to-one. We take the Laplace transform21 on both sides of Eqs. (63) and (64), thereby introducing the complex variable σ, setting x0 i = 0 for i ≥2, and obtain 12 416 M. Reis et al.  ∞ 0 e−σt dx1 dt dt =  ∞ 0 e−σtdx1 = e−σtx1(t) ... ∞ 0 + σ  ∞ 0 e−σtx1(t)dt = −x0 1 + σ ˜x1(σ) = −k1 ˜x1(σ), (65)  ∞ 0 e−σtdx2 = σ ˜x2(σ) = k1s Rα,1 α s Rβ,1 β s Rγ,1 γ ˜x1(σ) −k2 ˜x2(σ) ...  ∞ 0 e−σt dxn dt dt = σ ˜xn(σ) = kn−1s Rα,n−1 α s Rβ,n−1 β s Rγ,n−1 γ ˜xn−1(σ) −kn ˜xn(σ). (66)  ∞ 0 e−σt dx1 dt dt =  ∞ 0 e−σtdx1 = e−σtx1(t) ... ∞ 0 + σ  ∞ 0 e−σtx1(t)dt = −x0 1 + σ ˜x1(σ) = −k1 ˜x1(σ), (65)  ∞ 0 e−σtdx2 = σ ˜x2(σ) = k1s Rα,1 α s Rβ,1 β s Rγ,1 γ ˜x1(σ) −k2 ˜x2(σ) ..  ∞ 0 e−σt dx1 dt dt =  ∞ 0 e−σtdx1 = e−σtx1(t) ... 24 To see this, apply the integral triangular inequality. 23 See Boas (2006) for a review of the solution methods for this kind of integral. A.4 Solution of characteristic ODE system for decay chains ∞ 0 + σ  ∞ 0 e−σtx1(t)dt = −x0 1 + σ ˜x1(σ) = −k1 ˜x1(σ), (65) ∞ 1 1( ) = −k1 ˜x1(σ), (65) (65)  ∞ 0 e−σtdx2 = σ ˜x2(σ) = k1s Rα,1 α s Rβ,1 β s Rγ,1 γ ˜x1(σ) −k2 ˜x2(σ) .  ∞ 0 e−σt dxn dt dt = σ ˜xn(σ) = kn−1s Rα,n−1 α s Rβ,n−1 β s Rγ,n−1 γ ˜xn−1(σ) −kn ˜xn(σ). (66) = kn−1s Rα,n−1 α s Rβ,n−1 β s Rγ,n−1 γ ˜xn−1(σ) −kn ˜xn(σ). (66) (66) In Laplace domain, we solve a linear equation system and invert the Laplace transform ˜x(σ) to obtain x(t). The solution of Eqs. (65–66) is given by ˜x1(σ) = x0 1 k1 + σ , ˜x2(σ) = k1s Rα,1 α s Rβ,1 β s Rγ,1 γ k2 + σ ˜x2(σ), ... ˜xn(σ) = kn−1s Rα,n−1 α s Rβ,n−1 β s Rγ,n−1 γ kn + σ ˜xn−1(σ). (67) ˜xn(σ) = kn−1s Rα,n−1 α s Rβ,n−1 β s Rγ,n−1 γ kn + σ ˜xn−1(σ). (67) (67) The last Eq. (67) represents the starting point of a linear recurrence, which, if the initial conditions x2, . . . xn are set to zero,22 can be solved by ˜xn(σ) = &n−1 i=1 kis Rα,i α s Rβ,i β s Rγ,i γ &n i=1(ki + σ) x0 1. ˜xn(σ) = &n−1 i=1 kis Rα,i α s Rβ,i β s Rγ,i γ &n i=1(ki + σ) x0 1. Now, we go back from Laplace domain to the time domain, by performing the Bromwich integral Now, we go back from Laplace domain to the time domain, by performing the Bromwich integral xn(t) = 1 2πi  δ+i∞ δ−i∞ eσt ˜xn(σ)dσ. Here, δ defines an integration path parallel to the complex line that is situated right of all singularities of ˜xn(σ). We may choose δ = 0, since all poles are on the negative part General solution of the chemical master equation… General solution of the chemical master equation… If we set xm ̸= 0, we obtain for example xi(t) = x0 1  i−1  l=1 kls Rα,l α s Rβ,l β s Rγ,l γ  i l=1 e−klt &i j=1, j̸=l(k j −kl) +x0 m  i−1  l=m kls Rα,l α s Rβ,l β s Rγ,l γ  i l=m e−klt &l j=m, j̸=l(k j −kl) . Applying this idea to all x0 i , we conclude the solution of the rate equations with Applying this idea to all x0 i , we conclude the solution of the rate equations with xi(t) = x0 i e−kit + i−1  m=1 x0 m  i−1  l=m kls Rα,l α s Rβ,l β s Rγ,l γ  i l=m e−klt &i j=m, j̸=l(k j −kl) . References Anderson DF, Craciun G, Kurtz TG (2010) Product-form stationary distributions for deficiency zero chem- ical reaction networks. Bull Math Biol 72(8):1947–1970 Andreychenko A, Bortolussi L, Grima R, Thomas P, Wolf V(2017) Distribution approximations for the chemical master equation: comparison of the method of moments and the system size expansion. In: Modeling cellular systems. Springer, Cham, pp 39–66 Andreychenko A, Bortolussi L, Grima R, Thomas P, Wolf V(2017) Distribution approximations for the chemical master equation: comparison of the method of moments and the system size expansion. In: Modeling cellular systems. Springer, Cham, pp 39–66 Andreychenko A, Bortolussi L, Grima R, Thomas P, Wolf V(2017) Distribution approximations for the chemical master equation: comparison of the method of moments and the system size expansion. In: Modeling cellular systems. Springer, Cham, pp 39–66 g y p g pp Bateman H (1910) The solution of a system of differential equations occurring in the theory of radioactive transformations. Math Proc Camb Philos Soc 15(V):423–427 g y p g pp Bateman H (1910) The solution of a system of differential equations occurring in the theory of radioactive transformations. Math Proc Camb Philos Soc 15(V):423–427 ( ) Boas ML (2006) Mathematical methods in the physical sciences. Wiley, Hoboken Boas ML (2006) Mathematical methods in the physical sciences. Wiley, Hoboken annan JR (2010) Differential equations with boundary value problems: an introduction to modern methods and applications. Wiley, New York Brannan JR (2010) Differential equations with boundary value problems: an introduction to modern method and applications. Wiley, New York Constantino PH, Vlysidis M, Smadbeck P, Kaznessis YN (2016) Modeling stochasticity in biochemical reaction networks. J Phys D Appl Phys 49(9):093001 Constantino PH, Vlysidis M, Smadbeck P, Kaznessis YN (2016) Modeling stochasticity in biochemical reaction networks. J Phys D Appl Phys 49(9):093001 y pp y Deuflhard P, Hohmann A (2003) Numerical analysis in modern scientific computing. In: Texts in applied mathematics, vol 43. Springer, New York Deuflhard P, Hohmann A (2003) Numerical analysis in modern scientific computing. In: Texts in applied mathematics, vol 43. Springer, New York ans LC (2010) Partial differential equations. American Mathematical Society, Providence Evans LC (2010) Partial differential equations. American Mathematical Society, Providence Evans LC (2010) Partial differential equations. Friedman N, Cai L, Xie X (2006) Linking stochastic dynamics to population distribution: an framework of gene expression. Phys Rev Lett 97(16):1–4 Friedman N, Cai L, Xie X (2006) Linking stochastic dynamics framework of gene expression. General solution of the chemical master equation… General solution of the chemical master equation… 417 Fig. 11 Integration path used for the solution of the Bromwich integral Fig. 11 Integration path used for the solution of the Bromwich integral Fig. 11 Integration path used for the solution of the Bromwich integral of the real axis. By closing the integration path through the addition of a semicircle (see Fig. 11), we obtain an integral that can be solved by applying the residue theorem, since the contour is closed23: of the real axis. By closing the integration path through the addition of a semicircle (see Fig. 11), we obtain an integral that can be solved by applying the residue theorem, since the contour is closed23: 1 2πi 7 C eσt ˜xn(σ)dσ = 1 2πi  i∞ −i∞ eσt ˜xn(σ)dσ + lim ρ→∞ 1 2πi  3π/2 π/2 eρeiφt &n−1 i=1 kis Rα,i α s Rβ,i β s Rγ,i γ &n i=1(ki + ρeiφ) x0 1iρeiφdφ. Here, the contribution of the circular part to the value of the integral along C is zero.24 we may calculate xn(t) by the residue formula xn(t) = 1 2πi 7 C eσt ˜xn(σ)dσ = n  j=1 Res j(eσt ˜xn(σ)) , where the sum is over all poles. In our case, we have n simple poles and so the residue theorem yields where the sum is over all poles. In our case, we have n simple poles and so the residue theorem yields Res j(eσt ˜xn(σ)) = lim σ→−k j ((σ + k j)eσt ˜xn(σ)) = e−k jt &n−1 i=1 kis Rα,i α s Rβ,i β s Rγ,i γ &n i=1,i̸= j(ki −k j) x0 1 . 23 See Boas (2006) for a review of the solution methods for this kind of integral. 24 To see this, apply the integral triangular inequality. M. Reis et al. 418 Summing over all residues, we get Summing over all residues, we get ng over all residues, we get xn(t) = x0 1  n−1  i=1 kis Rα,i α s Rβ,i β s Rγ,i γ  n  j=1 e−kit &n i=1,i̸= j(ki −k j) . The only thing left to do is to generalize the initial conditions for x2, . . . xn−1. If we set xm ̸= 0, we obtain for example The only thing left to do is to generalize the initial conditions for x2, . . . xn−1. References Phys Rev Lett 97(16):1–4 ans PJ (1960) Open first-order stochastic processes. J Chem Phys 33(3):691 Gans PJ (1960) Open first-order stochastic processes. J Chem Phys 33(3):691 Gardiner C (2009) Stochastic methods. 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Proc Natl Acad Sci USA 105(45):17256–61 Thomas P, Popovi´c N, Grima R (2014) Phenotypic switching in gene regulatory networks. Proc Nat Acad Sci USA 111(19):6994–6999 Trcek T, Larson DR, Moldón A, Query CC, Singer RH (2011) Single-molecule mRNA decay measurements reveal promoter-regulated mRNA stability in yeast. Cell 147(7):1484–97 Trcek T, Larson DR, Moldón A, Query CC, Singer RH (2011) Single-molecule m reveal promoter-regulated mRNA stability in yeast. Cell 147(7):1484–97 Trcek T, Chao JA, Larson DR, Park HY, Zenklusen D, Shenoy SM, Singer RH (2012) Single-mRNA counting using fluorescent in situ hybridization in budding yeast. Nat Protoc 7(2):408–19 van Kampen NG (2011) Stochastic processes in physics and chemistry. North-Holland Personal Librar Amsterdam, London, New York u J, Minin VN (2015) Efficient transition probability computation for continuous-time branching processes via compressed sensing. arXiv:1503.02644 Zhang H, Liu Y, Li B (2014) Notes on discrete compound Poisson model with applications to risk theory. Insur Math Econ 59:325–336 Zheng Q (1999) Progress of a half century in the study of the Luria–Delbrück distribution. Math Biosci 162(1):1–32 Zheng Q (1999) Progress of a half century in the study of the Luria–Delbrück distribution. Math Biosci 162(1):1–32 123 123
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How Can We Provide Lifelong Support for People with Epilepsy to Reduce Their Self-stigma?
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Abstract Self-stigma denotes the internalization of negative societal attitudes, and it is commonly referred in patients with epilepsy (PWE). Higher levels of self-stigma have been linked to lower self-esteem. According to the author’s research, there is no significant correlation between knowledge level and self-stigma among PWE, and short-term treatment to control seizures alone proves inadequate for reducing self-stigma. These findings provide insights into the enduring nature of self-stig- matization, which appears to be influenced by cultural background, environmental factors, and personal experiences. Firstly, rectifying misinformation is crucial, as it promotes critical thinking and challenges cultural shame rather than accepting it. Secondly, cognitive approaches can be beneficial in helping individuals identify and overcome self-stigmatizing attitudes and beliefs. Recognizing the presence of social stigma and self-stigma are instrumental in reducing self-stigma. Lastly, facilitating individuals fostering a sense of agency in their lives can be accom- plished through decision-making and empowerment strategies. Assisting clients in identifying their strengths and enabling them to make autonomous life choices can foster improved self-esteem and decrease self-stigma. While the primary goal of epilepsy treatment is the reduction of seizures, it is crucial for all stakeholders to acknowledge the persistent nature of self-stigma throughout the lifelong treatment trajectory of PWE. Keywords: epilepsy, prejudice, self-stigma, quality of life, epilepsy self-stigma scale Keywords: epilepsy, prejudice, self-stigma, quality of life, epilepsy self-stigma scale Perspective Chapter: How Can We Provide Lifelong Support for People with Epilepsy to Reduce Their Self-Stigma? Izumi Kuramochi, Takayuki Iwayama and Sakie Shimotsu 1. Introduction When considering “epilepsy,” individuals often form mental associations or stereotypes about this medical condition. Stereotypes are constructed and established based on the prevailing social characteristics of the majority within a specific society, generally accepted as factual by most individuals in that society [1]. When individuals endorse negative stereotypes, it develops negative emotions and emotional responses, commonly known as “prejudice.” For instance, someone who believes that people 1 Epilepsy During the Lifespan – Beyond the Diagnosis and New Perspectives with epilepsy (PWE) pose a danger may experience fear toward individuals with this condition. The issue received significant attention in Japan following the media’s extensive coverage of traffic accidents involving individuals with epilepsy in 2011. As a result, the association between the public image of epilepsy and the concept of danger became more pronounced in 2013 [2]. These emotional reactions can manifest as discrimination, the behavioral response of harboring negative thoughts and feelings toward stigmatized groups. Due to fear (prejudice) and the acceptance of the associated stereotype, the general public may opt to maintain a physical and emotional distance from individuals with epilepsy. Epilepsy-related stigma is prevalent in numerous cultures [3, 4] and is widely recog- nized as one of the most significant factors negatively impacting the lives of PWE and their families [5–8]. Among those who feel “stigmatized” by society and live within that society are PWE themselves. Some PWE has stereotypes and self-stigmas about themselves. These include acceptance of self-stereotypes (e.g., “I am a danger to myself with epileptic seizures.”), prejudice (e.g., “I am afraid of myself”), and the accompanying self- discrimination (e.g., social isolation due to self-blame). Internalizing negative stereo- types can trigger adverse emotional reactions, such as low self-esteem and diminished self-efficacy. Self-discrimination, particularly self-isolation, has detrimental effects, including reduced healthcare utilization, poorer health outcomes, and decreased quality of life [9]. Low self-efficacy and self-esteem have also been associated with missed opportunities for employment and independent living [10, 11]. Link et al. referred to this as the modified labeling theory, highlighting that individuals who internalize the stigma of mental illness worsen their condition due to the harm caused by internalized experi- ences [12, 13]. Self-stigmatization entails a reduction in self-worth and a detrimental impact on one’s aspirations toward personal goals. Consequently, self-stigma emerges as an intrapersonal phenomenon that leads to adverse health outcomes and diminishes the quality of life experienced by individuals diagnosed with epilepsy (PWE) [9, 14]. 3.1 Self-stigma among epilepsy patients (results of qualitative analysis study) The authors used semi-structured interviews and their qualitative analysis to ana- lyze elements of self-stigma among Japanese PWE [19]. For historical reasons, epilepsy in Japan is treated by neurology, neurosurgery, and neuropsychiatry. In Japan’s admin- istrative classification, epilepsy is classified as a psychiatric disorder, necessitating the involvement of psychiatrists due to its distinctive features. Analysis of 206 verbatim data obtained from epilepsy patients attending outpatient clinics in Japan resulted in the extraction of 74 codes. Qualitative analysis of these codes identified 22 subcatego- ries of epilepsy-related self-stigma and three major categories: self-stigma, perceived social stigma, and actual distress/troubles. The subsequent paragraphs provide a detailed description of the self-stigma categories derived from the qualitative analysis. Concerning self-stigma among PWE, negative beliefs are not only negative cognitions about the disease and diagnosis of epilepsy, such as “I am not capable” and “I am a weak person,” but also “I have an image that epileptic seizures are something that makes me foam and fall” and “I will be a burden to others if I have an epileptic seizure.” It was found that these included negative perceptions about epileptic seizures, such as “I will be a burden to others” and “People will feel uncomfortable if they figure out I have epileptic seizures.” y g p p The findings additionally revealed the presence of “negative feelings about epilep- tic seizures,” exemplified by expressions such as “I do not want to have an epileptic sei- zure because I will burst into a bubble and collapse” and “It is painful to be seen having an epileptic seizure.” Furthermore, sentiments such as “It is embarrassing to be seen having an epileptic seizure,” “I hate going to a psychiatrist,” and “I have an aversion to seeing a psychiatrist” were also expressed within the dataset. Influenced by these adverse emotional experiences, the patients conveyed challenges encompassing vari- ous aspects of the disclosure, including “difficulties in revealing my epilepsy diagnosis to others,” “challenges in disclosing my condition to my family,” “obstacles in inform- ing my teachers about my epilepsy,” “struggles in divulging my epilepsy diagnosis to my workplace,” and “difficulties in communicating my condition to doctors from other medical departments,” among others. 2. Efforts to reduce the stigma of epilepsy Epilepsy has been documented as far back as 4000 B.C. and has had severe physi- cal, mental, and social effects on patients for centuries, with no difference in age, race, country, or geographical location [15]. In response to the history of prejudice due to lack of knowledge, preconceptions, and discrimination against epilepsy, in 1997, the International Epilepsy Congress and the WHO, in collaboration with the International League Against Epilepsy (ILAE) and the International Bureau of Epilepsy (IBE), launched an international campaign called “Out of the (Shadows [16]). The Intersectoral Global Action Plan (IGAP) on epilepsy and other neurological disorders aims to enhance accessibility to care and treatment for people with neurologi- cal disorders while concurrently preventing new cases and advocating brain health and development throughout the lifespan [17]. This comprehensive initiative spans 10 years, from 2022 to 2031. Its approval by 193 states on 27 May during the 75th World Health Assembly [17] signifies a decade-long commitment to prioritizing brain health by bolstering policy focus and governance; facilitating effective and timely diagnosis, treatment, and care; implementing strategies for promotion and prevention; fostering research and innovation; and fortifying information systems, as well as enhancing the public health approach to epilepsy [18]. However, to mitigate the societal stigma associ- ated with epilepsy, continued efforts will be necessary to sustain the campaign’s impact. 2 Perspective Chapter: How Can We Provide Lifelong Support for People with Epilepsy to Reduce... DOI: http://dx.doi.org/10.5772/intechopen.112136 Perspective Chapter: How Can We Provide Lifelong Support for People with Epilepsy to Reduce... DOI: http://dx.doi.org/10.5772/intechopen.112136 3. Lifelong support for epilepsy patients to reduce self-stigma What approaches help reduce self-stigma among people with epilepsy? To adequately facilitate the reduction of self-stigma, it is imperative to comprehend the intricacies involved in the formation of self-stigma within distinct cultural contexts. The authors have investigated the actual status of self-stigma in epilepsy patients in Japan, developed a measurement scale, and conducted group psychoeducation programs [19–21]. This paper therefore introduces the studies conducted by the authors and discusses the direction of clinical practice and future research aimed at reducing self-stigma in Japan. 3.1 Self-stigma among epilepsy patients (results of qualitative analysis study) The respondents admitted to behavioral changes such as “difficulty in telling my family that I have epilepsy,” “wanting to hide the fact that I have epilepsy,” “not wanting to take my medication in public,” and “wanting to hide the fact that I have epilepsy and have to go to the hospital for treatment.” The behavioral manifestations of these negative changes were identified to be more prominently evident within the cognitive component, characterized by a “negative evaluation of my life so far,” as opposed to the emotional component, which featured a “decreased general self-confidence.” Additionally, the negative behavioral changes, precisely the “abandonment of various activities due to the presence of epi- lepsy,” exhibited a lesser degree of recognition compared to the cognitive component. 3 Epilepsy During the Lifespan – Beyond the Diagnosis and New Perspectives These findings specifically examine the stigma of epilepsy in Japan, and it is cru- cial to note that not all of the identified factors may be applicable to other countries or cultures. In particular, the pronounced association with psychiatry may be influenced by the distinctive medical context of Japan. However, those individuals involved in epilepsy care need to acknowledge that patients with epilepsy may experience and endure these self-stigmas associated with the condition. q y y g p p y For each question, please choose the most appropriate number from the scale to the right of each sentence. 3.2 Creation of the epilepsy self-stigma scale The Epilepsy Self-Stigma Scale (ESSS) [21] (Table 1) was developed to measure self-stigma explicitly pertaining to “epilepsy” and to provide a convenient assessment scale suitable for use in a short consultation time. The development of the ESSS involved the utilization of results from qualitative analysis in Japan. Subsequently, the question- naire was formulated based on the results of a qualitative analysis, and an exploratory factor analysis was conducted based on data obtained from 100 epilepsy patients attend- ing outpatient clinics at multiple medical institutions. As a result, a final questionnaire comprising three factors (Internalization of stigma, Societal incomprehension, and Confidentiality) and eight items was derived. The ESSS items are rated on a four-point Likert-type scale: 1: Strongly Disagree, 2: Slightly agree, 3: Agree, 4: Strongly Agree. Total scores range from 8 to 32. The higher the total score, the greater is the extent of the self- stigma toward epilepsy. The calculation of scores for each factor enables a comprehensive assessment of the relative significance of different dimensions of self-stigma. In addition, high internal consistency has been obtained across the entire scale and all subscales, and high retest reliability and adequate construct validity concerning depressive symptoms Disagree Weakly Agree Agree Strongly Agree 1. When I hear news about traffic accidents related to epileptic seizures, I feel like I am being told about myself. 1 2 3 4 2. I feel discriminated against by others because of epilepsy. 1 2 3 4 3. I sometimes feel embarrassed about epilepsy. 1 2 3 4 4. I feel different from others because I have epilepsy. 1 2 3 4 5. Ordinary people do not understand my suffering from epilepsy and the worry of seizures. 1 2 3 4 6. Few people have the correct information about the disease of epilepsy. 1 2 3 4 7. It is hard to tell others that I have epilepsy. 1 2 3 4 8. I want to hide the fact that I go to the hospital to receive therapy for epilepsy. 1 2 3 4 This questionnaire will ask you about what you think about having epilepsy. For each question, please choose the most appropriate number from the scale to the right of each sentence. 4 Perspective Chapter: How Can We Provide Lifelong Support for People with Epilepsy to Reduce... DOI: http://dx.doi.org/10.5772/intechopen.112136 Perspective Chapter: How Can We Provide Lifelong Support for People with Epilepsy to Reduce... 3.3.1 Educational needs, psychosocial education programs The first step in reducing self-stigma is to provide accurate information about PWE and its supporters. Education and awareness can reduce misconceptions and prejudices about epilepsy and promote public understanding and empathy. Education should be targeted not only to PWE and others affected by the disease but also to the general public, teachers, and healthcare professionals. People with epilepsy must understand their condition and learn about appropriate treatment and daily living precautions. Psychosocial education programs on epilepsy have been conducted in various coun- tries, and their usefulness has been reported [22]. Although psychosocial and educa- tional programs took many forms depending on the country and context in which they were implemented, most interventions improved subjects’ knowledge about epilepsy. For example, the authors conducted the “Epi-school” program in Japan, which yielded notable outcomes [20]. Furthermore, other programs, such as MOSES [23], The Seizures and Epilepsy Education (SEE) Program [24, 25], FLIP & FLAP [26], Be Seizure Smart [27], Children’s (Epilepsy Program [28]), ACINDES [29], and A psycho- social self-management program for epilepsy [30], among others, have also showcased commendable effectiveness in this regard. The PEPE [31] and Ogata’s program in Japan [32] showed significant improvement in knowledge based on subjects’ subjec- tive ratings. The FAMOSES [33, 34] program for young PWE is being implemented in Germany. It is a psychosocial educational program for children with epilepsy and their families. It is imperative to teach PWE of young origin to know about epilepsy with a view to the future in order to support epilepsy treatment throughout their lives. pp p p y g The authors implemented a program named “Epi-school,” which targeted indi- viduals diagnosed with epilepsy (PWE) aged 16 years and older and their supporters within the outpatient psychiatry department of a university hospital. The program dealt with the epidemiology and fundamental knowledge of epilepsy, diagnosis and treatment of epilepsy, self-control and prognosis of epilepsy, and psychosocial aspects. The program was managed by two fixed trainers (an epilepsy specialist and a psychiatric nurse specialist) and a multidisciplinary team, including a pharmacist, a psychologist, and a mental health worker, depending on the content of each session. In addition to significant increases in knowledge about epilepsy for patients and relatives before and after the program, positive changes were observed in overall life satisfac- tion and psychological acceptance of epilepsy as an optimistic illness. 3.2 Creation of the epilepsy self-stigma scale DOI: http://dx.doi.org/10.5772/intechopen.112136 and self-esteem. Cronbach’s α for the total scale and each factor demonstrated good internal consistency (α = 0.76–0.87). The ESSS has undergone a prolonged development process, and its utilization in various studies has been relatively limited, resulting in a scarcity of comprehensive information. However, it is noteworthy that no significant correlation was observed between external criteria, such as an objective assessment of self-stigma conducted by the subject’s physician, and the total score on the ESSS, which contrasts with findings from comparable scales. This finding aligns with the conceptu- alization of self-stigma as internalized stigma and highlights the challenge of accurately assessing a patient’s self-stigma solely within the consultation setting. 3.3.1 Educational needs, psychosocial education programs In addition to enhancing disease-related knowledge and coping mechanisms, numerous participants also expressed observations regarding alterations in the psychological aspects of the disease. Further research is warranted to examine the changes in the self-stigmatiza- tion levels of PWE before and after the psychosocial education program. 5 Epilepsy During the Lifespan – Beyond the Diagnosis and New Perspectives 3.3.3 Support groups, self-help groups Support and self-help groups are places where people with the same illness can share information and experiences. For PWE, support and self-help groups can reduce feelings of isolation and anxiety by letting them know that others are going through similar expe- riences. Patients can increase their self-acceptance and reduce self-stigma by participat- ing in support groups. In the context of self-help groups dedicated to epilepsy worldwide, enormous organizations have been established in various countries, including notable examples such as the Japan Epilepsy Association [37], the Epilepsy Foundation [38] in the United States, Epilepsy Action [39] in the United Kingdom, and Epilepsy Action Australia [40] in Australia. These organizations offer a range of information, consulta- tion services and support activities pertaining to epilepsy. However, it is worth noting that the number of organizations operating on a global scale remains limited. From the authors’ survey, in Japan, the use of psychosocial support, that is, participating in epilepsy self-help groups and educational programs, was 5.8% [41]. As a reason for not participating in self-help groups, PWE in Japan worry that they might be discriminated against by society for participating in such groups. In order to effectively address and mitigate self-stigmatization experienced by PWE throughout their lifespans, it is vital to create an environment where PWE feel at ease engaging in self-help groups and study groups. Thus, constant efforts must be directed toward reducing the societal stigma associated with epilepsy and preventing the emergence of new self-stigmatizing beliefs among PWE. 3.3.2 Psychological support Epileptic seizures may cause emotional distress and social isolation in PWE. Psychological support is essential to reduce self-stigma, and psychotherapy and counseling, in particular, may promote patients’ mental health and reduce self- stigma. Psychological support includes counseling, acceptance and commitment therapy (ACT) [35, 36], cognitive behavioral therapy (CBT), relaxation methods, mindfulness, and motivational interviewing. Some psychosocial education programs mentioned in the previous section also offer psychosocial and psychological support. p p y p y g pp Psychological support may be provided within psychosocial education programs, but individual psychotherapy and psychotherapy may benefit PWE who experience self-stigma, even in settings where dedicated psychosocial programs are unavailable. Funding This work was supported by the 31st Ochiai Memorial Award Research Grant (2020) and JSPS KAKENHI (No. 21 K13709) and the Maruki Memorial Special Award Research Grant (2022, No.22-1A-01). The funding source was not involved in the design of this study, the writing of this report, and the decision to submit the manu- script for publication. 4. Conclusion This paper introduces the present situation regarding the stigma of epilepsy in Japan and attempts to reduce it, inspired by the authors’ research findings. Healthcare professionals involved in epilepsy care must take positive action to prevent the continuation of inadequate knowledge, superstition, and prejudice. Research on self-stigmatization in relation to epilepsy remains limited, underscoring the necessity to develop assessment tools and intervention strategies informed by cross-national findings while accounting for cultural nuances and the specific circumstances sur- rounding the management of epilepsy in individual countries. g g p p y It is imperative to acknowledge that Goffman, the pioneer of the stigma concept, emphasized the significance of understanding stigma as a “relationship” rather than a mere attribute of an individual. This perspective holds spectacular importance in 6 Perspective Chapter: How Can We Provide Lifelong Support for People with Epilepsy to Reduce... DOI: http://dx.doi.org/10.5772/intechopen.112136 Perspective Chapter: How Can We Provide Lifelong Support for People with Epilepsy to Reduce... DOI: http://dx.doi.org/10.5772/intechopen.112136 epilepsy, as it is insufficient to perceive epilepsy stigma solely as a characteristic of the individual with epilepsy. Consistent with Goffman’s original proposition, it is crucial to conduct research that delves into the process of stigma formation within the psychosocial context of interactions between individuals with epilepsy and their immediate environment and the broader society. y We hope that developing the Epilepsy Self-Stigma Scale (ESSS) and psychosocial education programs presented here will assist this effort and further expand research activities to reduce self-stigma in PWE. Declaration of competing interests None. None. 7 Author details Izumi Kuramochi1*, Takayuki Iwayama1,2 and Sakie Shimotsu3 1 Department of Psychiatry, Saitama Medical Center, Saitama Medical University, Japan 2 Department of Psychology, Showa Women‘s University, Japan 3 Faculty of Human Development and Education, Kyoto Women‘s University, Japan *Address all correspondence to: kizumi@saitama-med.ac.jp © 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 7 Author details Izumi Kuramochi1*, Takayuki Iwayama1,2 and Sakie Shimotsu3 1 Department of Psychiatry, Saitama Medical Center, Saitama Medical University, Japan 2 Department of Psychology, Showa Women‘s University, Japan 3 Faculty of Human Development and Education, Kyoto Women‘s University, Japan *Address all correspondence to: kizumi@saitama-med.ac.jp © 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Acknowledgements We appreciate Dr. Lester Luo’s invaluable assistance in editing this manuscript in English. Author details Izumi Kuramochi1*, Takayuki Iwayama1,2 and Sakie Shimotsu3 1 Department of Psychiatry, Saitama Medical Center, Saitama Medical University, Japan 3 Faculty of Human Development and Education, Kyoto Women‘s University, Japan © 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 7 Epilepsy During the Lifespan – Beyond the Diagnosis and New Perspectives Epilepsy During the Lifespan – Beyond the Diagnosis and New Perspectives References [1] Corrigan PW, Rao D. On the self-stigma of mental illness: Stages, disclosure, and strategies for change. Canadian Journal of Psychiatry. 2012;57(8):464-469. 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FLIP&FLAP: A training programme for children and adolescents with epilepsy, and their parents. Seizure. Sep 2009;18(7):478- 486. DOI: 10.1016/j.seizure.2009.04.007 [27] Austin JK, McNelis AM, Shore CP, Dunn DW, Musick B. A feasibility study of a family seizure management program: “Be Seizure Smart”. Journal of Neuroscience Nursing. 2002;34:30-37 [19] Kuramochi I, Horikawa N, Shimotsu S, et al. The self-stigma of patients with epilepsy in Japan: A qualitative approach. Epilepsy & Behavior. 2020;109:106994 [28] Lewis MA, Salas I, de la Sota A, Chiofalo N, Leake B. Randomized trial of a program to enhance the competencies of children with epilepsy. Epilepsia. 1990;31:101-109 [20] Kuramochi I, Oga K, Iwayama T, et al. Pilot trial of “Epi-school” group psychosocial education program for patients with epilepsy and their relatives in Japan. Epilepsy & Behavior. 2020;113:107545 [29] Tieffenberg JA, Wood EI, Alonso A, Tossutti MS, Vicente MF. A randomized field trial of ACINDES: A child- centered training model for children with chronic illnesses (asthma and epilepsy). Journal of Urban Health. 2000;77:280-297 [21] Kuramochi I, Iwayama T, Horikawa N, et al. Development and validation of the epilepsy self-stigma scale. Epilepsia Open. 2021;6(4):748-756 [21] Kuramochi I, Iwayama T, Horikawa N, et al. Development and validation of the epilepsy self-stigma scale. Epilepsia Open. 2021;6(4):748-756 [22] Mittan RJ. Psychosocial treatment programs in epilepsy: A review. Epilepsy & Behavior. 2009;16(3):371-380 [22] Mittan RJ. Psychosocial treatment programs in epilepsy: A review. Epilepsy & Behavior. 2009;16(3):371-380 [30] Pramuka M, Hendrickson R, Zinski A, Van Cott AC. A psychosocial self-management program for epilepsy: A randomized pilot study in adults. Epilepsy & Behavior. 2007;11(4):533-545 [30] Pramuka M, Hendrickson R, Zinski A, Van Cott AC. A psychosocial self-management program for epilepsy: A randomized pilot study in adults. Epilepsy & Behavior. 2007;11(4):533-545 [23] May TW, Pfafflin M. The efficacy of educational treatment program for patients with epilepsy (MOSES): Results of a controlled, randomized study. References 2005;4:171-178 [4] Jacoby A, Snape D, Baker GA. Epilepsy and social identity: The stigma of a chronic neurological disorder. Lancet Neurology. 2005;4:171-178 [14] Corrigan PW, Watson A, Barr L. The self-stigma of mental illness: Implications for self-esteem and self-efficacy. Journal of Social & Clinical Psychology. 2006;25(9):875-884 [15] World Health Organization (WHO). Epilepsy: A public health imperative. 2019. Available from: https://www. who.int/publications/i/item/epilepsy- a-public-health-imperative [accessed 2021-11-30] [15] World Health Organization (WHO). Epilepsy: A public health imperative. 2019. Available from: https://www. who.int/publications/i/item/epilepsy- a-public-health-imperative [accessed 2021-11-30] [7] Mclin WM, de Boer HM. Public perceptions about epilepsy. Epilepsia. 1995;36:957-959 [16] Reis R, Meinardi H. ILAE/WHO “Out of the Shadows Campaign” Stigma: Does the flag identify the cargo? Epilepsy & Behavior. 2002;3(6S2):33-37 [17] World Health Organization (WHO). Intersectoral global action plan on epilepsy and other neurological disorders 2022-2031. Available 8 Perspective Chapter: How Can We Provide Lifelong Support for People with Epilepsy to Reduce... DOI: http://dx.doi.org/10.5772/intechopen.112136 Perspective Chapter: How Can We Provide Lifelong Support for People with Epilepsy to Reduce... DOI: http://dx.doi.org/10.5772/intechopen.112136 aspects of epilepsy. Epilepsia. 1990;31:75-82 aspects of epilepsy. Epilepsia. 1990;31:75-82 aspects of epilepsy. Epilepsia. 1990;31:75-82 Epilepsia. 2002;43:539-549 [31] Huber B, Seidel M. PEPE: A psychoeducative programme for people with epilepsy and learning disabilities. In: Pfafflin M, Frasier RT, Specht U, Thorbeck R, editors. Comprehensive Care for People with Epilepsy. France: John Libbey Eurotext; 2001. pp. 155-162 [31] Huber B, Seidel M. PEPE: A psychoeducative programme for people with epilepsy and learning disabilities. In: Pfafflin M, Frasier RT, Specht U, Thorbeck R, editors. Comprehensive Care for People with Epilepsy. France: John Libbey Eurotext; 2001. pp. 155-162 [24] Helgeson DC, Mittan R, Tan SY, Chayasirisobhon S. Sepulveda Epilepsy education: the efficacy of a psychoeducational treatment program in treating medical and psychosocial 9 Epilepsy During the Lifespan – Beyond the Diagnosis and New Perspectives [32] Ogata A, Amano K. A psychosocial approach to epileptic patients. Epilepsia. 2000;41(Suppl. 9):36-38 [32] Ogata A, Amano K. A psychosocial approach to epileptic patients. Epilepsia. 2000;41(Suppl. 9):36-38 [41] Kuramochi I, Iwayama T, Oga K, Shiganami T, Umemura T, Kobayashi S, et al. A study of factors influencing self-stigma in people with epilepsy: A nationwide online questionnaire survey in Japan. Epilepsia Open. 2022;7(4):792- 801. DOI: 10.1002/epi4.12661 Epub 2022 Oct 25 [33] Wohlrab GC, Rinnert S, Bettendorf U, Fischbach H, Heinen G, Klein P, et al. Famoses Project Group. famoses: A modular educational program for children with epilepsy and their parents. Epilepsy & Behavior. 2007;10(1):44-48. DOI: 10.1016/j. yebeh.2006.10.005 Epub 2006 Nov 27 [34] Hagemann A, Pfäfflin M, Nussbeck FW, May TW. The efficacy of an educational program for parents of children with epilepsy (FAMOSES): Results of a controlled multicenter evaluation study. Epilepsy & Behavior. 2016;64(Pt A):143-151. DOI: 10.1016/j. yebeh.2016.09.027 Epub 2016 Oct 13 [35] Lundgren T, Dahl J, Yardi N, Melin L. Commitment therapy and yoga for drug- refractory epilepsy: A randomized controlled trial. Epilepsy & Behavior. 2008;13:102-108 [35] Lundgren T, Dahl J, Yardi N, Melin L. Commitment therapy and yoga for drug- refractory epilepsy: A randomized controlled trial. Epilepsy & Behavior. 2008;13:102-108 [36] Lundgren T, Dahl J, Melin L, Kies B. Evaluation of acceptance and commitment therapy for drug refractory epilepsy: A randomized controlled trial in South Africa—a pilot study. Epilepsia. 2006;47:2173-2179 [37] The Japan Epilepsy Association. Available from: https://jes-jp.org/en/ [accessed 2023-5-17] [38] The Epilepsy Foundation. Available from: https://www.jea-net.jp/ [accessed 2023-5-17] [39] Epilepsy Action. Available from: https://www.epilepsy.com/ [accessed 2023-5-17] [40] Epilepsy Action Australia. Available from: https://www.epilepsy.org.au/ [accessed 2023-5-17] [40] Epilepsy Action Australia. Available from: https://www.epilepsy.org.au/ [accessed 2023-5-17] 10
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The HER2- and Heregulin β1 (HRG)–Inducible TNFR Superfamily Member Fn14 Promotes HRG-Driven Breast Cancer Cell Migration, Invasion, and MMP9 Expression
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The WIRCam Ultra Deep Survey (WUDS) I. Survey overview and UV luminosity functions at z~5 and z~6
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⋆A copy of the data products is also available at the CDS via anony- mous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http: //cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/620/A51 The WIRCam Ultra Deep Survey (WUDS) I. Survey overview and UV luminosity functions at z ∼5 and z ∼6⋆ Sànchez, 38206 La Laguna, Tenerife, Spain 6 Departamento de Astrofísica y CC de la Atmósfera, Universidad Complutense de Madrid, Av Complutense s/n, 28040 Madrid, Spain 7 p 7 Department of Astronomy, University of Florida, 211 Bryant Space Science Center, Gainsville, FL 32611-2055, USA 8 Université Lyon 1, ENS de Lyon, CNRS, Centre de Recherche Astrophysique de Lyon (CRAL) UMR5574, 69230 Saint-Genis- Laval, France Department of Astronomy, University of Florida, 211 Bryan 7 Department of Astronomy, University of Florida, 211 Bryant Space Science Center, Gainsville, FL 32611-2055, USA 8 Université Lyon 1, ENS de Lyon, CNRS, Centre de Recherche Astrophysique de Lyon (CRAL) UMR5574, 69230 Saint-Genis- Laval, France 9 rvatory, Chemin des Maillettes 51, 1290 Versoix, Switzerla 9 Geneva Observatory, Chemin des Maillettes 51, 1290 Versoix, Switzerland 10 L b i d’A h i d M ill CNRS U i i é Ai M ill 10 Laboratoire d’Astrophysique de Marseille, CNRS – Université Aix-Marseille, 38 Rue Frédéric Joliot-Curie, 13388 Marseille Cedex 13, France 10 Laboratoire d’Astrophysique de Marseille, CNRS – Université Aix-Marseille, 38 Rue Frédéric Joliot-Curie, 13388 Marseille Cedex 13, France 11 Stockholm University, Department of Astronomy and Oskar Klein Centre for Cosmoparticle Physics, AlbaNova University Centre, 10691 Stockholm, Sweden 11 Stockholm University, Department of Astronomy and Oskar Klein Centre for Cosmoparticle Physics, AlbaNova University Centre, 10691 Stockholm, Sweden Received 11 October 2017 / Accepted 3 September 2018 Received 11 October 2017 / Accepted 3 September 2018 Received 11 October 2017 / Accepted 3 September 2018 ABSTRACT The aim of this paper is to introduce the WIRCam Ultra Deep Survey (WUDS), a near-IR photometric survey carried out at the CFH Telescope in the field of the CFHTLS-D3 field (Groth Strip). WUDS includes four near-IR bands (Y, J, H and Ks) over a field of view of ∼400 arcmin2. The typical depth of WUDS data reaches between ∼26.8 in Y and J, and ∼26 in H and Ks (AB, 3σ in 1.3′′ aperture), whereas the corresponding depth of the CFHTLS-D3 images in this region ranges between 28.6 and 29 in ugr, 28.2 in i and 27.1 in z (same S/N and aperture). The area and depth of this survey were specifically tailored to set strong constraints on the cosmic star formation rate and the luminosity function brighter or around L⋆in the z ∼6−10 redshift domain, although these data are also useful for a variety of extragalactic projects. This first paper is intended to present the properties of the public WUDS survey in details: catalog building, completeness and depth, number counts, photometric redshifts, and global properties of the galaxy population. We have also concentrated on the selection and characterization of galaxy samples at z ∼[4.5−7] in this field. For these purposes, we include an adjacent shallower area of ∼1260 arcmin2 in this region, extracted from the WIRCam Deep Survey (WIRDS), and observed in J, H and Ks bands. UV luminosity functions were derived at z ∼5 and z ∼6 taking advantage from the fact that WUDS covers a particularly interesting regime at intermediate luminosities, which allows a combined determination of M⋆and Φ⋆with increased accuracy. Our results on the luminosity function are consistent with a small evolution of both M⋆and Φ⋆between z = 5 and z = 6, irrespective of the method used to derive them, either photometric redshifts applied to blindly-selected dropout samples or the classical Lyman Break Galaxy color-preselected samples. Our results lend support to higher Φ⋆determinations at z = 6 than usually reported. The selection and combined analysis of different galaxy samples at z ≥7 will be presented in a forthcoming paper, as well as the evolution of the UV luminosity function between z ∼4.5 and 9. WUDS is intended to provide a robust database in the near-IR for the selection of targets for detailed spectroscopic studies, in particular for the EMIR/GTC GOYA Survey. Key words. surveys – galaxies: high-redshift – dark ages, reionization, first stars Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The WIRCam Ultra Deep Survey (WUDS) I. Survey overview and UV luminosity functions at z ∼5 and z ∼6⋆ I. Survey overview and UV luminosity functions at z ∼5 and z ∼6⋆ R. Pelló1, P. Hudelot2, N. Laporte3,1, Y. Mellier2, H. J. McCracken2, M. Balcells4, F. Boone1, N. Cardiel6, J. Gallego6, F. Garzón4,5, R. Guzmán7, J. F. Le Borgne1, M. Prieto4,5, J. Richard8, D. Schaerer9,1, L. Tresse8, S. Arnouts10, J. G. Cuby10, K. Disseau8, and M. Hayes11 R. Pelló1, P. Hudelot2, N. Laporte3,1, Y. Mellier2, H. J. McCracken2, M. Balcells4, F. Boone1, N. Cardiel6, J. Gallego6, F. Garzón4,5, R. Guzmán7, J. F. Le Borgne1, M. Prieto4,5, J. Richard8, D. Schaerer9,1, L. Tresse8, S. Arnouts10, J. G. Cuby10, K. Disseau8, and M. Hayes11 R. Pelló1, P. Hudelot2, N. Laporte3,1, Y. Mellier2, H. J. McCracken2, M. Balcells4, F. Boone1, N. Cardiel6, J. Gallego6, F. Garzón4,5, R. Guzmán7, J. F. Le Borgne1, M. Prieto4,5, J. Richard8, D. Schaerer9,1, L. Tresse8 S. Arnouts10, J. G. Cuby10, K. Disseau8, and M. Hayes11 1 Institut de Recherche en Astrophysique et Planétologie (IRAP), Université de Toulouse, CNRS, UPS, CNES, 14 Av. Edouard Belin, 31400 Toulouse, France e-mail: rpello@irap.omp.eu 2 I i d’A h i d P i UMR7095 CNRS U i i é Pi & M i C i 98 bi Bld A 75014 P i F 1 Institut de Recherche en Astrophysique et Planétologie (IRAP), Université de Toulouse, CNRS, UPS, CNES, 14 Av. Edouard Belin, 31400 Toulouse, France e-mail: rpello@irap.omp.eu e-mail: rpello@irap.omp.eu p p ophysique de Paris, UMR7095 CNRS, Université Pierre & Marie Curie, 98 bis Bld. Arago, 75014 Paris, France f Physics and Astronomy, University College London, Gower Street, London WC1E 6BT, UK p y y y g Instituto de Astrofísica de Canarias, C/ Via Lactea s/n, 38205 La Laguna, Tenerife, Spain 4 Instituto de Astrofísica de Canarias, C/ Via Lactea s/n, 38205 La Laguna, Tenerife, Spain 4 Instituto de Astrofísica de Canarias, C/ Via Lactea s/n, 38205 La Laguna, Tenerife, Spain 4 Instituto de Astrofísica de Canarias, C/ Via Lactea s/n, 38205 La Laguna, Tenerife, Spain 5 g p 5 Departamento de Astrofísica, Universidad de La Laguna, Avda. Astrofísico Fco. Sànchez, 38206 La Laguna, Tenerife, Spain 6 Departamento de Astrofísica y CC de la Atmósfera, Universidad Complutense de Madrid, Av Complutense s/n, 28040 Madrid, Spain 5 Departamento de Astrofísica, Universidad de La Laguna, Avda. Astrofísico Fco. HAL Id: hal-03000018 https://hal.science/hal-03000018v1 Submitted on 11 Nov 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Astronomy & Astrophysics Astronomy & Astrophysics A&A 620, A51 (2018) https://doi.org/10.1051/0004-6361/201732080 c⃝ESO 2018 A&A 620, A51 (2018) https://doi.org/10.1051/0004-6361/201732080 c⃝ESO 2018 A&A 620, A51 (2018) https://doi.org/10.1051/0004-6361/201732080 c⃝ESO 2018 1. Introduction The results obtained on the LF at z ≥7, as well as the evolution of the bright edge of the UV LF between z ∼4.5 and 9, will be presented in a separate paper (Laporte et al. 2018; hereafter Paper II). In this paper we have concentrated on the presentation of the survey, as well as on the selection and characterization of galaxy samples at z ∼[4.5−7] in this field. The results obtained on the LF at z ≥7, as well as the evolution of the bright edge of the UV LF between z ∼4.5 and 9, will be presented in a separate paper (Laporte et al. 2018; hereafter Paper II). ( p p ) In Sect. 2 we describe the WUDS observations. Section 3 is devoted to data processing and the construction of the image dataset. Section 4 presents the extraction of sources and the con- struction of photometric catalogs. The characterization of the photometric survey is given in Sect. 5, including the determi- nation of the depth and completeness of the survey, and number counts. Section 6 presents the quality achieved in the computa- tion of photometric redshifts up to z ∼1.5, and the global prop- erties of the galaxy population at low-z. Section 7 presents the selection of candidates in the redshift interval z ∼[4.5−7]. The properties of these samples of galaxies are studied, in particular the luminosity function (LF), and compared to previous findings. Conclusions and perspectives are given in Sect. 8. Figure 1 displays a comparison between the effective area versus depth for different representative “deep” NIR surveys available in the H-band (∼5σ). WUDS covers an interest- ing niche between wide (but still deep) surveys such as UltraVISTA (McCracken et al. 2012; Laigle et al. 2016), and ultra-deep pencil beam surveys such as the eXtreme Deep Field (XDF; Illingworth et al. 2013), or lensing clusters, such as CLASH (Postman et al. 2012) and the Hubble Frontier Fields (Lotz et al. 2017). In this respect, the region covered by WUDS is a single field in the northern hemisphere, with an area com- parable to the HST MCT CANDELS-N Survey (Grogin et al. 2011; Koekemoer et al. 2011). 1 http://www.astro.ufl.edu/GOYA/home.html 2 http://www.gtc.iac.es/instruments/emir/emir.php 1. Introduction Contrary to CANDELS WFC3 imaging, which is limited to wavelengths up to 1.6 µm (excepted for some Ks-band imaging on the CANDELS-S), WUDS also includes deep Ks-band imaging, and a complete and homoge- neous coverage in the visible domain from the CFHTLS-D3 field (Groth strip), that is a photometric catalog survey in nine filter- bands (ugrizYJHKs). Throughout this paper, a concordance cosmology is adopted, with ΩΛ = 0.7, Ωm = 0.3 and H0 = 70 km s−1 Mpc−1. All mag- nitudes are given in the AB system (Oke & Gunn 1983). Table 1 presents the conversion values between Vega and AB systems for our photometric dataset. p Data products described in this paper are available online3. WUDS was originally proposed by the Galaxy Origins and Young Assembly (GOYA1) team as part of the effort for the exploitation of the multiobject near-IR spectrograph EMIR at the GTC (Balcells 2003; Garzón et al. 2016), in particular to provide a robust selection of targets for observations with EMIR. EMIR is a wide-field, near-IR spectrograph commissioned in 2016 at the Nasmyth A focus of the Spanish GTC at Canary Islands2. EMIR is one of the first fully cryogenic multiobject spectro- graphs to be operated on a 10 m-class telescope, with a spectral resolution R ∼4000−5000, high enough to achieve an efficient OH-line suppression. It was specifically designed for the study of distant galaxies, in particular the GOYA project to be carried as part of the EMIR’s GTO program (see e.g., Guzman 2003; Balcells 2003; Garzón et al. 2007). http://wuds.irap.omp.eu/ 4 http://www.cfht.hawaii.edu/Instruments/Imaging/ WIRCam/ 1. Introduction 2012; Coe et al. 2013; McLure et al. 2013; Ono et al. 2018, and the references therein). y g p j Deep and/or wide-field surveys in the near-IR bands are recognized since the pioneering studies in the 90’s as key observations to understand the process of galaxy evolution at intermediate redshifts, in particular to address the process of stellar mass assembly at 1 ≤z ≤3 (see e.g., Cowie et al. 1994; Cimatti et al. 2002; Labbé et al. 2003; McCracken et al. 2012; Cassata et al. 2013; Papovich et al. 2015; Laigle et al. 2016, and the references therein). One of the main applica- tions of deep near-IR photometry is the selection of star-forming galaxies at z ∼6−10 based on their rest-frame UV contin- uum, a study conducted during the last decade in a context of international competition using the Hubble Space Telescope (HST) and various ground-based facilities, (see e.g., Kneib et al. 2004; Pelló et al. 2004; Bouwens et al. 2004, 2015; Bunker et al. 2010; Oesch et al. 2010; Finkelstein et al. 2012; Coe et al. 2013; McLure et al. 2013; Ono et al. 2018, and the references therein). The efficiency on the selection of high-z galaxies depends on the availability of ultra-deep multiwavelength data, using an appro- priate set of near-IR filters in combination with optical data. Although lensing clusters and ultra-deep pencil-beam surveys are more efficient to conduct detailed studies toward the faint- end of the LF, given the strong field to field variance in number counts in these regimes, observations of wide blank fields are mandatory and equally important to set reliable constraints on the brightest end of the UV LF. Fig. 1. Effective area versus depth for different NIR surveys available in the H-band (∼5σ), both in blank fields (UltraVISTA, CANDELS, HUDF and XDF) and lensing clusters (CLASH and Hubble Frontier Fields). The appropriate correction was applied to CLASH and Hub- ble Frontier Fields to account for lensing effects on the effective area beyond the limiting magnitude, assuming z ∼7 for the source plane. In this paper we have concentrated on the presentation of the survey, as well as on the selection and characterization of galaxy samples at z ∼[4.5−7] in this field. 1. Introduction strong constraints on the cosmic star-formation rate (SFR) and the UV luminosity function (hereafter LF) around or brighter than L⋆in the z ∼6−10 redshift domain, taking advantage from the large field of view and sensitivity of WIRCam. Determin- ing the precise contribution of star-forming sources at z ≥6 to the cosmic reionization remains an important challenge for modern cosmology. The study of their physical properties, start- ing with the spectroscopic confirmation of current photometric candidates, requires the use of the most efficient ground-based and space facilities presently available and, in practice, this This paper introduces the WIRCam Ultra Deep Survey (WUDS), a public near-IR photometric survey carried out at the CFH Tele- scope in the field of the CFHTLS-D3 field (Groth Strip). The area and depth of this survey were specifically tailored to set A51, page 1 of 16 A51, page 1 of 16 A&A 620, A51 (2018) Fig. 1. Effective area versus depth for different NIR surveys available in the H-band (∼5σ), both in blank fields (UltraVISTA, CANDELS, HUDF and XDF) and lensing clusters (CLASH and Hubble Frontier Fields). The appropriate correction was applied to CLASH and Hub- ble Frontier Fields to account for lensing effects on the effective area beyond the limiting magnitude, assuming z ∼7 for the source plane. exercise is limited to the brightest candidates. Although the moti- vation of WUDS is clearly focused on the high-z universe, these data are also useful for a variety of extragalactic projects. y g p j Deep and/or wide-field surveys in the near-IR bands are recognized since the pioneering studies in the 90’s as key observations to understand the process of galaxy evolution at intermediate redshifts, in particular to address the process of stellar mass assembly at 1 ≤z ≤3 (see e.g., Cowie et al. 1994; Cimatti et al. 2002; Labbé et al. 2003; McCracken et al. 2012; Cassata et al. 2013; Papovich et al. 2015; Laigle et al. 2016, and the references therein). One of the main applica- tions of deep near-IR photometry is the selection of star-forming galaxies at z ∼6−10 based on their rest-frame UV contin- uum, a study conducted during the last decade in a context of international competition using the Hubble Space Telescope (HST) and various ground-based facilities, (see e.g., Kneib et al. 2004; Pelló et al. 2004; Bouwens et al. 2004, 2015; Bunker et al. 2010; Oesch et al. 2010; Finkelstein et al. 3 http://wuds.irap.omp.eu/ 4 http://www.cfht.hawaii.edu/Instruments/Imaging/ WIRCam/ 3 http://wuds.irap.omp.eu/ 4 http://www.cfht.hawaii.edu/Instruments/Imaging/ 2. Observations Information given in this table: reference field, filter identification, filter effective wavelength, filter width, AB correction (mAB = mVega + CAB), total exposure time, 3σ limiting magnitudes (within 1.3′′ diameter aperture), 50% completeness level for point-like sources (in the regions with >50% of the total exposure time), average seeing for the final stack, and total area covered with >50% total exposure time. Table 2. WUDS observations summary table. 27' 29' 26' 47' WUDS (YJHKs ) WIRDS (extended JHKs) Fig. 2. Layout of the WUDS Survey showing the regions covered by the deep WUDS survey (red line) and the extended WIRDS area (green line) within the CFHTLS-D3 field (black area, ∼1 deg2). north is up and east is to the left. Table 2. WUDS observations summary table. Filter Number texp Total exposure of cubes (s) (h) Y (1) 561 80 12.5 J (1) 400 60 6.7 J (2) 589 45 7.3 H (1) 1616 15 6.7 H (2) 1017 15 4.3 Ks (1) 320 25 2.2 Ks (2) 874 20 5.0 Notes. (1) Data from May–July 2008 observations. (2) Data from the WIRDS survey on the same area. Notes. (1) Data from May–July 2008 observations. (2) Data from the WIRDS survey on the same area. included 16.6 h of exposure time on the same area, obtained by the WIRDS Survey in J, H and Ks (Bielby et al. 2012). Table 2 summarizes these observations, referenced as “WUDS” in Table 1, also corresponding to the area delimited in red in Fig. 2. Fig. 2. Layout of the WUDS Survey showing the regions covered by the deep WUDS survey (red line) and the extended WIRDS area (green line) within the CFHTLS-D3 field (black area, ∼1 deg2). north is up and east is to the left. g In order to obtain well sampled images given the goal see- ing conditions, and to achieve an optimum matching with the CFHTLS-D3 images, on-target observations were performed using the micro-dithering pattern of WIRCam. This consists of a 2 × 2 dithering pattern with offset positions separated by 0.5 pixels, constituting a “data-cube”. Each data-cube in Table 2 contains four such images. Table 2 also reports the individual exposure times and the number of exposures needed to com- plete the total exposures. 5 http://aegis.ucolick.org 6 http://terapix.iap.fr 6 http://terapix.iap.fr 5 http://aegis.ucolick.org 2. Observations Observations were carried out with WIRCam at the Canada France Hawaii Telescope. WIRCam4 is a wide-field near-IR camera with four HAWAII2-RG detectors, 2048 × 2048 pixels each, and a pixel scale of 0.306′′. The total WIRCam field of view is 21.5′ × 21.5′, with a gap of 45′′ between adjacent detec- tors. WUDS was carried out on the CFHTLS-D3 (Groth Strip), on a field centered around α = 14:18:15.3 δ = +52:38:45 (J2000), in order to avoid the presence of bright stars while max- imizing the coverage by other surveys, in particular the DEEP Groth Strip Survey (Vogt et al. 2005; Weiner et al. 2005a) and A51, page 2 of 16 R. Pelló et al.: The WIRCam Ultra Deep Survey (WUDS) Table 1. Photometric dataset used in this paper. Table 1. Photometric dataset used in this paper. Reference Filter λeff ∆λeff CAB texp m(3σ) m(50%) Seeing Area (>50%) (nm) (Å) (mag) (ksec) (mag) (mag) (′′) (arcmin2) CFHTLS-D3 u∗ 382 544 0.312 76.6 28.52 26.97 0.89 3224 CFHTLS-D3 g 490 1309 −0.058 79.6 28.94 26.79 0.84 3224 CFHTLS-D3 r 625 1086 0.176 142.8 28.57 26.30 0.78 3224 CFHTLS-D3 i 766 1330 0.404 249.4 28.24 25.95 0.76 3224 CFHTLS-D3 z 884 1033 0.525 175.4 27.09 25.46 0.69 3224 WUDS Y 1027 1077 0.632 44.9 26.78 26.26 0.63 392 WUDS J 1256 1531 0.949 50.4 26.69 26.17 0.60 396 WIRDS J 26.3 25.80 24.80 0.60 437 WUDS H 1636 2734 1.390 39.6 26.06 25.61 0.55 477 WIRDS H 15.5 25.73 24.80 0.55 681 WUDS Ks 2154 3071 1.862 25.9 25.93 25.46 0.56 450 WIRDS Ks 17.5 25.59 24.63 0.56 547 Notes. Information given in this table: reference field, filter identification, filter effective wavelength, filter width, AB correction (mAB = mVe CAB), total exposure time, 3σ limiting magnitudes (within 1.3′′ diameter aperture), 50% completeness level for point-like sources (in the reg with >50% of the total exposure time), average seeing for the final stack, and total area covered with >50% total exposure time. Notes. Information given in this table: reference field, filter identification, filter effective wavelength, filter width, AB correction (mAB = mVega + CAB), total exposure time, 3σ limiting magnitudes (within 1.3′′ diameter aperture), 50% completeness level for point-like sources (in the regions with >50% of the total exposure time), average seeing for the final stack, and total area covered with >50% total exposure time. Notes. 2. Observations Observations were performed with large dithering patterns (the equivalent of 1/2 of detector, the four detectors being separated by 45′′; see above) and large over- laps providing an optimized gap filling and also a better object removal for sky-subtraction (see below). the AEGIS Survey5 (Davis et al. 2007). The WUDS pointing was chosen in such a way that observations could be com- bined with public CFHTLS-Deep data on this field, obtained through five bands in the optical domain, namely u∗, g, r, i, and z (see TERAPIX6). Figure 2 presents the layout of WUDS showing the regions covered by the different data sets on the CFHTLS-D3 field. WUDS images were obtained in queue scheduling mode between May and July 2008, in the four broad-band filters of WIRCam, namely Y, J, H and Ks, covering a field of view of ∼400 arcmin2. This means a single shot with WIRCam, with nod- ding and dithering configurations allowing us to maximize the area covered with more than 75% of the total exposure time over the WIRCam field. In addition to these observations, we Exposure times in the near-IR were setup in such a way that a good signal-to-noise ration (S/N) is achieved for the detection of the rest-frame UV continuum of Lyman-Break Galaxy samples (hereafter LBG) given the depth of the CFHTLS-D3 images in the optical domain (see also Sect. 7.1). We have also extended the search for high-z candidates in Sect. 7 to an adjacent area of ∼1000 arcmin2 in the CFHTLS-D3 field, extracted from the WIRDS Survey in this A51, page 3 of 16 A&A 620, A51 (2018) Fig. 3. Schematic view of the advanced processing of WUDS data at Terapix. area (Bielby et al. 2012), and observed only in J, H and Ks bands. This dataset is referenced as “WIRDS” in Table 1, and corresponds to the area delimited in green in Fig. 2. The seeing in each individual image was determined as the median FWHM of four reference stars. The mean and standard deviation values measured across the sample pre- sented in Table 2 for the different filters are: 0.66′′ ± 0.09′′ (Y), 0.58′′ ± 0.07′′ (J), 0.57′′ ± 0.16′′ (H), and 0.54′′ ± 0.06′′ (Ks). The seeing distribution in Y and H is a little wider than in the two other filters, leading to a larger rms. 2. Observations In fact, more than ∼90% of the sample has a FWHM better than 0.75′′in these filters, leading to a mean and standard deviation of 0.65′′ ± 0.08′′ and 0.53′′ ± 0.06′′ in Y and H, respectively when excluding these extreme values. The seeing of individual images was included in the weighting process when building the final stacks, as explained in Sect. 3. Depending on the filter, the averaged seeing values achieved on the stacked images typically range between 0.55 and 0.63′′, as seen in Table 1. Table 1 summarizes the properties of the photometric dataset used in this paper, when combining the whole WUDS, WIRDS and CFHTLS-D3 observations in this field. The total effec- tive area covered by this survey with >50% and >75% of the total exposure time is also given in Table 1 for the dif- ferent bands. The maximum intersection with all nine filter- bands and >50% of exposure time is limited by the Y-band (i.e., ∼390 arcmin2), whereas it is ∼440 arcmin2 with eight filter- bands in the extended (WIRDS) area (outside the WUDS region, that is ∼830 arcmin2 in total on the CFHTLS-D3 field of view covered at least by JHKs bands with >50% of the total exposure time). Fig. 3. Schematic view of the advanced processing of WUDS data at Terapix. 7. Final stack. Before combining the frames into a final stack, we applied weight values to individual images optimized to improve the detectability of faint compact sources in this way: weight ∝(ZP × var × s2)−1, where ZP and s correspond to zero-point and seeing values respectively, and var is the pixel-to-pixel variance derived in a reference clean area. 7. Final stack. Before combining the frames into a final stack, we applied weight values to individual images optimized to improve the detectability of faint compact sources in this way: weight ∝(ZP × var × s2)−1, where ZP and s correspond to zero-point and seeing values respectively, and var is the pixel-to-pixel variance derived in a reference clean area. Automatic preprocessing at CFHT included steps from 1 to 3 above. It was done with the ’I’iwi IDL Interpretor of WIRCam Images (see details at the WIRCam home page at CFHT). Astrometry included the detection of stars using SExtractor, the computation of a full mosaic WCS linear solution, followed by a detector-by-detector refinement using IMWCS. 3. Data processing Data processing was performed at CFHT (preprocessing phase) and Terapix/IAP (advanced processing). A two-step approach was adopted for sky-subtraction and image stacking mostly inspired from the reduction of near-IR observations by Labbé et al. (2003) and Richard et al. (2006) with similar goals. The main steps are the following: 1. Detrending process of raw images, performed at the CFHT. This includes flagging the saturated pixels, correcting for non-linearity, reference pixels subtraction, dark subtraction, dome flat-fielding, bad-pixels masking, and guide-window masking. These steps are described in details at the WIRCam home page at CFHT. p The advanced processing at Terapix started from detrended images, therefore including steps from 2 to 7 above. Figure 3 presents a schematic view of this process. Two particular prob- lems needed a specific solution to improve the final stack. Firstly, a large fraction of images suffered from horizontal stripe-like residuals due to the detector amplifier, with a small amplitude (typically 10 counts over ∼10 000 counts for the sky back- ground). They were successfully removed from the individual images by subtracting a model obtained from the horizontal pro- jection of thin stripes (∼1/4 of the amplifier width), after object masking. The second correction was performed to suppress large-scale gradient residuals from the background sky. This correction was obtained through SExtractor background sub- traction, using a large mesh-size (256 pixels) on images where objects had been previously masked. It is worth to note the highly time-consuming process of manual quality-assessment of individual images (using QualityFITS), given the huge num- ber of images in the stacks (∼560 in Y, ∼1420 in J, ∼3670 in H and ∼1920 in Ks), and the fact that the whole pro- cess was performed twice. Also weight maps were obtained 2. First sky-subtraction. Given the fast variations of the sky- background on large and small spatial scales, we used all the science images taken between ∼10 min before and after the actual image to produce a “sky” background, by median- ing these adjacent exposures. The efficiency of this process strongly depends on the dithering strategy, that is using larger offset-paths provides better results. 3. Astrometry and photometric calibration (standard prepro- cessing; see below). 4. First image stack. Images were sky-subtracted using the pre- vious “local” backgrounds and then registered and combined together into a first stack. 5. Object mask. 2. Observations For each filter- band, photometric calibration was performed by WCS match- ing all stars detected by SExtractor to the 2MASS photom- etry, on a detector by detector basis. For the J, H and Ks bands, ZPs were derived from reference 2MASS stars in the Vega system (Skrutskie et al. 2006), converted into AB magni- tudes using the conversion values in Table 1. For the Y band, a ZP was estimated using reference spectrophotometric stars. The first images obtained in this way suffered from several problems described below, and were used only for tests as first-epoch data. 5.1. Completeness and depth This section presents the extraction of sources and the construc- tion of the photometric catalogs, publicly available online8. The completeness of the WUDS survey has been estimated through simulations of stars and galaxies based on the STUFF and SKYMAKER softwares. Simulated samples of stars/galaxies have been randomly added to real images after masking of objects detected in the stacks, with the appropriate PSF convolution. These sources are then detected with SEx- tractor, using the same extraction parameters as for science images, therefore the completeness levels are directly obtained as a function of magnitude in the different bands. These values are reported in Table 1. The difference between the complete- ness levels in regions with the highest exposure time (>90%) and regions with smaller exposure times, for instance regions with >70% and >50% exposure time, is typically 0.08 and 0.13 mag worse respectively. Therefore, given the geometry of the survey, the search for optical dropouts is limited in practice to regions with at least 50% of the total exposure time in the final stack (see Table 1). This means that the detection level does not change dramatically across the surveyed field. To built the final catalogs, WUDS data were combined with public CFHTLS-Deep data on this field, obtained through five bands in the optical domain, namely u∗, g, r, i, and z. Therefore, WUDS catalogs include nine filter bands with full wavelength coverage between ∼0.35 and 2.3 µm. Hereafter, we refer to this ensemble as WUDS (or WUDS + WIRDS) data. Two near-IR-selected catalogs were built for the needs of this project. The first one (hereafter C1) is based on the Y + J detec- tion image. It was primarily intended to be used for the identi- fication of i and z dropouts in this paper (i.e., z ∼6 and z ∼7 candidates respectively). The second one (C2) is based on the H + Ks detection image, as it was intended to be used for the identification of Y and J dropouts (i.e., z ∼7−11 candidates). This later selection will be presented in Paper II. We also com- puted a catalog based on the i + z detection image to be used for the identification of r-dropouts (i.e., z ∼5 candidates) in addi- tion to the near-IR selected samples. All these catalogs are used in Sect. 7 for the selection of galaxies at z ∼[4.5−7]. 7 http://www.astromatic.net/software/ 8 http://wuds.irap.omp.eu/ 5. Characterization of the photometric survey In this section we characterize the properties of the WUDS pho- tometric catalogs in different ways. Completeness and depth are estimated based on realistic simulations of stars and galaxies. Number counts are obtained in the near-IR bands and compared to previous findings. 3. Data processing Sources were detected with SExtractor (Bertin & Arnouts 1996) in the first image stacks in order to create an object mask. 6. Second sky-subtraction. The second step was repeated using the object mask to reject pixels located on detected sources when computing the sky value. A second sky-subtraction was applied to the data using these new backgrounds. A51, page 4 of 16 R. Pelló et al.: The WIRCam Ultra Deep Survey (WUDS) during this phase, using the WeightWatcher software developed by Marmo & Bertin (2008). original images (without any seeing matching or rescaling), within apertures of the same physical size as for flux measure- ments (either aperture or MAG_AUTO magnitudes). Errors in colors were derived by quadratically adding the corresponding errors in magnitude. Astrometric calibration was performed with SCAMP7. The accuracy of internal astrometry ranges between 0.02 and 0.035′′at 1σ level for all filters. Images were combined using the weighting scheme given in point 7 above, with the SWARP software. The combination was a sigma-clipped mean with a 3σ rejection threshold. The final stacks were matched to the CFHTLS-D3 T0006 images and pixel scale (0.186′′). The astrometric solution was computed by SCAMP including inter- nal constraints (overlapping frames) and external references (CFHTLS-D3 objects catalog). The accuracy of astrometry in this case is typically σ = 0.05′′, with a systematic offset of less than 0.005′′. Figure 4 displays the exposure-time maps for the ∼1 × 1◦ CFHTLS-D3 field of view, and for the different near-IR fil- ters. We note the difference in the total exposure times between the WUDS (deep) region and the extended region of WIRDS. Table 3 summarizes the number of sources detected in the differ- ent areas, for the different filters and detection images. Table 1 also presents the 3σ limiting magnitudes achieved in the differ- ent filters within 1.3′′ diameter aperture (point sources). Com- pleteness and depth achieved by WUDS are discussed below (see Sect. 5.1). Regarding the accuracy achieved in the determination of ZPs, it ranges from 0.021 to 0.023 mag in J, H and Ks bands in the WUDS survey alone (it is between 0.03 and 0.04 when combining WUDS and WIRDS data on the whole field). It is 0.067 mag in the Y-band for WUDS (0.075 for WUDS + WIRDS) due to indirect recalibration. These estimates are based the comparison between individual detections of sev- eral 104 stars in each band. A51, page 5 of 16 5.1. Completeness and depth The typical depth of the WUDS data reaches between ∼26.8 in Y and J, and ∼26 in H and Ks (AB, 3σ in 1.3′′ aperture), for a completeness level of ∼80% at Y ∼26 and H and Ks ∼25.2, and excellent seeing values for the final stacks (ranging between 0.55 and 0.65 arcsec). The corresponding depth of the CFHTLS-D3 images in this region ranges between 28.6 and 29 in ugr, 28.2 in i and 27.1 in z (same S/N and aperture). Sources were detected with the SExtractor package version 2.8 (Bertin & Arnouts 1996), using the weight maps mentioned in Sect. 3. Extraction was performed using a very low detection threshold of 0.8 sigma (SExtractor definition) for a minimum number of four pixels above the threshold, in order to optimize the detection of compact and faint sources. The price to pay for the low detection threshold used in this survey is an enhanced fraction of spurious detections, increas- ing with magnitude. As explained in Sect. 5.2 below, we have estimated this contamination using the same detection scheme on “negative” images and, in addition, all high-z candidates have been manually inspected. Based on these results, we have esti- mated that our catalogs are robust (in the sense that they are not dominated by false-positive detections in the near-IR images) up to AB ∼25.5 in Y and J, and AB ∼24.75 in H and Ks, irre- spective of the detection image. The number of sources detected in the different areas, with magnitudes brighter than these lim- its are reported in Table 3, for the different filters and detection images. A background mesh of 64 pixels was used for background subtraction. Magnitudes and fluxes were measured in all images with the SExtractor “double-image” mode using the correspond- ing detection images (i + z, Y + J or H + Ks). Total magnitudes and fluxes were computed based on SExtractor MAG_AUTO magnitudes. Also aperture magnitudes were derived within 14 different apertures ranging from 1.3 to 5′′diameter, on the orig- inal images, and also on images matched to the u∗-band see- ing using a simple Gaussian convolution. Photometric errors were measured using the typical background variance of the A51, page 5 of 16 A&A 620, A51 (2018) A&A 620, A51 (2018) Fig. 4. 9 This index ranges between 0.0 for extended sources and 1.0 for unre- solved ones (see Bertin & Arnouts 1996). 5.1. Completeness and depth Exposure-time maps for the CFHTLS-D3 field of view in Y (top left panel), J (top right panel), H (bottom left panel) and Ks bands (bottom right panel). Gray levels display in linear scale the regions where near-IR data are available. Note the different exposure times between the WUDS (deep) region and the wide field extended region of WIRDS. Fig. 4. Exposure-time maps for the CFHTLS-D3 field of view in Y (top left panel), J (top right panel), H (bottom left panel) and Ks bands (bottom right panel). Gray levels display in linear scale the regions where near-IR data are available. Note the different exposure times between the WUDS (deep) region and the wide field extended region of WIRDS. 5.2. Number counts erature (e.g., Cristóbal-Hornillos et al. 2003, 2009; Laigle et al. 2016), without any correction for incompleteness. The detection image was Y + J for Y and J bands, whereas it was H + Ks for H and Ks. The 50% completeness levels for point sources coin- cide with the drop in number counts in the WUDS area. Com- pact sources with magnitudes brighter than 17.7 in Y, 18.1 in J, 17.2 in H and 17.6 in Ks are affected by saturation. Although there is some scatter toward the bright end (AB ≲20), there is a good agreement with previously published results, in particular Bielby et al. (2012). Galaxy number counts have been obtained in the four bands of WUDS and compared to the literature, as a consistency check for the calibration of the present data set. The separation between stars and galaxies is based on the SExtractor stellarity index9. Since the reliability of this index for galaxies diminishes toward the faintest magnitudes, we have applied this selection up to a S/N ∼10 in the detection images, with galaxies selected by imposing a SExtractor stellarity index <0.9. These threshold val- ues are based on straightforward simulations, using the same approach as for the determination of the completeness levels in Sect. 5.1. The detection scheme described above was optimized to identify faint and compact sources (see Sect. 5.1). Therefore, a large fraction of spurious detections was expected in the near- IR bands, increasing toward the faintest magnitudes. In order to evaluate the incidence of this effect, we have applied an identi- cal scheme for source detection as described in Sect. 5.1 to neg- ative images obtained by multiplying the original stacks by −1, to blindly extract these spurious non-astronomical signal. The Figure 5 displays the resulting number counts in the four bands of WUDS, as compared to previous findings from the lit- A51, page 6 of 16 R. Pelló et al.: The WIRCam Ultra Deep Survey (WUDS) Table 3. Summary of detections in the main survey (WUDS) and extended area (WIRDS), for the different near-IR filters and detection images (1). 5.2. Number counts Area Y J H Ks JHKs YJHKs (1) AB < 25.5 AB < 25.5 AB < 24.75 AB < 24.75 WUDS Y + J 77 715 91 340 59 552 66 623 34 832 29 841 H + Ks 66 479 79 526 66 627 72 960 35 791 30 655 i + z 49 920 57 574 46 185 49 089 31 900 28 088 WIRDS Y + J – 82 200 35 999 46 225 21 721 – H + Ks – 86 985 48 063 65 858 21 706 – i + z – 41 637 31 759 36 056 18 371 – Notes. Detection images reported in Col. (1) are the same for a given row. A column is provided for each filter reporting the total number of sources brighter than the magnitude given in the second row (see discussion in Sect. 5.1). The last two columns refer to objects simultaneously detected in the three filters JHKs and the four filters YJHKs respectively. etections in the main survey (WUDS) and extended area (WIRDS), for the different near-IR filters and detection ima Table 3. Summary of detections in the main survey (WUDS) and extended area (WIRDS), for the different near- (1). Table 3. Summary of detections in the main survey (WUDS) and extended area (WIRDS), for the different near-IR filters and detection images (1). Notes. Detection images reported in Col. (1) are the same for a given row. A column is provided for each filter reporting the total number of sources brighter than the magnitude given in the second row (see discussion in Sect. 5.1). The last two columns refer to objects simultaneously detected in the three filters JHKs and the four filters YJHKs respectively. Fig. 5. Galaxy number counts in the four bands of WUDS. Black filled circles display the results in the WUDS field. Results from Cristóbal-Hornillos et al. (2009; open stars), Cristóbal-Hornillos et al. (2003; filled stars) and Laigle et al. (2016; red points) are also shown for comparison. Open circles display the results obtained on the negative images, as an indication of contamination by spurious sources. Error bars correspond to 1σ Poissonian errors. Vertical solid and dashed lines indicate the 50% completeness levels for point sources in WUDS and extended (WIRDS) areas respectively. Fig. 5. Galaxy number counts in the four bands of WUDS. 6. Data properties at low-z In this section we assess the quality achieved in the computa- tion of photometric redshifts based on these data, up to z ∼1.5. We also use the SED-fitting approach to derive the properties of the near-IR selected galaxy population in this field in terms of redshift distribution and stellar masses. Regarding the comparison with previous findings on near-IR counts, our results are fully consistent with Cristóbal-Hornillos et al. (2003, 2009) and Laigle et al. (2016) at m > 20, whereas a larger dispersion is observed for brighter sources as expected. WUDS is still deeper than UltraVISTA-DR2 survey in the Ks-band (Laigle et al. 2016). The change in the slope of the near-IR counts at AB ∼19.5–20.0 is clearly visible in the Ks-band, whereas it is less obvious 10 http://userpages.irap.omp.eu/~rpello/newhyperz/ 5.2. Number counts Black filled circles display the results in the WUDS field. Results from Cristóbal-Hornillos et al. (2009; open stars), Cristóbal-Hornillos et al. (2003; filled stars) and Laigle et al. (2016; red points) are also shown for comparison. Open circles display the results obtained on the negative images, as an indication of contamination by spurious sources. Error bars correspond to 1σ Poissonian errors. Vertical solid and dashed lines indicate the 50% completeness levels for point sources in WUDS and extended (WIRDS) areas respectively. in the other bands, and not present in the optical bands (e.g., Cristóbal-Hornillos et al. 2009; Eliche-Moral et al. 2006). This trend has been identified by several authors using models (e.g., Eliche-Moral et al. 2010; Prieto & Eliche-Moral 2015, and the references therein) as the result of the late assembly by major-mergers of a substantial fraction of present-day massive early-type galaxies at 0.7 < z < 1.2, and inconsistent with a simple passive evolution since z ∼2. result of this procedure is also shown in Fig. 5 (open dots). The structure of the noise in these negative images is somewhat dif- ferent with respect to the astronomical ones, in the sense that an excess of faint and compact sources appears toward the faintest magnitudes. As seen in Fig. 5, this systematic trend and the dom- inance of false positives start close to the 50% completeness lev- els in WUDS. The reason for this trend, which is also observed in other similar surveys (e.g., public CLASH data from HST), is not clear. It could be due to the drizzling and resampling procedure. For this reason, we did not try to use these negative counts to correct our results, but as an indication of the flux level at which severe contamination is expected. In practice, we have limited the detection samples to magnitudes reported in Table 3, where contamination is not expected to dominate. In addition, all the high-z candidates presented in this paper and in Paper II have been manually inspected to remove obvious spurious sources. 6.1. Photometric redshifts New-Hyperz performs a χ2 minimization in the parameter space in the first pass, followed by a small correction for sys- tematics trends as a function of zphot obtained through the poly- nomial fit of the residuals between the best-fit redshift above and the true value for the spectroscopic sample described below, excluding outliers. These residuals encode our lack of precise knowledge on the overall system transmission as a function of wavelength. The procedure yields the best fit zphot and model template for each source, as well as a number of fitting byproducts (e.g., abso- lute magnitudes in the different bands, normalized redshift prob- ability distribution, zphot error bars, secondary solutions, ...). An interesting indicator of the goodness of the fit is provided by the integrated probability Pint between zphot ± 0.1, where zphot stands for the best fit redshift, with the probability distribution normalized between z = [0, 12]. Among the fitting byproducts is a rough classification of the rest-frame SED of galaxies into five different spectral types, according to their best fit with the simplest empirical templates given by Coleman et al. (1980) and Kinney et al. (1996): (1) E/S0, (2) Sbc, (3) Scd, (4) Im and (5) S (starbursts). Fig. 6. Comparison between photometric and spectroscopic redshifts in this survey for the H + K detection image across the entire field (top panel), and for the WUDS area only (bottom panel). Dot-dashed lines display the locus of z(phot) = z(spec) ± 0.1(1 + z) to guide the eye. value H = 21.9. Results based on H + K detection are the same as for the Y + J-based catalog. We have also consid- ered the 3828 galaxies extracted form the i + z detection image. Figure 6 displays the comparison between photometric and spec- troscopic redshifts based on the H+K detection image across the entire field, and the same restricted to the WUDS area, that is with photometry including Y-band data and 1651 spectroscopic sources. ( ) The photometric redshift accuracy has been estimated through a direct comparison between zphot and secure spec- troscopic samples publically available in the CFHTLS-D3 field, extracted from the DEEP Groth Streep Survey DR3 (Weiner et al. 2005b; Davis et al. 2003, 2007). Photometric and spectroscopic catalogs were blindly matched in ALPHA and DEC positions. 6.1. Photometric redshifts Photometric redshifts have been computed with the version v12 of the public code Hyperz (New-Hyperz10), originally devel- opped by Bolzonella et al. (2000). This method is based on the A51, page 7 of 16 A&A 620, A51 (2018) A51 (2018) Fig. 6. Comparison between photometric and spectroscopic redshifts in this survey for the H + K detection image across the entire field (top panel), and for the WUDS area only (bottom panel). Dot-dashed lines display the locus of z(phot) = z(spec) ± 0.1(1 + z) to guide the eye. fitting of the photometric Spectral Energy Distributions (SED) of galaxies. The accuracy of photometric redshifts (zphot) is used here as a consistency check for the calibration of the whole data set, as well as for the characterization of the different high-z sam- ples in WUDS. p The template library used in this paper includes 14 templates: eight evolutionary synthetic SEDs computed with the last ver- sion of the Bruzual & Charlot code (Bruzual & Charlot 2003), with Chabrier IMF (Chabrier 2003) and solar metallicity, match- ing the observed colors of local galaxies from E to Im types (namely a delta burst -SSP-, a constant star-forming system, and six τ-models with exponentially decaying SFR); a set of four empirical SEDs compiled by Coleman et al. (1980), and two starburst galaxies from the Kinney et al. (1996) library. Inter- nal extinction is considered as a free parameter following the Calzetti et al. (2000) extinction law, with AV ranging between 0 and 3.0 mag (E(B −V) in the range ∼[0,0.75] mag). Photometric redshifts have been computed in the range z = [0, 12] using two different priors in luminosity. The first one is the usual flat luminosity prior in redshift, that is a simple cut in the permitted range of luminosities for extragalactic sources, with absolute magnitudes in the range MB = [−14, −23]. The second one is a “soft” probability distribution as a function of redshift and magnitude, following Benítez (2000), encom- passing the B-band luminosity function derived by Ilbert et al. (2006b). This new option of New-Hyperz computes a smooth probability distribution prior for each object as a function of red- shift, the absolute magnitude MB being derived from the appar- ent magnitude m which is closer to the rest-frame B-band. The final probability distribution is given by the usual Hyperz P(z) combined with the prior. 6.2. Properties of the galaxy population We have taken advantage from the SED-fitting byproducts obtained by New-Hyperz when deriving zphot, as described in Sect. 6.1, to characterize the properties of the galaxy popula- tion in the WUDS survey. These quantities have been computed based on the catalogs used to select high-z sources in Sect. 7 below. Hereafter in this section, we limit the sample to sources fainter than the saturation limits in all WUDS filters, with SEx- tractor stellarity index <0.9, and detected in at least two near-IR filters with magnitudes brighter than AB ∼25.5 in Y and J, and AB ∼24.75 in H and Ks. As discussed in Sect. 5.2, these limits ensure that the sample is not dominated by spurious sources. The sample presented here contains ∼110(118) × 103 sources in both catalogs based on Y + J(H + Ks) detection images. The availability of near-IR filters helps improving the zphot accuracy beyond z ∼1.3, where the 4000 Å break goes out of the z′ filter and the Lyman break is not yet detectable in the u∗band. The main impact when including Y-band data is on the percentage of outliers. Unfortunately, only ∼3% of the spec- troscopic control sample is found at z ≥1.3. Results obtained with a flat luminosity prior are not significantly different from those achieved using a more aggressive prior based on the LF. Results based on seeing-matched aperture magnitudes taking the u∗-band as a reference are significantly worse than those based on MAG_AUTO, with a dispersion increasing by 0.01–0.02 with respect to the σ values displayed in Table 4. The reason for this is that the improved sampling in the SED obtained for seeing- matched apertures is compensated by a worse S/N in the photom- etry as compared to MAG_AUTO, the net effect being a lower quality in the photometric redshifts. g g Figure 7 displays the photometric redshift distribution obtained for different Ks-band selected samples in this survey. As expected, the distribution extends to higher redshifts with increasing magnitudes, with a clear drop in the distribution of galaxies at z ≳3.5, when the 4000 Å break enters the Ks-band. Stellar masses are among the quantities derived by the SED- fitting procedure, based on the best-fit model obtained with the Bruzual & Charlot code (Bruzual & Charlot 2003), with Chabrier IMF (Chabrier 2003) and solar metallicity. The param- eter space is precisely the same used for zphot. 6.1. Photometric redshifts Only objects with the highest spectroscopic red- shift quality (ZQUALITY ≥3) were considered in this compar- ison, that is 3424(3409) galaxies in the entire WUDS + WIRDS area based on H + K(Y + J) detection images. Magnitudes in this sample range between i = 18 and 24.4, with median value i = 22.4, corresponding to H = ∼[17.0−24.8] with median Table 4 presents a summary of the zphot quality achieved in this survey based on the usual statistical indicators, namely σ(∆z/(1 + z)), σ(|∆z/(1 + z)|), the median of (∆z/(1 + z)), the normalized median absolute deviation (defined as σz,MAD = 1.48 × median (|∆z|/(1 + z)), which is less sensi- tive to outliers), and the percentage of outliers. These results A51, page 8 of 16 R. Pelló et al.: The WIRCam Ultra Deep Survey (WUDS) Table 4. Summary of the zphot quality achieved with New-Hyperz on the WUDS/CFHTLS D3 field. Detection H + K (All) i + z (All) (1) (2) (1) (2) σ(∆z/(1 + z)) 0.047 0.048 0.051 0.050 σ(|∆z/(1 + z)|) 0.031 0.031 0.032 0.032 Median (3) 0.0016 0.0017 −0.0017 −0.0012 σz,MAD 0.043 0.043 0.046 0.045 Outliers 4.4% 4.4% 4.9% 4.9% Detection H + K (WUDS) i + z (WUDS) (1) (2) (1) (2) σ(∆z/(1 + z)) 0.047 0.047 0.049 0.049 σ(|∆z/(1 + z)|) 0.030 0.030 0.031 0.031 Median (3) 0.0005 0.0007 0.0007 0.0007 σz,MAD 0.042 0.042 0.044 0.045 Outliers 2.9% 2.8% 3.8% 3.9% Notes. (1) Flat prior. (2) LF prior. (3) Median (∆z/(1 + z)). Fig. 7. Comparison between the photometric redshift distribution obtained for different Ks-band selected samples in WUDS, from top to bottom Ks < 24, 23, 22, 21 and 20. Table 4. Summary of the zphot quality achieved with New-Hyperz on the WUDS/CFHTLS D3 field. Table 4. Summary of the zphot quality achieved with New-Hyperz on the WUDS/CFHTLS D3 field. Notes. (1) Flat prior. (2) LF prior. (3) Median (∆z/(1 + z)). Fig. 7. Comparison between the photometric redshift distribution obtained for different Ks-band selected samples in WUDS, from top to bottom Ks < 24, 23, 22, 21 and 20. are based on SExtractor MAG_AUTO magnitudes. Outliers are defined here as sources with |z(spec) −z(phot)| > 0.15(1 + z(spec)). 6.1. Photometric redshifts As shown in the table, the dispersion is below or on the order of ∼0.05(1 + z) in all cases based on the usual indi- cators, and the percentage of outliers ranges between 4 and 5% for the entire field, improving to 3 and 4% for the WUDS area, the results being slightly better for the near-IR selected samples. The same trends are also seen in Fig. 6, although the difference is small and hardly significant. The correction for systematic trends mentioned above is included in the results presented in Table 4 and Fig. 6. When this correction is not included, the results on the dispersion are worse by ∼0.002–0.006 depending on the sample and indicator, whereas the median bias (∆z/(1+z)) is up to a factor of 10 larger. 7.1. Selection criteria Several trends in Fig. 8 deserve a specific comment. There is a systematic trend in the sense that the dispersion within the sample increases with redshift for a given spectral type. Uncer- tainties in the determination of individual stellar masses due to various degeneracies in the parameter space are expected to be typically below 0.2 dex up to z ∼3.5, that is when the observed SED includes the 4000 Å break, but the determination becomes hazardous beyond this redshift (see e.g., Pforr et al. 2013; Mitchell et al. 2013, for a detailed discussion). As seen in Figures 7 and 8, the population of galaxies beyond z ∼3.5 strongly diminishes in our near-IR selected catalog, essentially because the region of the SED beyond the 4000 Å break, tracing the “old” stellar population and the stellar mass, progressively moves beyond the reddest band (Ks). This trend is expected given the limited wavelength coverage of WUDS (the reddest band is Ks). For this reason, the completeness limit displayed in Fig. 8 for early-type galaxies stops at z = 3.5, whereas it is given as an indication only for late-type galaxies beyond this limit, given the uncertainties associated to the stellar mass deter- mination beyond this redshift. We have used two different methods to select high-z galaxies. The first one is the usual three-band dropout technique applied to a combination of WUDS data and deep optical data from the CFHTLS-D3, that is a photometric catalog including the nine filter-bands (ugrizYJHKs). Different redshift intervals have been defined using an appropriate combination of filters. The second method is based on pure photometric redshifts and probability distributions, taking full advantage from the wide wavelength coverage. In all cases, a S/N higher than 5σ was requested in the fil- ters encompassing the rest-frame UV, irrespective of the detec- tion image, together with a non-detection (<2σ level) in all filters bluewards from the Lyman limit. In this respect, CFHTLS-D3 data are particularly useful to provide robust non- detection constraints for candidates at z ∼[4.5−7]. Detailed selection criteria are provided below. In the subsequent sec- tions we compare the samples selected and the global properties derived when using different approaches. Regarding the dropout technique, three selection windows have been used to cover the z ∼[4.5−7] interval, as shown in Fig. 6.2. Properties of the galaxy population As discussed by Davidzon et al. (2013), stellar masses derived in this way depend very weakly on the parameter space used for SED-fitting, in par- ticular the detailed star-formation histories. The final quality achieved for WUDS without further refine- ment is within the requirements for large cosmological surveys (e.g., Euclid, Laureijs et al. 2011), and it is expected to be rep- resentative of the general behavior for other SED-fitting zphot codes applied to these data. Indeed, the dispersion and the per- centage of outliers are comparable to the ones obtained with a similar number of filters in this redshift domain, such as for the 22.5 < i < 24.0 -selected sample in the VIMOS VLT Deep Sur- vey (VVDS; Ilbert et al. 2006a), the Hubble Ultra Deep Field (Coe et al. 2006), or the COSMOS field (Mobasher et al. 2007), whereas a better accuracy could be achieved by using a wider filter set (see e.g., Ilbert et al. 2009). A rough classification for the rest-frame SED of galaxies is provided by New-Hyperz using five arbitrary spectral types, according to their best fit with the simplest empirical templates in the local universe, namely (1) E/S0, (2) Sbc, (3) Scd, (4) Im and (5) Starbursts (see Sect. 6.1). We consider here as gen- uine “early-type” all galaxies with best-fit type (1), and all the others are considered as “late-type”. Following Pozzetti et al. (2010), we have determined the completeness in mass for the early and late-type galaxies respectively for a sample limited to Ks ≤24.75. This was done by computing the mass it would have an early/late type galaxy at the center of each redshift bin A51, page 9 of 16 A&A 620, A51 (2018) A&A 620, A51 (2018) Fig. 8. Distribution of stellar masses as a function of redshift for the catalog based on H + K detection image. Colors encode the different spectral types of galaxies, from (1) E/S0 to (5) Starbursts. Black squares and diamonds represent the completeness limits in mass up to Ks = 24.75, for early and late-type galaxies respectively. Error bars represent the dispersion within the sample for a typical Ks = 24.75 galaxy (see text for details). Fig. 8. Distribution of stellar masses as a function of redshift for the catalog based on H + K detection image. Colors encode the different spectral types of galaxies, from (1) E/S0 to (5) Starbursts. 6.2. Properties of the galaxy population Black squares and diamonds represent the completeness limits in mass up to Ks = 24.75, for early and late-type galaxies respectively. Error bars represent the dispersion within the sample for a typical Ks = 24.75 galaxy (see text for details). to log(M∗/M⊙) > 9.9 ± 0.60. Beyond this limit in redshift, stellar masses cannot be properly determined. if its apparent magnitude was Ks = 24.75. Figure 8 displays the distribution of stellar masses measured in the WUDS field as a function of redshift and galaxy types, together with the limiting mass corresponding to Ks = 24.75 for early and late-type galax- ies. Error bars in this figure represent the dispersion within the sample for a typical Ks = 24.75 galaxy. 7.1. Selection criteria Photometric redshifts and associated probability distributions have several advantages with respect to the three-band dropout technique (see e.g., McLure et al. 2009; Finkelstein et al. 2015). Although the later have proven to be successful in isolat- ing high-z galaxies, a significant fraction of the whole pop- ulation could have been excluded due to different reasons (e.g., older stellar populations, redder colors, ...). SED fit- ting analysis is particularly useful for objects lying close to the boundaries of the color–color selection boxes. The selec- tion criteria in this case are simply given by magnitude- selected catalogs (at least 5σ in the filter encompassing the rest-frame UV), and a detection below 2σ level in all filters bluewards with respect to 912 Å rest-frame. We have adopted the redshift probability distributions (hereafter P(z)) obtained when applying the procedure described in Sect. 6.1. Given the selection based on rest-frame UV, the final sample is still expected to be biased toward star-forming and low-reddening galaxies. 7.1. Selection criteria In all cases we compare the samples extracted from the different detection images and corresponding catalogs, namely i + z, C1 (J + Y) and C2 (H + Ks), and we restrict the selection area to the region covered by all filters involved in the detection with at least 50% of the total exposure time, excluding noisy areas (e.g., around bright stars). The following samples have been selected: ( ) d l A S/N hi h h 5 i d i i d Table 5. Number of sources included in the different redshift bins, for the different selection criteria and input catalogs. Table 5. Number of sources included in the different redshift bins, for the different selection criteria and input catalogs. Fig. 9. Color–color diagrams showing the position expected for spec- tral templates with redshifts z ∼0−7.0: E-type galaxies (CWW; red solid line), Scd (CWW; blue lines), Im (CWW; cyan lines), and star- burst templates of Kinney et al. (1996; magenta and green lines). Red stars show the expected colors of typical stars based on the Pickles library (Pickles 1998). Black lines delimit the selection windows for the riz (z ∼[4.5−5.3]; top panel), izY (z ∼[5.3−6.4]; mid panel), and zYJ (z ∼[6.3−7.2]; bottom panel) dropouts. To guide the eye, black dots indicate the redshifts from z = 4.5 to 5.5 (top panel), z = 5.0 to 6.5 (mid panel), and z = 6.0 to 7.2 (bottom panel), with ∆z = 0.1, for a typical star-forming galaxy. C1 C2 (i + z) (Y + J) (H + Ks) Criteria N N N r-dropout (raw) 2016 2591 2797 z ∼[4.5−5.3] CC window 863 1085 1205 CC window corrected 817 711 – i-dropout (raw) 166 134 i-dropout (corrected) 91 71 z ∼[5.3−6.4] CC window 98 66 CC window corrected 48 32 z-dropout (raw) 212 256 z-dropout (corrected) 142 132 z ∼[6.3−7.2] CC window 36 30 CC window corrected 14 11 (c) z−Y > 1.0 and z−Y > 4(Y−J)−1.0. This window is intended to select z-band dropouts in the z ∼[6.3−7.2] interval, as shown in the bottom panel of Fig. 9. The field of view in this case is limited to the WUDS region (see Table 1 and Fig. 4). The use of deep Y-band images is particularly useful in this redshift interval and it is rarely found in the literature excepted for HST data (see e.g., Bouwens et al. 2011, 2015). 7.2. Samples of galaxies at z ∼[4.5−7] Fig. 9. Color–color diagrams showing the position expected for spec- tral templates with redshifts z ∼0−7.0: E-type galaxies (CWW; red solid line), Scd (CWW; blue lines), Im (CWW; cyan lines), and star- burst templates of Kinney et al. (1996; magenta and green lines). Red stars show the expected colors of typical stars based on the Pickles library (Pickles 1998). Black lines delimit the selection windows for the riz (z ∼[4.5−5.3]; top panel), izY (z ∼[5.3−6.4]; mid panel), and zYJ (z ∼[6.3−7.2]; bottom panel) dropouts. To guide the eye, black dots indicate the redshifts from z = 4.5 to 5.5 (top panel), z = 5.0 to 6.5 (mid panel), and z = 6.0 to 7.2 (bottom panel), with ∆z = 0.1, for a typical star-forming galaxy. The results obtained when applying the selection criteria described in Sect. 7.1 are presented in this section. Table 5 sum- marizes these results for the different redshift bins and selection criteria. In all cases we compare the samples extracted from the different detection images and corresponding catalogs, namely i + z, C1 (J + Y) and C2 (H + Ks), and we restrict the selection area to the region covered by all filters involved in the detection with at least 50% of the total exposure time, excluding noisy areas (e.g., around bright stars). The following samples have been selected: (b) i −z > 0.7 and i −z > 1.7(z −Y) + 0.35, a window selecting i-dropout candidates in the z ∼[5.3−6.4] interval, as shown in the mid panel of Fig. 9. This selection window is anal- ogous to the one used in other studies previous (see e.g., Bouwens et al. 2007, 2015), but less strict than in the selec- tion conducted by Willott et al. (2013). (b) i −z > 0.7 and i −z > 1.7(z −Y) + 0.35, a window selecting i-dropout candidates in the z ∼[5.3−6.4] interval, as shown in the mid panel of Fig. 9. This selection window is anal- ogous to the one used in other studies previous (see e.g., Bouwens et al. 2007, 2015), but less strict than in the selec- tion conducted by Willott et al. (2013). ) r-dropout sample: A S/N higher than 5σ is requested in i and z, together with a non-detection at less than 2σ level in u∗and g, and r−i > 1.2. 7.1. Selection criteria 9: Although WUDS catalogs were built to fulfill the needs of our primary science goal around the z ≥4.5 population, they could also be advantageously used for studies at low and mid- z, in particular for the stellar-mass preselection of galaxies for spectroscopic studies. Up to z ∼3.5, early-type galaxies can be reliably identified and used for statistical purposes down to log(M∗/M⊙) > 10.5 ± 1.0 (completeness for a typical Ks = 24.75 galaxy), whereas we expect to detect late-type galaxies down z ∼[4.5−7] interval, as shown in Fig. 9: g (a) r−i > 1.2, i−z < 0.7 and r−i > 1.0(i−z)+1.0. This window selects r-dropout candidates in the z ∼[4.5−5.3] interval, as shown in the top panel of Fig. 9. This selection window is analogous to the one used in the literature to isolate RIz LBGs in the same redshift range from foreground interlopers and galactic stars (see e.g., Ouchi et al. 2004; Yoshida et al. 2006; Bouwens et al. 2015; Ono et al. 2017). A51, page 10 of 16 R. Pelló et al.: The WIRCam R. Pelló et al.: The WIRCam Ultra Deep Survey (WUDS) Fig. 9. Color–color diagrams showing the position expected for spec- tral templates with redshifts z ∼0−7.0: E-type galaxies (CWW; red solid line), Scd (CWW; blue lines), Im (CWW; cyan lines), and star- burst templates of Kinney et al. (1996; magenta and green lines). Red stars show the expected colors of typical stars based on the Pickles library (Pickles 1998). Black lines delimit the selection windows for the riz (z ∼[4.5−5.3]; top panel), izY (z ∼[5.3−6.4]; mid panel), and zYJ (z ∼[6.3−7.2]; bottom panel) dropouts. To guide the eye, black dots indicate the redshifts from z = 4.5 to 5.5 (top panel), z = 5.0 to 6.5 (mid panel), and z = 6.0 to 7.2 (bottom panel), with ∆z = 0.1, for a typical star-forming galaxy. (b) i 0 7 d i 1 7( ) 0 35 i d l i Table 5. Number of sources included in the different redshift bins, for the different selection criteria and input catalogs. 7.1. Selection criteria C1 C2 (i + z) (Y + J) (H + Ks) Criteria N N N r-dropout (raw) 2016 2591 2797 z ∼[4.5−5.3] CC window 863 1085 1205 CC window corrected 817 711 – i-dropout (raw) 166 134 i-dropout (corrected) 91 71 z ∼[5.3−6.4] CC window 98 66 CC window corrected 48 32 z-dropout (raw) 212 256 z-dropout (corrected) 142 132 z ∼[6.3−7.2] CC window 36 30 CC window corrected 14 11 (c) z−Y > 1.0 and z−Y > 4(Y−J)−1.0. This window is intended to select z-band dropouts in the z ∼[6.3−7.2] interval, as shown in the bottom panel of Fig. 9. The field of view in this case is limited to the WUDS region (see Table 1 and Fig. 4). The use of deep Y-band images is particularly useful in this redshift interval and it is rarely found in the literature excepted for HST data (see e.g., Bouwens et al. 2011, 2015). Photometric redshifts and associated probability distributions have several advantages with respect to the three-band dropout technique (see e.g., McLure et al. 2009; Finkelstein et al. 2015). Although the later have proven to be successful in isolat- ing high-z galaxies, a significant fraction of the whole pop- ulation could have been excluded due to different reasons (e.g., older stellar populations, redder colors, ...). SED fit- ting analysis is particularly useful for objects lying close to the boundaries of the color–color selection boxes. The selec- tion criteria in this case are simply given by magnitude- selected catalogs (at least 5σ in the filter encompassing the rest-frame UV), and a detection below 2σ level in all filters bluewards with respect to 912 Å rest-frame. We have adopted the redshift probability distributions (hereafter P(z)) obtained when applying the procedure described in Sect. 6.1. Given the selection based on rest-frame UV, the final sample is still expected to be biased toward star-forming and low-reddening galaxies. 7.2. Samples of galaxies at z ∼[4.5−7] The results obtained when applying the selection criteria described in Sect. 7.1 are presented in this section. Table 5 sum- marizes these results for the different redshift bins and selection criteria. 7.2. Samples of galaxies at z ∼[4.5−7] As in the previous case, only the WUDS region is used for this selection. When these criteria are blindly applied, 655(627) objects are selected in C1(C2). However, there is a difference between this sam- ple and the previous ones due to the depth in the z-band fil- ter, which is ∼0.5 mag shallower than the i-band, as shown in Table 1. A robust z-dropout selection based on z −Y is only achieved for objects with Y < 25.50. When introduc- ing this additional constraint, the final sample reduces to 212(256) objects in C1(C2) over ∼390 arcmin2, and among them only 36(30) objects are included within the color–color window for the selection of candidates at z ∼[6.3−7.2]. An average(median) value of zphot = 6.83(6.87) is found for this sample. Absolute magnitudes in the UV at ∼1500 Å (M1500) are derived from the photometric SED datapoints overlapping this wavelength for a given (photometric) redshift. We have checked that there is no significant difference in the LF results when using the closest filter-band, assuming a flat continuum, or the precise flux at rest-frame 1500 Å for the best-fit model instead. Number densities have been corrected for photometric incompleteness depending on the selection bands, according to Sect. 5.1. For samples selected in LBG windows, an additional multiplicative correction was applied to include the effect of color selection as a function of redshift and magnitude (S/N) in the detection filter encompassing the 1500 Å rest-frame. The shape of this later correction was obtained through sim- ulated catalogs, each one containing ∼4 × 105 objects, fully covering the redshift windows [4.5,5.5] and [5.5,6.5]. These cat- alogs are based on the same templates used to define the selec- tion windows, with magnitudes and corresponding photometric errors sorted to uniformly sample the actual range of magnitudes in the WUDS survey. We have then applied to these simulated catalogs the same color selection as for real data. The correction factor in a given redshift and magnitude bin is simply derived as the ratio between the number of galaxies in the input sample and number actually retrieved by the selection process. Also the redshift interval defined by r-dropout and i-dropout selections is actually narrower than the nominal ∆z = 1. In order to facilitate the comparison between the different samples, we have used a fixed ∆z = 1 in all cases assuming a uniform number density of sources. 7.2. Samples of galaxies at z ∼[4.5−7] We have also corrected for obvious spurious sources in the above catalogs by visual inspection carried out by two different observers. The percentage of spurious sources in the r-dropout sample is smaller than for the i- and z-dropouts, reaching only ∼5% for the i + z detection image. This trend was somewhat expected because a good S/N was requested in both i and z, that is at least two visible bands, together with a detection on the near- IR images for C1 and C2, making the selection of spurious signal highly unlikely. On the contrary, the contamination is expected to be much higher when the selection is essentially based on near-IR images, with extraction in double-image mode and a low detec- tion threshold, together with a poor detection or not-detection in the optical bands. The presence of spurious sources is indeed larger in this case, reaching between ∼30 and 60% of the sample, depending on the detection image and selection window. There- fore we have visually inspected and validated all objects used in the subsequent analysis. Corrected counts are reported in Table 5 together with the raw counts, to illustrate this effect. A detailed study of the z-dropout sample is presented in Paper II, in partic- ular the contamination affecting the z ∼7 sample. Therefore, in the following, number densities are given for redshift bins [4.5,5.5] and [5.5,6.5] respectively for convenience, and also to facilitate the comparison with other surveys. LF values have been computed using regularly spaced bins in luminosity of ∆M1500 = ±0.125 at z = 5 and ∆M1500 = ±0.250 at z = 6, excepted for the first (brightest) bins, arbitrarily set to ∆M1500 = 0.25 mag or 0.50 to improve statistics. Note that the number of sources used in the blind photometric redshift approach is larger than in the LBG color–color window (because less restrictive; see Table 5), allowing us to better sample the LF at z = 6. The fact that no source was detected at magnitudes brighter than M1500 ≤−22.4 and −21.9 respectively for r and i-dropouts has been used to determine upper limits for the bright end of the LF. 7.2. Samples of galaxies at z ∼[4.5−7] This blind selection provides ∼2600(2800) sources in C1(C2) respectively, over the ∼1200 arcmin2 field covered by near-IR data. Among them, ∼1100(1200) are included within the color–color window for the selection of r-dropout sample: A S/N higher than 5σ is requested in i and z, together with a non-detection at less than 2σ level in u∗and g, and r−i > 1.2. This blind selection provides ∼2600(2800) sources in C1(C2) respectively, over the ∼1200 arcmin2 field covered by near-IR data. Among them, ∼1100(1200) are included within the color–color window for the selection of A51, page 11 of 16 A51, page 11 of 16 A&A 620, A51 (2018) candidates in the z ∼[4.5−5.3] interval. When using the i +z detection image instead, ∼2000 sources are found over the same area, 863 of them within the color–color window. An average(median) value of zphot = 4.82(4.80) is found for this sample. Although the number of sources is smaller in the later case, the i + z-selected sample contains a smaller frac- tion of false positives, as discussed below. This i + z-selected sample will be used to derive the LF at z ∼5. r-dropout and i-dropout catalogs, corrected for spurious detections, without any additional color-selection, are used to compute the LF based on photometric redshifts probability dis- tributions (P(z)). On the other hand, we have used the (corrected) subsamples included within the corresponding LBG color–color selection windows. These two different approaches and samples are widely used in the literature. Number density values in luminosity bins have been com- puted using the 1/Vmax method (see e.g., Schmidt 1968). The bootstrap approach developed by Bolzonella et al. (2002) has been adopted to compute the LF data points, that is number density values and confidence intervals. This method is based on blind photometric redshifts and associated probability distri- butions P(z). For a given redshift interval, 1000 realizations of each catalog have been performed; for each realization of the catalog and for each object, a random value of the photomet- ric redshift was sorted out according to its P(z). This procedure takes into account by construction the existence of degenerate solutions in redshift for a given source. The number of realiza- tions of each catalog is large enough to ensure that the LF results do not depend on the number of realizations. 7.2. Samples of galaxies at z ∼[4.5−7] (b) (b) i-dropout sample: A S/N higher than 5σ is requested in z and Y, together with a non-detection at less than 2σ level in u∗, g and r, and i −z > 0.7. In this case the selection is only applied to the deep WUDS region covered by the Y band. These criteria blindly select 166(134) sources in C1(C2) over ∼390 arcmin2, and among them 98(66) objects included within the color–color window for the selection of candidates at z ∼[5.3−6.4]. An average(median) value of zphot = 5.84(5.89) is found for this sample. (b) i-dropout sample: A S/N higher than 5σ is requested in z and Y, together with a non-detection at less than 2σ level in u∗, g and r, and i −z > 0.7. In this case the selection is only applied to the deep WUDS region covered by the Y band. These criteria blindly select 166(134) sources in C1(C2) over ∼390 arcmin2, and among them 98(66) objects included within the color–color window for the selection of candidates at z ∼[5.3−6.4]. An average(median) value of zphot = 5.84(5.89) is found for this sample. (c) phot ( ) p (c) z-dropout sample: A S/N higher than 5σ is requested in Y and J, together with a non-detection at less than 2σ level in u∗, g, r and i, and z −Y > 1.0. As in the previous case, only the WUDS region is used for this selection. When these criteria are blindly applied, 655(627) objects are selected in C1(C2). However, there is a difference between this sam- ple and the previous ones due to the depth in the z-band fil- ter, which is ∼0.5 mag shallower than the i-band, as shown in Table 1. A robust z-dropout selection based on z −Y is only achieved for objects with Y < 25.50. When introduc- ing this additional constraint, the final sample reduces to 212(256) objects in C1(C2) over ∼390 arcmin2, and among them only 36(30) objects are included within the color–color window for the selection of candidates at z ∼[6.3−7.2]. An average(median) value of zphot = 6.83(6.87) is found for this sample. p p (c) z-dropout sample: A S/N higher than 5σ is requested in Y and J, together with a non-detection at less than 2σ level in u∗, g, r and i, and z −Y > 1.0. A51, page 12 of 16 7.3. Luminosity functions at z ∼5 and z ∼6 y i-drop photometric redshifts LBG color window M1500 Φ ∆Φ M1500 Φ ∆Φ (AB mag) (Mpc−3 mag−1) (Mpc−3 mag−1) (AB mag) (Mpc−3 mag−1) (Mpc−3 mag−1) −22.375 ± 0.50 <4.19 × 10−7 −22.375 ± 0.50 <4.19 × 10−7 −22.75 ± 0.25 2.25 × 10−6 2.41 × 10−6 −22.25 ± 0.50 9.07 × 10−6 4.0 × 10−6 −22.25 ± 0.25 8.56 × 10−6 5.08 × 10−6 −21.50 ± 0.25 3.51 × 10−5 1.24 × 10−5 −21.75 ± 0.25 1.80 × 10−5 0.82 × 10−5 −20.87 ± 0.25 8.72 × 10−5 2.62 × 10−5 −21.25 ± 0.25 1.15 × 10−4 0.30 × 10−4 −20.75 ± 0.25 1.82 × 10−4 0.47 × 10−4 Notes. ∆Φ includes the 68% (1σ) confidence level intervals from the bootstrap procedure, Poisson uncertainties and field-to-field variance. It should be noted that results obtained at z = 5 strongly depend on the detection image used for the r-dropout selection. In the following and for a sake of consistency, we use the cata- logs derived from detection images encompassing the rest-frame 1500 Å (see discussion below), that is i + z at z = 5 and C1 at z = 6. Tables 6 and 7 summarize the LF data points at z = 5 and z = 6 respectively, for the different detection catalogs. In all cases, error bars corresponding to 68% (1σ) confidence lev- els resulting from the bootstrap process are combined together with an estimate of Poisson uncertainties and field-to-field vari- ance derived from the public cosmic variance calculator by Trenti & Stiavelli (2008). These later contributions clearly dom- inate the error budget, excepted for the brightest M1500 ≤−22 luminosity bins. based on a simple least-squares χ2 minimization, assuming that the Schechter function provides a good representation of data. The fit has been obtained separately for data points derived through the classical LBG color-selection window and blind photometric redshifts to facilitate the comparison with previous findings. Our data being essentially sensitive to the normalization and M⋆, we have studied the influence of the slope α in two differ- ent ways: by adding data points from the literature toward the faintest luminosities, and by setting the slope of the LF. First, we completed our step-wise data points toward the faint edge using the LF estimates by Bouwens et al. (2015) at z = 5 and z = 6, based on deep HST imaging. 7.3. Luminosity functions at z ∼5 and z ∼6 In this section we derive the UV LF at 1500 Å in two different red- shift bins around z ∼5 and z ∼6, based on the near-IR-selected samples of WUDS reported in Table 5. The results obtained on the LF at z ≥7, as well as the evolution of the UV LF between z ∼4.5 and 9, are presented and discussed in Paper II. For each redshift bin, two different approaches and samples have been used to derive the LF. In one hand, the complete A51, page 12 of 16 R. Pelló et al.: The WIRCam Ultra Deep Survey (WUDS) Table 6. Luminosity function at z = 5 in the WUDS field. Table 6. Luminosity function at z = 5 in the WUDS field. r- drop photometric redshifts LBG color window M1500 Φ ∆Φ M1500 Φ ∆Φ (AB mag) (Mpc−3 mag−1) (Mpc−3 mag−1) (AB mag) (Mpc−3 mag−1) (Mpc−3 mag−1) −22.875±0.500 <1.23 × 10−7 −22.875 ± 0.500 <1.23 × 10−7 −22.125 ± 0.250 1.86 × 10−5 0.77 × 10−5 −22.125 ± 0.250 2.57 × 10−5 0.97 × 10−5 −21.625 ± 0.250 5.86 × 10−5 2.12 × 10−5 −21.625 ± 0.250 7.90 × 10−5 2.10 × 10−5 −21.250 ± 0.125 1.73 × 10−4 0.44 × 10−4 −21.250 ± 0.125 1.58 × 10−4 0.42 × 10−4 −21.000 ± 0.125 2.71 × 10−4 0.68 × 10−4 −21.000 ± 0.125 2.59 × 10−4 0.61 × 10−4 −20.750 ± 0.125 7.86 × 10−4 1.60 × 10−4 −20.750 ± 0.125 4.13 × 10−4 0.87 × 10−4 −20.500 ± 0.125 9.51 × 10−4 1.94 × 10−4 −20.500 ± 0.125 7.19 × 10−4 1.36 × 10−4 Notes. ∆Φ includes the 68% (1σ) confidence level intervals from the bootstrap procedure, Poisson uncertainties and field-to-field variance. Table 7. Luminosity function at z = 6 in the WUDS field. r- drop photometric redshifts 68% (1σ) confidence level intervals from the bootstrap procedure, Poisson uncertainties and field-to-field variance. Notes. ∆Φ includes the 68% (1σ) confidence level intervals from the bootstrap procedure, Poisson uncertainties and field-to-field variance. Table 7. Luminosity function at z = 6 in the WUDS field. Table 7. Luminosity function at z = 6 in the WUDS field. Φ(M)dM = Φ⋆ 1500 ln(10) 2.5  10−0.4(M−M∗)α+1 exp  −10−0.4(M−M∗) dM (1) 7.3. Luminosity functions at z ∼5 and z ∼6 A good-quality fit was obtained at z = 5, with stable and consistent results for both the LBG color- selection window and blind photometric redshifts, also consis- tent with the slope value derived by Bouwens et al. (2015). On the contrary, at z = 6 a good-quality fit could not be achieved for the enlarged data set based on the LBG color-selection win- dow, but only for the photometric redshift LF data (for which the sampling of the LF is better, as shown in Table 7). Therefore, in a second step, we imposed a constant slope for the LF following Bouwens et al. (2015; also based on LBG color window) while leaving the normalization and M⋆free. In this case, a good fit was also achieved at z = 6, yielding consistent results for both the LBG color-selection window and blind photometric redshifts. The effect of imposing a constant slope for the LF was studied by using also the steeper value from the Finkelstein (2016) review and from Livermore et al. (2017) in lensing fields, both studies being based on photometric redshifts. As seen in Table 8, our results for Φ⋆and L⋆are in good agreement with those previ- ously found by Finkelstein (2016) and Livermore et al. (2017) when imposing the same value of α (see below). Tables 6 and 7 present the LF points derived at z = 5 and z = 6 using the two approaches/samples to compute the LF, namely photometric redshifts applied to blindly selected dropout sam- ples, and the classical LBG color-preselected samples. Results are found to be consistent, in general at better than ∼1σ level, the largest deviations being observed for the faintest luminosities at z = 6. As discussed below, these results are also in agreement with previous findings at the same redshifts, as if photometric redshifts and associated probability distributions could be safely used for these purposes on the dropout samples, without intro- ducing more sophisticated and to a certain extent dangerous cuts in color space. This statement requires some additional discus- sion (see below). A subsequent maximum-likelihood fit of data points has been performed to the analytic Schechter function (Schechter 1976) as follows: Φ(M)dM = Φ⋆ 1500 ln(10) 2.5  10−0.4(M−M∗)α+1 exp  −10−0.4(M−M∗) dM (1) Φ(M)dM = Φ⋆ 1500 ln(10) 2.5  10−0.4(M−M∗)α+1 exp  −10−0.4(M−M∗) dM (1) A51, page 13 of 16 A&A 620, A51 (2018) Table 8. 7.3. Luminosity functions at z ∼5 and z ∼6 When quadratically com- bining these differences with the current error bars, as if they were the expression of a systematic error, we find that the fit results for the LF are very slightly modified (e.g., ∼0.01 for the slope when it is let free, ∼0.02 for M1500, and a negligible amount for the normalization, the error bars on these parameters increas- ing by only ∼10%). This means that there is indeed a systematic effect to take into account, but it is not strong enough here to modify the conclusions of this study. Our best fit to WUDS data-sets yields the results summa- rized in Table 8, namely Φ⋆ 1500 = (8.20+0.3 −0.5) × 10−4 Mpc−3, M⋆= −20.98 ± 0.03 mag, and α = −1.74 ± 0.02 for z = 5, and Φ⋆ 1500 = (7.5+2.5 −2.5) × 10−4 Mpc−3 and M⋆= −20.58 ± 0.022 mag with fixed α = −1.87 for z = 6, for the LBG color-selection win- dow. Figures 10 and 11 display the data points adopted for the LBG color-selection window for z ∼5 and z ∼6 respectively, together with the best-fit representation by a Schechter function as discussed above. Our results confirm a slight evolution in the UV LF between z = 5 and z = 6, consistent with a dimming of both Φ⋆and L⋆. Oddly, when comparing our results at z ∼6 with previ- ous findings in Fig. 11, at first glance they are found to be more consistent with Bouwens et al. (2015) for the blind pho- tometric redshift approach, whereas they are more consistent with Finkelstein et al. (2015) for the LBG color-selection win- dow, while the opposite trend would have been expected. In reality, as seen in Table 8, the results of the Schechter fit derived from both the color-selection window and the photo- metric redshift approaches are fully consistent within the error bars with Bouwens et al. (2015) when imposing the same slope α = −1.87, the larger differences being in the normalization. When imposing a steeper slope α ∼−2, as found by Finkelstein (2016) or Livermore et al. (2017) based on the photometric red- shift approach, then the results of the fit are fully consistent with their findings regarding Φ⋆and L⋆. 7.3. Luminosity functions at z ∼5 and z ∼6 Comparison between the LF parameters at z = 5 and z = 6 in the recent literature z = 5 References α M⋆ Φ⋆ 1500 (FUV AB mag) (10−4 Mpc−3) WUDS (LBG color window) −1.74 ± 0.02 −20.98 ± 0.03 8.20 +0.3 −0.5 WUDS (Photometric redshifts) −1.74 ± 0.04 −21.02 ± 0.08 8.00 +0.8 −0.6 McLure et al. (2009) −1.66 ± 0.06 −20.73 ± 0.11 9.4 ± 0.19 Finkelstein et al. (2015) −1.67 ± 0.06 −20.81+0.13 −0.12 8.95+1.92 −1.31 Bouwens et al. (2015) −1.76 ± 0.06 −21.17 ± 0.12 7.4+1.8 −1.4 Ono et al. (2018) −1.60 ± 0.06 −20.95 ± 0.06 10.7+1.3 −1.1 z = 6 WUDS (LBG color window) −1.87 (fixed) −20.58 ± 0.22 7.5+2.5 −2.5 WUDS (Photometric redshifts) −1.84 ± 0.09 −20.77 ± 0.20 6.5+2.0 −1.6 WUDS (Photometric redshifts) −1.87 (fixed) −20.83 ± 0.22 5.8+1.8 −1.4 WUDS (Photometric redshifts) −1.91 (fixed) −20.80 ± 0.30 5.7 +2.6 −2.1 WUDS (Photometric redshifts) −2.10 (fixed) −21.20 +0.10 −0.15 2.45+0.59 −0.40 McLure et al. (2009) −1.71 ± 0.11 −20.04 ± 0.12 1.80 ± 0.50 Su et al. (2011) −1.87 ± 0.14 −20.25 ± 0.23 1.77+0.62 −0.49 Finkelstein et al. (2015) −2.02 ± 0.10 −21.13+0.25 −0.31 1.86+0.94 −0.80 Bouwens et al. (2015) −1.87 ± 0.10 −20.94 ± 0.20 5.0+2.2 −1.6 Bowler et al. (2015) −1.88+0.15 −0.14 −20.77+0.18 −0.19 5.7+2.7 −2.0 Finkelstein (2016) −1.91+0.04 −0.03 −20.79+0.05 −0.04 4.26+0.52 −0.38 Livermore et al. (2017) −2.10 ± 0.03 −20.826+0.051 −0.040 2.254+0.20 −0.16 Ono et al. (2018) −1.86 ± 0.07 −20.90 ± 0.07 5.5+0.9 −0.9 Table 8. Comparison between the LF parameters at z = 5 and z = 6 in the recent literature for a review), meaning that the final errors on the LF points and fits could be systematically underestimated. In this study, the LF points derived at z ∼5 are fully consistent within the error bars between the two approaches, making the LF parameters particu- larly robust. At z ∼6, the difference between the two approaches is negligible for the brightest region, that is smaller than ∼30% up to M1500 ≤−21.5 (similar or smaller than the error bars), whereas at lower luminosities the photometric redshift approach yields ∆log Φ ∼0.3 higher in average. A51, page 14 of 16 7.3. Luminosity functions at z ∼5 and z ∼6 In other words, the results obtained on L⋆and the normalization seem to be more sen- sitive to the imposed value for the slope α rather than to the approach/method used to select the samples. 7.4. Discussion. Comparison with previous findings 7.4. Discussion. Comparison with previous findings 7.4. Discussion. Comparison with previous findings Pelló et al.: The WIRCam Ultra Deep Survey (WUDS) Fig. 10. Comparison of the WUDS luminosity function at z = 5 (based on i + z detection) with equivalent estimates by Finkelstein et al. (2015; black crosses, dot-dashed line), Bouwens et al. (2015; blue diamonds, dashed line), and Ono et al. (2018; gray stars, dotted line). The best Schechter fit to WUDS (LBG color selection) + extended data is plotted by a solid line. Fig. 10. Comparison of the WUDS luminosity function at z = 5 (based on i + z detection) with equivalent estimates by Finkelstein et al. (2015; black crosses, dot-dashed line), Bouwens et al. (2015; blue diamonds, dashed line), and Ono et al. (2018; gray stars, dotted line). The best Schechter fit to WUDS (LBG color selection) + extended data is plotted by a solid line. z = 6 with current findings in the literature, in complement to Figs. 10 and 11. As expected by construction, the best-fit values when the slope α is let free are fully consistent with Bouwens et al. (2015) because we used their data points to complete the LF toward the faintest luminosities. It should be mentioned however that, at z = 6, our faintest bins are in agreement with Bouwens et al. (2015) for the photometric redshift “blind” approach, whereas a better agreement is found with Finkelstein et al. (2015) when using the LBG color-window, as seen in Fig. 11, while the oppo- site trend would have been expected. As discussed in Sect. 7.3, given the error bars, the results obtained on Φ⋆and L⋆seem to be more sensitive to the value imposed for the slope α rather than to the method used to select the samples. Fig. 11. Comparison of the WUDS luminosity function at z = 6 with the equivalent estimates by Finkelstein et al. (2015; black crosses, dot- dashed line), Bouwens et al. (2015; blue diamonds, dashed line), and Ono et al. (2018; gray stars, dotted line) for the LBG color-selected sample (top panel) and photometric redshifts applied to the i-drop sam- ple (bottom panel). The best Schechter fits to WUDS + extended data are plotted by solid lines. For the LBG color-selected sample presented in this figure, the slope has been fixed as in Bouwens et al. (2015; see Sect. 7.3). 7.4. Discussion. Comparison with previous findings With the clarifications given above, our results are consistent with a small evolution of both M⋆and Φ⋆between z = 5 and z = 6. There is also a good agreement in general between our M⋆and Φ⋆ 1500 values and recently published results within the error bars. At z = 6 this is true provided that the same value for the slope α is imposed. between the Schechter parameters on one hand, and the differ- ences in the methods and samples used to build the LF on the other hand. p p At z = 5, the present combined determination of M⋆and Φ⋆is more accurate then previous findings due to a better statistics in the intermediate-luminosity regime. Regarding the value of the slope at this redshift, we confirm the value pro- posed by Bouwens et al. (2015), and we are marginally con- sistent with Finkelstein et al. (2015) and Ono et al. (2017). Our results lend support to higher Φ⋆ 1500 determinations than usu- ally reported at z = 6, but still consistent with the latest find- ings by Bouwens et al. (2015), Finkelstein (2016), and Ono et al. (2017). Of particular interest is the comparison with Willott et al. (2013) at z = 6 (Table 3 in their paper), because it was based on CFHTLS-Deep data partly covering the WIRDS area. Even though the selection criteria are not the identical (see Sect. 7.1), and their LF was obtained at 1350 instead of 1500 Å the global fit to our data as well as the number densities obtained in the present paper are consistent with their results. 7.4. Discussion. Comparison with previous findings 11, while the oppo- site trend would have been expected. As discussed in Sect. 7.3, given the error bars, the results obtained on Φ⋆and L⋆seem to be more sensitive to the value imposed for the slope α rather than to the method used to select the samples. With the clarifications given above, our results are consistent with a small evolution of both M⋆and Φ⋆between z = 5 and z = 6. There is also a good agreement in general between our M⋆and Φ⋆ 1500 values and recently published results within the error bars. At z = 6 this is true provided that the same value for the slope α is imposed. At z = 5, the present combined determination of M⋆and Φ⋆is more accurate then previous findings due to a better statistics in the intermediate-luminosity regime. Regarding the value of the slope at this redshift, we confirm the value pro- posed by Bouwens et al. (2015), and we are marginally con- Fig. 11. Comparison of the WUDS luminosity function at z = 6 with the equivalent estimates by Finkelstein et al. (2015; black crosses, dot- dashed line), Bouwens et al. (2015; blue diamonds, dashed line), and Ono et al. (2018; gray stars, dotted line) for the LBG color-selected sample (top panel) and photometric redshifts applied to the i-drop sam- ple (bottom panel). The best Schechter fits to WUDS + extended data are plotted by solid lines. For the LBG color-selected sample presented in this figure, the slope has been fixed as in Bouwens et al. (2015; see Sect. 7.3). between the Schechter parameters on one hand, and the differ- ences in the methods and samples used to build the LF on the other hand. 8 Conclusions and perspectives Ultra Deep Survey (WUDS) Fig. 11. Comparison of the WUDS luminosity function at z = 6 with the equivalent estimates by Finkelstein et al. (2015; black crosses, dot- dashed line), Bouwens et al. (2015; blue diamonds, dashed line), and Ono et al. (2018; gray stars, dotted line) for the LBG color-selected sample (top panel) and photometric redshifts applied to the i-drop sam- ple (bottom panel). The best Schechter fits to WUDS + extended data are plotted by solid lines. For the LBG color-selected sample presented in this figure, the slope has been fixed as in Bouwens et al. (2015; see Sect. 7.3). R. 7.4. Discussion. Comparison with previous findings The LF at z = 5 and z = 6 has been the subject of various studies during the last ten years (see e.g., Yoshida et al. 2006; Bouwens et al. 2007, 2012, 2015; McLure et al. 2009; Su et al. 2011; Willott et al. 2013; Finkelstein et al. 2015; Finkelstein 2016; Livermore et al. 2017; Ono et al. 2017). Although detailed results on the evolution of the LF will be presented in Paper II, the comparison between the present results and previous find- ings is important to assess the quality of the survey. WUDS cov- ers an interesting region in luminosity between the exponential and power-law-dominated regimes of the LF. WUDS has been designed to provide constraints on the brightest part of the LF at high-z, in particular on M⋆and Φ⋆, and it is much less sensi- tive to the value of α, as discussed in Sect. 7.3. We compare in Table 8 the LF parameters obtained in this survey at z = 5 and It is worth to mention that the difference between the LF points derived from the two samples/approaches defined within the same WUDS field provides an estimate of the systematic uncertainties related to the selection function, that are not neces- sarily taken into account in the literature (see Finkelstein 2016, R. Pelló et al.: The WIRCam Ultra Deep Survey (WUDS) R. Pelló et al.: The WIRCam Ultra Deep Survey (WUDS) Fig. 10. Comparison of the WUDS luminosity function at z = 5 (based on i + z detection) with equivalent estimates by Finkelstein et al. (2015; black crosses, dot-dashed line), Bouwens et al. (2015; blue diamonds, dashed line), and Ono et al. (2018; gray stars, dotted line). The best Schechter fit to WUDS (LBG color selection) + extended data is plotted by a solid line. z = 6 with current findings in the literature, in complement to Figs. 10 and 11. 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E., Papovich, C., et al. 2015, ApJ, 810, 71 Garzón, F., Abreu, D., Barrera, S., et al. 2006, Proc. SPIE, 6269, 18 Garzón, F., Abreu, D., Barrera, S., et al. 2007, in RM&AC, 29, 12 Garzón, F., Castro, N., Insausti, M., et al. 2016, in Proc. SPIE, 9908, 99081J Grogin, N. A., Kocevski, D. D., Faber, S. M., et al. 2011, ApJS, 197, 35 p bal-Hornillos, D., Balcells, M., Prieto, M., et al. 2003, ApJ, 595, 7 Cristóbal-Hornillos, D., Balcells, M., Prieto, M., et al. 2003, ApJ, 595, 71 Cristóbal-Hornillos, D., Aguerri, J. A. L., Moles, M., et al. 2009, ApJ, 696, 155 Davidzon, I., Bolzonella, M., Coupon, J., et al. 2013, A&A, 558, A23 Davis M Faber S M & Newman J 2003 Proc SPIE 4834 161 Cristóbal-Hornillos, D., Aguerri, J. A. L., Moles, M., et al. 2009, ApJ, 696, 1554 The selection and combined analysis of different galaxy sam- ples at z ≥7 will be presented in a forthcoming paper, as well as the evolution of the UV luminosity function between z ∼4.5 and 9 (Laporte et al. 2018; Paper II). 8. Conclusions and perspectives In this paper we have introduced and characterized the WIRCam Ultra Deep Survey (WUDS), a 4-band near-IR photometric sur- vey covering ∼400 arcmin2 on the CFHTLS-D3 field (Groth Strip). This public survey was specifically tailored to set strong constraints on the cosmic SFR and the UV luminosity function brighter or around L⋆in the z ∼6−10 domain. Regarding the properties of the data at low-z, and accord- ing to the estimates presented in this article, WUDS should allow the users to detect early-type galaxies with stellar masses down to log(M⋆/M⊙) > 10.5 ± 1.0, and late-type galaxies down to log(M⋆/M⊙) > 9.9 ± 0.60 up to z ∼3.5. The quality of the photometric redshifts achieved for the WUDS survey is compa- rable to the one obtained by other large surveys when using a similar number of filters and a similar depth. Given the error bars, our results are consistent with an evolu- tion in L⋆, as expected if the UV luminosity of galaxies follows the assembly of host dark matter halos (see e.g., Bouwens et al. 2008), without excluding an evolution on the global normaliza- tion, as reported for instance by Bouwens et al. (2015). A direct comparison is recognized to be difficult given the degeneracy As part of this effort, we have focused in this article on the selection of galaxy samples at z ∼[4.5−7] and the determination A51, page 15 of 16 A&A 620, A51 (2018) of the UV LF at z = 5 and z = 6, taking advantage from the deep optical data available from the CFHTLS-Deep Survey. We have also extended the research to an adjacent shallower area of ∼1000 arcmin2 extracted from the WIRDS Survey, and observed in three near-IR bands. Using two different approaches, the clas- sical LBG color-selection technique and photometric redshifts blindly applied to dropout samples, we have selected high-z galaxies and computed the UV LF. At z = 5, the combined deter- mination of M⋆and Φ⋆is more accurate then previous results due to a better statistics in the intermediate-luminosity regime. The evolution in the UV LF between z = 5 and z = 6 is consistent with a small dimming in both Φ⋆and L⋆. These results are con- sistent with previous findings in terms of the M⋆and Φ⋆values, knowing that WUDS covers a particularly interesting interval at intermediate luminosities. Bouwens, R. J., Illingworth, G. D., Franx, M., & Ford, H. 8. Conclusions and perspectives 2015, ApJ, 803, 26 Pelló, R., Schaerer, D., Richard, J., Le Borgne, J.-F., & Kneib, J.-P. 2004, A&A, 416, L35 Pforr, J., Maraston, C., & Tonini, C. 2013, MNRAS, 435, 1389 Pickles, A. 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Conclusions and perspectives This work has been carried out thanks to the support of the OCEVU Labex (ANR-11-LABX-0060) and the A*MIDEX project (ANR-11-IDEX-0001-02) funded by the “Investissements d’Avenir” French government program managed by the ANR. NL acknowledges finan- cial support from European Research Council Advanced Grant FP7/669253. JG and NC acknowledge financial support from Spanish MINECO (AYA2016- 75808-R). MB acknowledges support from AYA grant 2009-11139. This paper is based on observations obtained with WIRCam, a joint project of CFHT, Taiwan, Korea, Canada, France, and the Canada-France-Hawaii Telescope (CFHT) which is operated by the National Research Council (NRC) of Canada, the Institute National des Sciences de l’Univers of the Centre National de la Recherche Sci- entifique of France, and the University of Hawaii. Funding for the DEEP2 survey has been provided by NSF grants AST95-09298, AST-0071048, AST-0071198, AST-0507428, and AST-0507483 as well as NASA LTSA grant NNG04GC89G. Laporte, N., Pelló, R., Hudelot, P., et al. 2018, A&A, submitted (Paper II) Laureijs, R., Amiaux, J., & Arduini, S., et al. 2011, ArXiv e-prints [arXiv:1110.3193] Livermore, R. C., Finkelstein, S. L., & Lotz, J. M. 2017, ApJ, 835, 113 Lotz, J. M., Koekemoer, A., Coe, D., et al. 2017, ApJ, 837, 97 Marmo, C., & Bertin, E. 2008, in ASP Conf. Ser., 394, 619 McCracken, H. J., Milvang-Jensen, B., Dunlop, J., et al. 2012, A&A, 544, A156 McLure, R. J., Cirasuolo, M., Dunlop, J. S., Foucaud, S., & Almaini, O. 2009, MNRAS, 395, 2196 McLure, R. J., Dunlop, J. S., Bowler, R. A. A., et al. 2013, MNRAS, 432, 2696 Mitchell, P. D., Lacey, C. G., Baugh, C. M., & Cole, S. 2013, MNRAS, 435, 87 Mobasher, B., Capak, P., Scoville, N. Z., et al. 2007, ApJS, 172, 117 Oesch, P. A., Bouwens, R. J., Illingworth, G. 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This work recieved support from Agence Nationale de la recherche bearing the reference ANR-09-BLAN-0234, and from the ECOS SUD Program C16U02. This work has been carried out thanks to the support of the OCEVU Labex (ANR-11-LABX-0060) and the A*MIDEX project (ANR-11-IDEX-0001-02) funded by the “Investissements d’Avenir” French government program managed by the ANR. NL acknowledges finan- cial support from European Research Council Advanced Grant FP7/669253. JG and NC acknowledge financial support from Spanish MINECO (AYA2016- 75808-R). MB acknowledges support from AYA grant 2009-11139. This paper is based on observations obtained with WIRCam, a joint project of CFHT, Taiwan, Korea, Canada, France, and the Canada-France-Hawaii Telescope (CFHT) which is operated by the National Research Council (NRC) of Canada, the Institute National des Sciences de l’Univers of the Centre National de la Recherche Sci- entifique of France, and the University of Hawaii. 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GOYA is a scientific pro- gram to be developed mainly using the guaranteed time of the international consortium building EMIR, a wide-field, near-IR spectrograph currently installed in the Nasmyth focus of the Spanish 10.4 m GTC at Canary Islands11 (Garzón et al. 2006). The GOYA project addresses the formation and evolution of galaxies, in particular the structure, dynamics and integrated stellar populations of galaxies at high redshift (see e.g., Guzman 2003; Balcells 2003, 2007; Domínguez-Palmero et al. 2008). Ilbert, O., Arnouts, S., McCracken, H. J., et al. 2006a, A&A, 457, 841 Ilbert, O., Lauger, S., Tresse, L., et al. 2006b, A&A, 453, 809 Ilbert, O., Capak, P., Salvato, M., et al. 2009, ApJ, 690, 1236 Illingworth, G. D., Magee, D., Oesch, P. A., et al. 2013, ApJS, 209, 6 Kinney, A. L., Calzetti, D., Bohlin, R. 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Bisherige Theorien zur Erklärung islamistischer Gewalt
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4 Bisherige Theorien zur Erklärung islamistischer Gewalt Im Folgenden werden Theorien und Erklärungsmodelle vorgestellt, die den Anspruch erheben, die Begehung islamistischer Gewalttaten im Westen (Europa, Nordamerika und Australien/Neuseeland) erklären zu können. Der Fokus der Betrachtung soll sich dabei vor allem darauf richten, ob diese Erklärungsansätze die im vorherigen Kapitel formulierten Anforderungen erfüllen. 4.1 Allgemeine Theorien Drei allgemeine kriminologische Theorien wurden bislang auf den islamistischen Terrorismus im Westen angewendet: Situational Action Theory Einen vielversprechenden Versuch, terroristische Gewalt zu erklären, stellt die Situational Action Theory dar (vgl. Bouhana & Wikström 2011). Denn sie legt nicht nur dar, wie eine Bereitschaft zur Begehung terroristischer Gewalthandlungen entsteht, sondern nennt auch einige Bedingungen, wann diese in tatsächliches Verhalten umschlägt. Die Gewaltbereitschaft basiert dieser Theorie zufolge auf moral rules supportive of terrorism (vgl. Bouhana & Wikström 2011, S. 70; Wikström 2010, S. 1004). Diese formen einen moralischen Filter, durch den Terrorismus zu einer möglichen Verhaltensoption für die jeweilige Person wird (vgl. Bouhana & Wikström 2011, S. 35). Für die Entstehung einer solchen Haltung muss die Person einerseits wiederholt in Kontakt mit terrorism-supportive moral contexts kommen und andererseits vulnerabel für die dortigen Einflüsse sein (vgl. Bouhana & Wikström 2011, S. 70). Diese Beeinflussbarkeit kann auf einer kognitiven Vulnerabilität, also einem Defizit im Umgang mit Stress und belastenden Situationen, © Der/die Autor(en) 2023 M.-T. Lohmann, Islamistische Gewalttaten in Westeuropa, https://doi.org/10.1007/978-3-658-39285-7_4 27 28 4 Bisherige Theorien zur Erklärung islamistischer Gewalt und/oder einer moralischen Vulnerabilität, also einer schwachen Bindung an konventionelle moralische Regeln, fußen (vgl. Bouhana & Wikström 2011, S. 72). Hinsichtlich der Bedingungen für das Umschlagen der Gewaltbereitschaft in eine Handlung führt die Theorie zwei Auslöser an: temptations, also Gelegenheiten zur Erfüllung von Wünschen, Bedürfnissen oder Verpflichtungen, sowie provocations, also vom Täter ungewollte Beeinträchtigungen, die Aufregung oder Zorn erzeugen (vgl. Bouhana & Wikström 2011, S. 6 f.; Wikström 2015, S. 181). Danach folgt ein Entscheidungsprozess darüber, ob eine Tat begangen werden soll oder nicht, der durch eine Interaktion zwischen Person und sozialem Kontext bestimmt wird (vgl. Wikström 2010, S. 1005; Wikström & Treiber 2009, S. 91). In Fällen, wo die Moralvorstellungen der Person und des Kontexts übereinstimmen, kommt es, laut Theorie, wahrscheinlich zur Tatausführung. Bei einem Widerspruch hingegen, beispielsweise wenn ein Täter einer Terrorzelle am geplanten Tatvorhaben zweifelt, die anderen Gruppenmitglieder ihn aber zum Weitermachen bestärken, ist die Fähigkeit der jeweiligen Person zur Selbstkontrolle ausschlaggebend dafür, ob es zur Tat kommt (vgl. Wikström 2010, S. 1005 f.; Wikström & Treiber 2009, S. 92). Die Situational Action Theory benennt verschiedene Tatbedingungen (z. B. Auslöser und Entscheidungsprozesse). Allerdings befasst sie sich nur unzureichend mit jenen Faktoren, die verständlich machen, warum gerade diese ideologie-basierte Gewaltform, also die Begehung eines Terroranschlags im Westen, vom Täter gewählt wurde. Denn es gibt stets ein breites Spektrum an möglichen Handlungsoptionen. Zwar begünstigt die Disposition des moralischen Filters, dass terroristische Gewalt als Mittel gewählt wird, doch empirisch zeigt sich, dass die meisten gewaltbereiten Islamisten, die tatsächlich gewaltsam aktiv werden, keine Tat in Westeuropa verüben, sondern anstelle dessen in ein Kriegsgebiet (z. B. Afghanistan, Irak oder Syrien) ausreisen, um sich im Rahmen des Jihads zu engagieren (vgl. Hegghammer 2013). Es scheinen demnach noch weitere Einflüsse, außer der Disposition, relevant dafür zu sein, dass sich eine Person dazu entscheidet, die Begehung einer islamistischen Gewalttat in Westeuropa gegenüber anderen Mitteln vorzuziehen. General Strain Theory Gemäß der General Strain Theory sind kollektive Strains, die 1. ein hohes Ausmaß an Leid in der Zivilbevölkerung verursachen, die 2. als ungerecht bewertet werden und für die 3. mächtigere Fremdgruppen als verantwortlich wahrgenommen werden, grundlegend für die Entstehung von Terrorismus (vgl. Agnew 2010, S. 136). Beispiele für kollektive Strains mit diesen Merkmalen 4.1 Allgemeine Theorien 29 sind z. B. Gewaltviktimisierungen, Enteignungen/Vertreibungen oder systematische Diskriminierungen. Größte Herausforderung für diese Theorie ist es zu begründen, warum die meisten Personen, die von solchen Belastungen betroffen sind, nicht zum Terrorismus greifen. Die Theorie benennt aus diesem Grund mehrere Bedingungen, die begünstigen bzw. unwahrscheinlicher machen, dass solche Belastungen in terroristischer Gewalt gipfeln. Angeführt werden u. a. Bewältigungsstrategien, soziale Unterstützung, moralische Überzeugungen sowie Kosten-Nutzen-Bewertungen hinsichtlich terroristischer Gewalthandlungen (vgl. Agnew 2010, S. 143 ff.). Die General Strain Theory formuliert somit zwar verschiedene Faktoren, die das Auftreten von Terrorismus wahrscheinlicher machen, nennt aber keine Bedingungen, wann es zu einer konkreten terroristischen Gewalthandlung kommt. Transformative Learning Theory Der Transformative Learning Theory zufolge sind terroristische Gewalthandlungen das Resultat eines Radikalisierungsprozesses, der auf einer Transformation der eigenen Identität und eigenen Verhaltensbereitschaften basiert (vgl. Wilner & Dubouloz 2010, S. 34 f.). Der Radikalisierungsprozess wird dabei in drei aufeinanderfolgende Phasen unterteilt: trigger phase, changing phase und outcome phase (vgl. Wilner & Dubouloz 2010, S. 48). In der trigger phase kann der Radikalisierungsprozess durch drei verschiedene individuelle Ausgangslagen, die empfänglich für islamistische Ideologien machen, ausgelöst werden: 1) einer mangelnden gesellschaftlichen Integration, 2) einer Desorientierung in Folge der Globalisierung und 3) einer Unzufriedenheit mit der Außenpolitik des Westens (vgl. Wilner & Dubouloz 2010, S. 37 ff.). Während der changing phase kommt es unter dem Einfluss der Ideologie dann zu einer Rekonstruktion des eigenen Selbst und zu veränderten Einstellungen bzgl. verschiedener Verhaltensweisen (vgl. Wilner & Dubouloz 2010, S. 48). In der outcome phase kann es nun in Folge dieser Veränderungen zu einer terroristischen Gewalthandlung kommen (vgl. Wilner & Dubouloz 2010, S. 48). Defizit der Transformative Learning Theory ist, dass sie keine Bedingungen benennt, wann es tatsächlich zu einer solchen Gewalthandlung kommt. Dieses Manko trifft auf viele Theorien in diesem Bereich zu. Dies ist insbesondere deshalb fatal, da die allermeisten Personen, die einen Radikalisierungsprozess durchlaufen haben, nicht gewalttätig werden (vgl. Borum 2014, S. 298; Schuurman & Taylor 2018). Es bedarf daher offensichtlich zusätzlicher Bedingungen, damit es zu einer Gewalttat kommt. 30 4.2 4 Bisherige Theorien zur Erklärung islamistischer Gewalt Spezielle Theorien und Modelle Neben diesen allgemeinen Kriminalitätstheorien existieren zudem Theorien und Erklärungsmodelle, die speziell für den Bereich islamistischer Gewalt im Westen entwickelt wurden. Die folgenden Erklärungsansätze werden besonders häufig in der Literatur erwähnt (vgl. z. B. King and Taylor 2011; Köhler 2017; Logvinov 2014b; McCauley & Moskalenko 2017): Staircase-Metapher Die Staircase-Metapher von Fathali M. Moghaddam (2005) zeichnet den Weg hin zu einer terroristischen Gewalttat im Rahmen eines 6-stufigen Prozesses nach. Umso weiter eine Person die einzelnen Stufen hinaufsteigt, desto weniger Handlungsoptionen nimmt sie wahr, bis sie schließlich nur noch Gewalt als letzte Möglichkeit sieht (vgl. Moghaddam 2005, S. 161). Den potentiellen Nährboden für eine Radikalisierung bilden Gefühle von Benachteiligung (vgl. Moghaddam 2005, S. 162 f.). Allerdings fühlen sich viele Personen benachteiligt. Ausgangspunkt für eine Radikalisierung ist daher, ob sich die Absicht entwickelt, etwas gegen diese Schlechterstellung zu unternehmen (1. Stufe; vgl. Moghaddam 2005, S. 163). Auf der 2. Stufe wird dann in Folge der Beeinflussung von Mobilisierungsakteuren bestimmten Fremdgruppen die Schuld für die eigene Benachteiligung zugeschrieben (vgl. Moghaddam 2005, S. 164). Drittens entsteht die Überzeugung, dass der Einsatz von Gewalt zur Lösung der Probleme legitim ist (vgl. Moghaddam 2005, S. 165). Viertens kommt es zu einer Verfestigung des Wir-gegen-sie-Denkens (vgl. Moghaddam 2005, S. 165 f.). Auf der 5. Stufe begeht die Person schließlich eine terroristische Gewalttat (vgl. Moghaddam 2005, S. 166). Im Erklärungsansatz von Moghaddam (2005) werden beiläufig mehrere Bedingungen erwähnt, die dazu führen, dass es zu einer konkreten Gewalthandlung kommt. So wird etwa die tatauslösende Wirkung von extremistischen Organisationen genannt (vgl. Moghaddam 2005, S. 162) sowie auf tatbegünstigende Gruppenprozesse in Terrorzellen hingewiesen (vgl. Moghaddam 2005, S. 166). Eine systematische Behandlung dieser Tatbedingungen erfolgt allerdings nicht. Integrated Framework for the Analysis of Group Risk for Terrorism Eine wesentlich systematischere Thematisierung dieser Faktoren erfolgt in dem Modell von Jerrold M. Post et al. (2002), welches sich um die Risikobewertung, ob eine extremistische Gruppe möglicherweise zu Gewalt greifen wird, dreht (vgl. Post et al. 2002, S. 73 f.). Das Modell listet verschiedene gewaltbegünstigende sowie –hemmende Einflussfaktoren auf die Gruppe auf: 1) kulturelle und 4.2 Spezielle Theorien und Modelle 31 gesellschaftliche Faktoren (z. B. eine gesellschaftliche Tradition der Gewalt), 2) Einflüsse anderer Akteure (z. B. Angriffe durch die Regierung oder Hilfe von Unterstützungsgruppen), 3) Entwicklungen in der Gruppe selbst (z. B. veränderte Kosten-Nutzen-Bewertungen hinsichtlich Gewalt) und 4) die gegenwärtige Situation der Gruppe (vgl. Post et al. 2002, S. 77 ff.). Im Rahmen dieser vierten Faktorengruppe erwähnt das Modell mehrere Bedingungen für das Umschlagen in konkrete Gewalthandlungen. Es beschränkt sich dabei jedoch lediglich auf die Nennung verschiedener Gewaltauslöser, wie z. B. physische Angriffe auf Gruppe (vgl. Post et al. 2002, S. 97 f.). Solche Tatauslöser sind aber nicht die einzigen relevanten Bedingungsfaktoren für eine Gewalttat. Beispielsweise werden vermutlich viele gewaltbereite Islamisten nach der Veröffentlichung der Mohammed-Karikaturen in westlichen Medien den Entschluss gefasst haben, sich an den Verantwortlichen gewaltsam zu rächen, aber die Entscheidung nach einiger Zeit wieder verworfen haben. Einige wenige Personen haben ihren Tatentschluss aber nicht aufgegeben und sind bis zur Tatrealisierung vorangeschritten. Eine Theorie muss daher zusätzlich die Bedingungen benennen, die verantwortlich dafür sind, dass einige Personen ihren Tatentschluss auch tatsächlich realisieren und wieder andere nicht. Radikalisierungsmodell von Wiktorowicz Quintan Wiktorowicz (2005b) entwickelt sein Erklärungsmodell am Beispiel von Al-Muhajiroun, einer anfangs legalistisch agierenden Islamistenorganisation in Großbritannien mit einer ambivalenten Haltung zum gewaltsamen Jihad. Das Modell dreht sich um drei Fragen: 1) wie entsteht ein Interesse am Islamismus, 2) warum schließt sich eine Person ausgerechnet Al-Muhajiroun an und 3) wie kommt es zu einem eigenem Engagement der Person? Die Hinwendung zum Islamismus ist die Folge einer kognitiven Offenheit (cognitive opening) für die Ideologie (vgl. Wiktorowicz 2005b, S. 5). Grund hierfür können einerseits persönliche Krisen, wie z. B. Diskriminierungserfahrungen oder traumatische Ereignisse, sein (vgl. Wiktorowicz 2005b, S. 20). Andererseits können islamistische Organisationen eine kognitive Empfänglichkeit auch gezielt bei einer Person verursachen, etwa indem Geschichten über Gewalttaten gegen Muslim*innen präsentiert werden (vgl. Wiktorowicz 2005b, S. 20 f.). Diese unterschiedlichen Erfahrungen können eine Sinnsuche auslösen, die dazu führen kann, dass sich eine Person dem Islamismus zuwendet (vgl. Wiktorowicz 2005b, S. 5). Ausschlaggebend dafür, dass sich eine Person aus dem breiten Angebotsspektrum islamistischer Organisationen gerade Al-Muhajiroun anschließt, ist, dass die Organisation von ihr als der beste Weg wahrgenommen wird, um Probleme zu lösen (vgl. Wiktorowicz 2005b, S. 24 f.). Diese Einschätzung gründet auf der 32 4 Bisherige Theorien zur Erklärung islamistischer Gewalt wahrgenommenen Legitimität sowie der Reputation der Organisation und ihrer Anführer (vgl. Wiktorowicz 2005b, S. 6). Damit eine Person schließlich auch selbst aktiv wird und sich für Al-Muhajiroun engagiert, muss die Organisation die jeweilige Person davon überzeugen, dass ein eigenes Handeln notwendig ist, um religiöses Heil im Jenseits zu erlangen (vgl. Wiktorowicz 2005b, S. 6). Das Besondere an diesem Ansatz ist, dass er Bedingungen für eigenes Handeln thematisiert. Das Spektrum an Möglichkeiten, sich zu engagieren, ist allerdings breit und reicht von legalen und gewaltfreien bis hin zu gewaltsamen Optionen. Das Modell spezifiziert nicht, warum sich einige Personen ausgerechnet gewaltsam engagieren und wieder andere sich ausschließlich auf gewaltfreie Mittel beschränken. Radikalisierungsmodell des NYPD Das Radikalisierungsmodell des New York City Police Departments beschreibt den Radikalisierungsprozess durch vier aufeinanderfolgende Phasen (vgl. Silber & Bhatt 2007, S. 6). Die 1. Stufe, die Prä-Radikalisierungsphase, umfasst die Lebenssituation vor einer Radikalisierung (vgl. Silber & Bhatt 2007, S. 22). Es wird davon ausgegangen, dass bestimmte Lebenslagen die Anfälligkeit für eine islamistische Ideologie erhöhen. Verschiedene Arten von ökonomischen, sozialen, politischen oder persönlichen Krisen (z. B. ein Jobverlust, Diskriminierungserfahrungen oder der Verlust von Angehörigen) können eine Selbstidentifikation mit der Ideologie auslösen, die dann in einer Übernahme ihrer Inhalte mündet (2. Phase: Selbstidentifikationsphase) (vgl. Silber & Bhatt 2007, S. 6 f.). Im Zuge einer zunehmenden Radikalisierung kann die Person schließlich zu der Überzeugung gelangen, dass ein eigenes gewaltsames Engagement nötig ist (3. Phase: Indoktrinisierungsphase) (vgl. Silber & Bhatt 2007, S. 7). In der letztendlichen Jihadismus-Phase beginnt eine Person eine islamistische Gewalttat im Westen zu planen und vorzubereiten (vgl. Silber & Bhatt 2007, S. 7). Gemäß Modell, steigt nicht jede Person bis zur höchsten Stufe auf (vgl. Silber & Bhatt 2007, S. 6). Der Erklärungsansatz spezifiziert aber nicht, unter welchen Bedingungen eine Person zur nächsten Stufe aufsteigt, insbesondere nicht, wann eine Person beginnt, eine konkrete Gewalttat vorzubereiten. 4.3 Defizite bisheriger Theorien und Modelle Die vorherigen Abhandlungen sollten ersichtlich gemacht haben, dass ein Hauptversäumnis vieler bisheriger Theorien ist, dass sie sich primär auf Radikalisierungsprozesse beschränken und die Bedingungen, unter denen eine radikalisierte 4.3 Defizite bisheriger Theorien und Modelle 33 Person zum Gewaltäter wird, eher stiefmütterlich behandelt werden. So thematisieren einige der Theorien etwa spezifische Auslöser für Gewalt, führen ihre Beschreibungen dann aber nicht weiter aus (vgl. z. B. Moghaddam 2005; Post et al. 2002). Wie noch gezeigt werden wird, reichen solche Auslöser allein allerdings nicht dazu aus, Gewalt hervorzurufen. Sie führen nicht per se zu Gewalthandlungen, hierfür müssen noch weitere Faktoren hinzukommen, die allerdings von bisherigen Theorien unberücksichtigt bleiben. Im Zuge der Vorbereitung eines terroristischen Anschlags können z. B. Zweifel bzgl. der Tatbegehung in der Tatgruppe aufkommen, die in einem Abbruch des geplanten Gewaltvorhabens resultieren können. Keiner der Theorien spezifiziert, wie Gruppen mit solchen Zweifeln umgehen. In den wenigen Fällen, wo Theorien Tatbedingungen ausführlicher behandeln, zeigen sich aber ebenfalls Defizite. Beispielsweise können sie nicht verständlich machen, warum gerade dieses Mittel aus einer breiten Palette von möglichen Optionen gewählt wurde. D. h., sie berücksichtigen nicht ausreichend, dass es stets alternative Handlungsmöglichkeiten gibt, es also auch anders hätte verlaufen können. In Anbetracht dieser Vielzahl von Unzulänglichkeiten erscheint es ratsam, ein neues Erklärungsmodell zu entwickeln, die diesen Anforderungen gerecht werden kann. Dies soll in den folgenden Kapiteln erfolgen. Open Access Dieses Kapitel wird unter der Creative Commons Namensnennung 4.0 International Lizenz (http://creativecommons.org/licenses/by/4.0/deed.de) veröffentlicht, welche die Nutzung, Vervielfältigung, Bearbeitung, Verbreitung und Wiedergabe in jeglichem Medium und Format erlaubt, sofern Sie den/die ursprünglichen Autor(en) und die Quelle ordnungsgemäß nennen, einen Link zur Creative Commons Lizenz beifügen und angeben, ob Änderungen vorgenommen wurden. Die in diesem Kapitel enthaltenen Bilder und sonstiges Drittmaterial unterliegen ebenfalls der genannten Creative Commons Lizenz, sofern sich aus der Abbildungslegende nichts anderes ergibt. Sofern das betreffende Material nicht unter der genannten Creative Commons Lizenz steht und die betreffende Handlung nicht nach gesetzlichen Vorschriften erlaubt ist, ist für die oben aufgeführten Weiterverwendungen des Materials die Einwilligung des jeweiligen Rechteinhabers einzuholen.
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Test sensitivity is secondary to frequency and turnaround time for COVID-19 screening
Science advances
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†daniel.larremore@colorado.edu ‡mmina@hsph.harvard.edu §roy.parker@colorado.edu . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint Test sensitivity is secondary to frequency and turnaround time for COVID-19 surveillance Daniel B. Larremore†1,2, Bryan Wilder3, Evan Lester6,5, Soraya Shehata4,5, James M. Burke6, James A. Hay7,8, Milind Tambe3, Michael J. Mina‡7,8,9,*, and Roy Parker§4,6,10,2,* 1Department of Computer Science, University of Colorado Boulder 2BioFrontiers Institute, University of Colorado at Boulder 3Center for Research on Computation & Society, Harvard John A Paulson School of Engineering and Applied Sciences, Harvard University 1Department of Computer Science, University of Colorado Boulder 2BioFrontiers Institute, University of Colorado at Boulder 3Center for Research on Computation & Society, Harvard John A Paulson School of Engineering and Applied Sciences, Harvard University 4Department of Molecular, Cellular and Developmental Biology, University of Colorado 5Medical Scientist Training Program, University of Colorado Anschutz Medical Campus 6Department of Biochemistry, University of Colorado Boulder 7 Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health 9Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School 10Howard Hughes Medical Institute *These authors contributed equally. 8Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health 9Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School 10Howard Hughes Medical Institute *These authors contributed equally. Introduction Successful surveillance testing of SARS-CoV-2 depends on understanding both the dynamics of spread between individuals and the dynamics of the virus within the human body. Critically, the ability of SARS- CoV-2 to spread from individuals who are pre-symptomatic, symptomatic, or essentially asymptomatic [1, 2, 3] means that diagnosis and isolation based on symptoms alone will be unable to prevent ongoing spread [4, 5] A th f ill t ti t id tif i f ti i di id l t ibl Successful surveillance testing of SARS-CoV-2 depends on understanding both the dynamics of spread between individuals and the dynamics of the virus within the human body. Critically, the ability of SARS- CoV-2 to spread from individuals who are pre-symptomatic, symptomatic, or essentially asymptomatic [1, 2, 3] means that diagnosis and isolation based on symptoms alone will be unable to prevent ongoing spread [4, 5]. As a consequence, the use of surveillance testing to identify infectious individuals presents one possible means to break enough transmission chains to suppress the ongoing pandemic and reopen societies, with or without a vaccine. Successful surveillance testing of SARS-CoV-2 depends on understanding both the dynamics of spread between individuals and the dynamics of the virus within the human body. Critically, the ability of SARS- 5]. As a consequence, the use of surveillance testing to identify infectious individuals presents one possible means to break enough transmission chains to suppress the ongoing pandemic and reopen societies, with or without a vaccine. The reliance on testing as a means to safely reopen societies has placed a microscope on the analytical sensitivity of virus assays, with a gold-standard of quantitative real-time polymerase chain reaction (qPCR). These assays have analytical limits of detection that are usually within around 103 viral RNA copies per ml (cp/ml) [6]. However, qPCR remains expensive and as a laboratory based assay often have sample-to-result times of 24-48 hours. New developments in SARS-CoV-2 diagnostics have the potential to reduce cost significantly, allowing for expanded testing or greater frequency of testing and can reduce turnaround time to minutes [7, 8, 9]. These assays however largely do not meet the gold standard for analytical sensitivity, which has encumbered the widespread use of these assays [10]. Abstract The COVID-19 pandemic has created a public health crisis. Because SARS-CoV-2 can spread from individuals with pre-symptomatic, symptomatic, and asymptomatic infections, the re-opening of soci- eties and the control of virus spread will be facilitated by robust surveillance, for which virus testing will often be central. After infection, individuals undergo a period of incubation during which viral titers are usually too low to detect, followed by an exponential viral growth, leading to a peak viral load and infectiousness, and ending with declining viral levels and clearance. Given the pattern of viral load kinet- ics, we model surveillance effectiveness considering test sensitivities, frequency, and sample-to-answer reporting time. These results demonstrate that effective surveillance depends largely on frequency of testing and the speed of reporting, and is only marginally improved by high test sensitivity. We there- fore conclude that surveillance should prioritize accessibility, frequency, and sample-to-answer time; analytical limits of detection should be secondary. 1 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint Introduction Three features of the viral increase, infectivity, and decline during SARS-CoV-2 infection led us to hypoth- esize that there might be minimal differences in effective surveillance using viral detection tests of different sensitivities, such as RT-qPCR with a limit of detection (LOD) at 103 cp/ml [6] compared to often cheaper or faster assays with higher limits of detection (i.e., around 105 cp/ml [7, 8, 9]) such as point-of-care nucleic acid LAMP and rapid antigen tests (Figure 1A). First, since filtered samples collected from patients display- ing less than 106 N or E RNA cp/ml contain minimal or no measurable infectious virus [11, 12, 13], either class of test should detect individuals who are currently infectious. The absence of infectious particles at viral RNA concentrations < 106 cp/ml is likely due to (i) the fact that the N and E RNAs are also present in abundant subgenomic mRNAs, leading to overestimation of the number of actual viral genomes by ∼100- 1000X [14], (ii) technical artifacts of RT-PCR at Ct values > 35 due to limited template [15, 16], and (iii) the production of non-infectious viral particles as is commonly seen with a variety of RNA viruses [17]. Second, during the exponential growth of the virus, the time difference between 103 and 105 cp/ml is short, allowing only a limited window in which only the more sensitive test could diagnose individuals. For qPCR, this corresponds to the time required during viral growth to go from Ct values of 40 to ∼34. While this time window for SARS-CoV-2 is not yet rigorously defined [18], for other respiratory viruses such as influenza, and in ferret models of SARS-CoV-2 transmission, it is on the order of a day [19, 20]. Finally, high-sensitivity screening tests, when applied during the viral decline accompanying recovery, are unlikely to substantially impact transmission because such individuals detected have low, if any, infectiousness [14]. Indeed, a recent review by Cevik et al [18] notes that no study to date has successfully cultured live virus more than 9 days after the onset of symptoms. Impact of surveillance on individuals To examine how surveillance testing would reduce the average infectiousness of individuals, we first mod- eled the viral loads and infectiousness curves of 10,000 simulated individuals using the predicted viral trajectories of SARS-CoV-2 infections based on key features of latency, growth, peak, and decline identified 2 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint 0 2 4 6 8 10 12 14 16 18 20 days since exposure 2 3 4 5 6 7 8 9 10 11 log10 viral load LOD 103 LOD 105 A viral load positive test & dx returned no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% infectiousness removed C self-isolation LOD 103 LOD 105 0 2 4 6 8 10 12 14 16 18 20 days since exposure 0.00 0.25 0.50 0.75 1.00 rel. infectiousness 65% infectiousness isolated & removed B infectious infectious but isolated no test daily 3 days weekly 14 days 0 0.2 0.4 0.6 0.8 1.0 indiv. infectiousness arbitrary units D missed / caught late dx via test, LOD 103 dx via test, LOD 105 Figure 1: Surveillance testing effectiveness depends on frequency. (A) An example viral load trajectory is shown with LOD thresholds of two tests, and a hypothetical positive test on day 6, two days after peak viral load. 20 other stochastically generated viral loads are shown to highlight trajectory diversity (light grey; see Methods). (B) Relative infectiousness for the viral load shown in panel A pre-test, totaling 35% (blue) and post-isolation, totaling 65% (black). (C) Surveillance programs using tests at LODs of 103 and 105 at frequencies indicated were applied to 10, 000 individuals’ trajectories of whom 35% would undergo symptomatic isolation near their peak viral load if they had not been tested and isolated first. Impact of surveillance on individuals infectiousness arbitrary units D missed / caught late dx via test, LOD 103 dx via test, LOD 105 days since exposure 0 2 4 6 8 10 12 14 16 18 20 days since exposure 0.00 0.25 0.50 0.75 1.00 rel. infectiousness 65% infectiousness isolated & removed B infectious infectious but isolated B Figure 1: Surveillance testing effectiveness depends on frequency. (A) An example viral load trajectory is shown with LOD thresholds of two tests, and a hypothetical positive test on day 6, two days after peak viral load. 20 other stochastically generated viral loads are shown to highlight trajectory diversity (light grey; see Methods). (B) Relative infectiousness for the viral load shown in panel A pre-test, totaling 35% (blue) and post-isolation, totaling 65% (black). (C) Surveillance programs using tests at LODs of 103 and 105 at frequencies indicated were applied to 10, 000 individuals’ trajectories of whom 35% would undergo symptomatic isolation near their peak viral load if they had not been tested and isolated first. Total infectiousness removed during surveillance (colors) and self isolation (hatch) are shown for surveillance as indicated, relative to total infectiousness with no surveillance or self-isolation. (D) The impact of surveillance on the infectiousness of 100 individuals is shown for each surveillance program and no testing, as indicated, with each individual colored by test if their infection was detected during infectiousness (medians, black lines) or colored blue if their infection was missed by surveillance or detected positive after their infectious period (medians, blue lines). Units are arbitrary and scaled to the maximum infectiousness of sampled individuals. Figure 1: Surveillance testing effectiveness depends on frequency. (A) An example viral load tra in the literature (Figure 1A; see Methods). Accounting for these within-host viral kinetics, we calculated what percentage of their total infectiousness would be removed by surveillance and isolation (Figure 1B) with tests at LOD of 103 and 105, and at different frequencies. Here, infectiousness was taken to be pro- portional to the logarithm of viral load in excess of 106 cp/ml (with alternative assumptions addressed in sensitivity analyses; see Supplemental Materials), consistent with the observation that pre-symptomatic pa- tients are most infectious just prior to the onset of symptoms [21], and evidence that the efficiency of viral transmission coincides with peak viral loads, which was also identified during the related 2003 SARS out- break [22, 23]. Impact of surveillance on individuals Total infectiousness removed during surveillance (colors) and self isolation (hatch) are shown for surveillance as indicated, relative to total infectiousness with no surveillance or self-isolation. (D) The impact of surveillance on the infectiousness of 100 individuals is shown for each surveillance program and no testing, as indicated, with each individual colored by test if their infection was detected during infectiousness (medians, black lines) or colored blue if their infection was missed by surveillance or detected positive after their infectious period (medians, blue lines). Units are arbitrary and scaled to the maximum infectiousness of sampled individuals. 0 2 4 6 8 10 12 14 16 18 20 days since exposure 2 3 4 5 6 7 8 9 10 11 log10 viral load LOD 103 LOD 105 A viral load positive test & dx returned infectiousness removed C no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% infectiousness removed C self-isolation LOD 103 LOD 105 D infectiousness removed C A 0 2 4 6 8 10 12 14 16 18 20 days since exposure 2 3 4 5 6 7 8 9 10 11 log10 viral load LOD 103 LOD 105 A viral load positive test & dx returned no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% infectiousness removed C self-isolation LOD 103 LOD 105 0 2 4 6 8 10 12 14 16 18 20 days since exposure 0.00 0.25 0.50 0.75 1.00 rel. infectiousness 65% infectiousness isolated & removed B infectious infectious but isolated no test daily 3 days weekly 14 days 0 0.2 0.4 0.6 0.8 1.0 indiv. infectiousness arbitrary units D missed / caught late dx via test, LOD 103 dx via test, LOD 105 0 2 4 6 8 10 12 14 16 18 20 days since exposure 2 3 LOD 10 no test daily 3 days weekly 14 days 0% inf 0 2 4 6 8 10 12 14 16 18 20 days since exposure 0.00 0.25 0.50 0.75 1.00 rel. infectiousness 65% infectiousness isolated & removed B infectious infectious but isolated no test daily 3 days weekly 14 days 0 0.2 0.4 0.6 0.8 1.0 indiv. Impact of surveillance on individuals We considered that 35% of patients would undergo symptomatic isolation within three days of their peak viral load if they had not been tested and isolated first, and 65% would have sufficiently mild or no symptoms such that they would not isolate unless they were detected by surveillance testing. Based on recent results, we modeled asymptomatic and symptomatic infections as having the same initial viral loads [1, 24, 25, 26], but with faster clearance among asymptomatics [24, 26, 27, 28, 29] (see Methods). This analysis demonstrated that there was little difference in averting infectiousness between the two classes of test. Dramatic reductions in total infectiousness of the individuals were observed by testing daily or every third day, ∼65% reduction when testing weekly, and < 50% under biweekly testing (Figure 1C). Because viral loads and infectiousness vary across individuals, we also analyzed the impact of different surveillance regimens on the distribution of individuals’ infectiousness, revealing that more sporadic testing leads to an in the literature (Figure 1A; see Methods). Accounting for these within-host viral kinetics, we calculated what percentage of their total infectiousness would be removed by surveillance and isolation (Figure 1B) with tests at LOD of 103 and 105, and at different frequencies. Here, infectiousness was taken to be pro- portional to the logarithm of viral load in excess of 106 cp/ml (with alternative assumptions addressed in sensitivity analyses; see Supplemental Materials), consistent with the observation that pre-symptomatic pa- tients are most infectious just prior to the onset of symptoms [21], and evidence that the efficiency of viral transmission coincides with peak viral loads, which was also identified during the related 2003 SARS out- break [22, 23]. We considered that 35% of patients would undergo symptomatic isolation within three days of their peak viral load if they had not been tested and isolated first, and 65% would have sufficiently mild or no symptoms such that they would not isolate unless they were detected by surveillance testing. Based on recent results, we modeled asymptomatic and symptomatic infections as having the same initial viral loads [1, 24, 25, 26], but with faster clearance among asymptomatics [24, 26, 27, 28, 29] (see Methods). This analysis demonstrated that there was little difference in averting infectiousness between the two classes of test. Impact of surveillance on individuals daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R fully mixed model A LOD 103 LOD 105 no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model C daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R agent based model B no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections agent based model D daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R fully mixed model A LOD 103 LOD 105 B B Figure 2: Surveillance testing affects disease dynamics. Both the fully-mixed compartmental model (top row) and agent based model (bottom row) are affected by surveillance programs. (A, B) More frequent testing reduces the effective reproductive number R, shown as the percentage by which R0 is reduced, 100 × (R0 −R)/R0. Values of R were estimated from 50 independent simulations of dynamics (see Methods). (C, D) Relative to no testing (grey bars), surveillance suppresses the total number of infections in both models when testing every day or every three days, but only partially mitigates total cases for weekly or bi-weekly testing. Error bars indicate inner 95% quantiles of 50 independent simulations each. fully-mixed compartmental model (top row) and increased likelihood that individuals will test positive after they are no longer infectious or be missed by testing entirely (Figure 1D). Impact of surveillance on individuals Dramatic reductions in total infectiousness of the individuals were observed by testing daily or every third day, ∼65% reduction when testing weekly, and < 50% under biweekly testing (Figure 1C). Because viral loads and infectiousness vary across individuals, we also analyzed the impact of different surveillance regimens on the distribution of individuals’ infectiousness, revealing that more sporadic testing leads to an 3 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R fully mixed model A LOD 103 LOD 105 no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model C daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R agent based model B no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections agent based model D Figure 2: Surveillance testing affects disease dynamics. Both the fully-mixed compartmental model (top row) and agent based model (bottom row) are affected by surveillance programs. (A, B) More frequent testing reduces the effective reproductive number R, shown as the percentage by which R0 is reduced, 100 × (R0 −R)/R0. Values of R were estimated from 50 independent simulations of dynamics (see Methods). (C, D) Relative to no testing (grey bars), surveillance suppresses the total number of infections in both models when testing every day or every three days, but only partially mitigates total cases for weekly or bi-weekly testing. Error bars indicate inner 95% quantiles of 50 independent simulations each. Impact of surveillance on a population (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint relative to no surveillance (Figures 2C and D). Testing frequency was found to be the primary driver of population-level epidemic control, with only a small margin of improvement provided by using a more sensitive test. Direct examination of simulations showed that with no surveillance or biweekly testing, infections were uncontrolled, whereas surveillance testing weekly with either LOD = 103 or 105 effectively attenuated surges of infections (examples shown in Figure S1). relative to no surveillance (Figures 2C and D). Testing frequency was found to be the primary driver of population-level epidemic control, with only a small margin of improvement provided by using a more sensitive test. Direct examination of simulations showed that with no surveillance or biweekly testing, infections were uncontrolled, whereas surveillance testing weekly with either LOD = 103 or 105 effectively attenuated surges of infections (examples shown in Figure S1). The relationship between test sensitivity and the frequency of testing required to control outbreaks in both the fully mixed model and the agent-based model generalize beyond the examples shown in Figure 2 and are also seen at other testing frequencies, sensitivities, and asymptomatic fractions. We simulated both models at LODs of 103, 105, and 106, and for testing ranging from daily to every 14 days. For those, we measured each surveillance policy’s impact on total infections (Figure S2A and B) and on R (Figure S2C and D). In Figure 2, we modeled infectiousness as proportional to log10 of viral load. To address whether these finding are sensitive to this modeled relationship, we performed similar simulations with infectiousness proportional to viral load (Figure S3), or uniform above 106/ml (Figure S4). We found that results were robust to these large variations in the modeled relationship between infectiousness and viral load. To further address whether our results depended on the exact 35% fraction of individuals assumed to be behaviorally symptomatic, we performed sensitivity analyses with fewer (20%) or more (50%) symptomatic individuals and found no meaningful difference in results (Figure S5). Impact of delayed test results An important variable in surveillance testing is the time between a test’s sample collection and the reporting of a diagnosis. To examine how time to reporting affected epidemic control, we re-analyzed both the re- duction in individuals’ infectiousness, as well as the epidemiological simulations, comparing the results of instantaneous reporting (reflecting a rapid point-of-care assay), one day delay, and two day delay (Figure 3A and B). Delays in reporting dramatically decreased the reduction in infectiousness in individuals as seen by the total infectiousness removed (Figure 3C), the distribution of infectiousness in individuals (Figure 3D), or the dynamics of the epidemiological models (Figure 4). This result was robust to the modeled relation- ship between infectiousness and viral load in both simulation models and for various test sensitivities and frequencies (Figure S6). These results highlight that delays in reporting lead to dramatically less effective control of viral spread and emphasize that fast reporting of results is critical in any surveillance testing. These results also reinforce the relatively smaller benefits of improved limits of detection. Impact of surveillance on a population Above, we assumed that each infection was independent. To investigate the effects of surveillance testing strategies at the population level, we used simulations to monitor whether epidemics were contained or became uncontrolled, while varying the frequencies at which the test was administered, ranging from daily testing to testing every 14 days, and considering tests with LOD of 103 and 105, analogous to RT-qPCR and RT-LAMP / rapid antigen tests, respectively. We integrated individual viral load trajectories into two different epidemiological models to ensure that important observations were independent of the specific modeling approach. The first model is a simple fully mixed model representing a population of 20,000, similar to a large university setting, with a constant rate of external infection approximately equal to one new import per day. The second model is a previously described agent-based model with both within- household and age-stratified contact structure based on census microdata in a city representative of New York City [30], which we initialized with 100 cases without additional external infections. Individual viral loads were simulated for each infection, and individuals who received a positive test result were isolated, but contact tracing and monitoring was not included to more conservatively estimate the impacts of surveillance alone [31, 32]. Model details and parameters are fully described in Methods. We observed that a surveillance program administering either test with high frequency limited viral spread, measured by both a reduction in the reproductive number R (Figures 2A and B; see Methods for calculation procedure) and by the total infections that persisted in spite of different surveillance programs, expressed 4 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Generality of findings to changes in modeling assumptions Communities vary in their transmission dynamics, due to difference in rates of imported infections and in the basic reproductive number R0, both of which will influence the frequency and sensitivity with which surveillance testing must occur. We performed two analyses to illustrate this point. First, we varied the rate of external infection in our fully mixed model, and confirmed that when the external rate of infection is higher, more frequent surveillance is required to prevent outbreaks (Figure S7A). Second, we varied the reproductive number R0 between infected individuals in both models, and confirmed that at higher R0, more frequent surveillance is also required (Figure S7B and C). This may be relevant to institutions like college campuses or military bases wherein frequent classroom setting or dormitory living are likely to increase contact rates. Thus, the specific strategy for successful surveillance will depend on the current community infection prevalence and transmission rate. of our findings to different epidemiological parameters (Figure S7), relationships between The generality of our findings to different epidemiological parameters (Figure S7), relatio 5 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint 0 2 4 6 8 10 12 14 16 18 20 days since exposure 2 3 4 5 6 7 8 9 10 11 log10 viral load L=103 L=105 A viral load test sample positive dx no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% infectiousness removed 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 C self-isolation LOD 103 LOD 105 0 2 4 6 8 10 12 14 16 18 20 days since exposure 0.00 0.25 0.50 0.75 1.00 rel. Generality of findings to changes in modeling assumptions (D) The impact of surveillance with delays in returning diagnosis of 0, 1, or 2 days (small text beneath axis) on the infectiousness of 100 individuals is shown for each surveillance program and no testing, as indicated, with each individual colored by test if their infection was detected during infectiousness (medians, black lines) or colored blue if their infection was missed by surveillance or diagnosed positive after their infectious period (medians, blue lines). Units are arbitrary and scaled to the maximum infectiousness of sampled individuals. Figure 3: Effectiveness of surveillance testing is compromised by delays in reporting. (A) An example viral load trajectory is shown with LOD thresholds of two tests, and a hypothetical positive test on day 6, but with results reported on day 8. 20 other stochastically generated viral loads are shown to highlight trajectory diversity (light grey; see Methods). (B) Relative infectiousness for the viral load shown in panel A pre-test (totaling 35%; blue) and post- test but pre-diagnosis (totaling 34%; green), and post-isolation (totaling 31%; black). (C) Surveillance programs using tests at LODs of 103 and 105 at frequencies indicated, and with results returned after 0, 1, or 2 days (indicated by small text beneath bars) were applied to 10, 000 individuals trajectories of whom 35% were symptomatic and self-isolated after peak viral load if they had not been tested and isolated first. Total infectiousness removed during surveillance (colors) and self isolation (hatch) are shown, relative to total infectiousness with no surveillance or self-isolation. Delays substantially impact the fraction of infectiousness removed. (D) The impact of surveillance with delays in returning diagnosis of 0, 1, or 2 days (small text beneath axis) on the infectiousness of 100 individuals is shown for each surveillance program and no testing, as indicated, with each individual colored by test if their infection was detected during infectiousness (medians, black lines) or colored blue if their infection was missed by surveillance or diagnosed positive after their infectious period (medians, blue lines). Units are arbitrary and scaled to the maximum infectiousness of sampled individuals. viral load and infectiousness (Figures S3 and S4), and proportion of symptomatic individuals (Figure S5) led us to ask whether a more general mathematical formula could predict R without requiring epidemiological simulation. Generality of findings to changes in modeling assumptions We derived such a formula (Supplemental Text S1) and found that its predicted values of R were nearly perfectly correlated with simulation-estimated values (Pearson’s r = 0.998, p < 10−6; Figure S8), providing a mathematical alternative to simulation-based sensitivity analyses. viral load and infectiousness (Figures S3 and S4), and proportion of symptomatic individuals (Figure S5) led us to ask whether a more general mathematical formula could predict R without requiring epidemiological simulation. We derived such a formula (Supplemental Text S1) and found that its predicted values of R were nearly perfectly correlated with simulation-estimated values (Pearson’s r = 0.998, p < 10−6; Figure S8), providing a mathematical alternative to simulation-based sensitivity analyses. Generality of findings to changes in modeling assumptions 0 2 4 6 8 10 12 14 16 18 20 days since exposure 2 3 4 5 6 7 8 9 10 11 log10 viral load L=103 L=105 A viral load test sample positive dx i f ti d C no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% infectiousness removed 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 C self-isolation LOD 103 LOD 105 D 1 infectiousness removed C 1 d C A days since exposure 0 2 4 6 8 10 12 14 16 18 20 days since exposure 0.00 0.25 0.50 0.75 1.00 rel. infectiousness 31% infectiousness isolated & removed 34% infectiousness awaiting dx B infectious infectious (awaiting dx) infectious but isolated B D no test daily 3 days weekly 14 days 0 0.2 0.4 0.6 0.8 1.0 indiv. infectiousness arbitrary units 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 D missed / caught late diagnosed, LOD 103 diagnosed, LOD 105 Figure 3: Effectiveness of surveillance testing is compromised by delays in reporting. (A) A Figure 3: Effectiveness of surveillance testing is compromised by delays in reporting. (A) An example viral load trajectory is shown with LOD thresholds of two tests, and a hypothetical positive test on day 6, but with results reported on day 8. 20 other stochastically generated viral loads are shown to highlight trajectory diversity (light grey; see Methods). (B) Relative infectiousness for the viral load shown in panel A pre-test (totaling 35%; blue) and post- test but pre-diagnosis (totaling 34%; green), and post-isolation (totaling 31%; black). (C) Surveillance programs using tests at LODs of 103 and 105 at frequencies indicated, and with results returned after 0, 1, or 2 days (indicated by small text beneath bars) were applied to 10, 000 individuals trajectories of whom 35% were symptomatic and self-isolated after peak viral load if they had not been tested and isolated first. Total infectiousness removed during surveillance (colors) and self isolation (hatch) are shown, relative to total infectiousness with no surveillance or self-isolation. Delays substantially impact the fraction of infectiousness removed. Generality of findings to changes in modeling assumptions infectiousness 31% infectiousness isolated & removed 34% infectiousness awaiting dx B infectious infectious (awaiting dx) infectious but isolated no test daily 3 days weekly 14 days 0 0.2 0.4 0.6 0.8 1.0 indiv. infectiousness arbitrary units 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 D missed / caught late diagnosed, LOD 103 diagnosed, LOD 105 Figure 3: Effectiveness of surveillance testing is compromised by delays in reporting. (A) An example viral load trajectory is shown with LOD thresholds of two tests, and a hypothetical positive test on day 6, but with results reported on day 8. 20 other stochastically generated viral loads are shown to highlight trajectory diversity (light grey; see Methods). (B) Relative infectiousness for the viral load shown in panel A pre-test (totaling 35%; blue) and post- test but pre-diagnosis (totaling 34%; green), and post-isolation (totaling 31%; black). (C) Surveillance programs using tests at LODs of 103 and 105 at frequencies indicated, and with results returned after 0, 1, or 2 days (indicated by small text beneath bars) were applied to 10, 000 individuals trajectories of whom 35% were symptomatic and self-isolated after peak viral load if they had not been tested and isolated first. Total infectiousness removed during surveillance (colors) and self isolation (hatch) are shown, relative to total infectiousness with no surveillance or self-isolation. Delays substantially impact the fraction of infectiousness removed. (D) The impact of surveillance with delays in returning diagnosis of 0, 1, or 2 days (small text beneath axis) on the infectiousness of 100 individuals is shown for each surveillance program and no testing, as indicated, with each individual colored by test if their infection was detected during infectiousness (medians, black lines) or colored blue if their infection was missed by surveillance or diagnosed positive after their infectious period (medians, blue lines). Units are arbitrary and scaled to the maximum infectiousness of sampled individuals. Surveillance testing to mitigate an ongoing epidemic The impact of surveillance testing on transmission dynamics led us to hypothesize that surveillance testing could be used as an active tool to mitigate an ongoing epidemic. To test this idea, we simulated an outbreak situation using both the fully-mixed and agent-based models but with three additional conditions. First, we assumed that in an ongoing pandemic, other mitigating interventions would cause the reproductive number to be lower, though nevertheless larger than one. Second, we considered that not all individuals would want to or be able to participate in a SARS-CoV-2 surveillance program. Third, we assumed that the collection 6 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 A LOD 103 LOD 105 no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 C daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 B dx delay none 1 day 2 days no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 D Figure 4: Delays in reporting decrease the epidemiological impact of surveillance-driven isolation. Surveillance testing to mitigate an ongoing epidemic The eff Figure 4: Delays in reporting decrease the epidemiological impact of surveillance-driven isolation. The effective- ness of surveillance programs are dramatically diminished by delays in reporting in both the fully-mixed compartmen- tal model (top row) and agent based model (bottom row). (A, B) The impact of surveillance every day, 3 days, weekly, or biweekly, on the reproductive number R, calculated as 100 × (R0 −R)/R0, is shown for LODs 103 and 105 and delays of 0, 1, or 2 days (small text below axis). Values of R were estimated from 50 independent simulations of dy- namics (see Methods). (C, D) Relative to no testing (grey bars), surveillance suppresses the total number of infections in both models when testing every day or every three days, but delayed results lead to only partial mitigation of total cases, even for testing every day or 3 days. Error bars indicate inner 95% quantiles of 50 independent simulations each. Figure 4: Delays in reporting decrease the epidemiological impact of surveillance-driven isolation. The effective- ness of surveillance programs are dramatically diminished by delays in reporting in both the fully-mixed compartmen- tal model (top row) and agent based model (bottom row). (A, B) The impact of surveillance every day, 3 days, weekly, or biweekly, on the reproductive number R, calculated as 100 × (R0 −R)/R0, is shown for LODs 103 and 105 and delays of 0, 1, or 2 days (small text below axis). Values of R were estimated from 50 independent simulations of dy- namics (see Methods). (C, D) Relative to no testing (grey bars), surveillance suppresses the total number of infections in both models when testing every day or every three days, but delayed results lead to only partial mitigation of total cases, even for testing every day or 3 days. Error bars indicate inner 95% quantiles of 50 independent simulations each. of samples for testing, if performed on a large scale, could result in imperfect sample collection, causing an increase in the false negative rate, independent of an assay’s analytical sensitivity. These modifications are fully described in Methods. We simulated epidemics in which surveillance testing began only at the point when uncontrolled infections reached 4% prevalence. Surveillance testing to mitigate an ongoing epidemic no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 C D daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 A LOD 103 LOD 105 daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 A LOD 103 LOD 105 no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 C daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 B dx delay none 1 day 2 days no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 D Fi 4 D l i i d h id i l i l i f ill d i i l i Th ff i B aily 3 days weekly 14 days no test daily 3 days weekly 14 days aily 3 days weekly 14 days 0 0 0 0 0 0 0 1 1 1 1 1 1 1 2 2 2 2 2 2 2 dx delay none 1 day 2 days no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 D Delays in reporting decrease the epidemiological impact of surveillance-driven isolation. Surveillance testing to mitigate an ongoing epidemic The effective- ness of surveillance programs are dramatically diminished by delays in reporting in both the fully-mixed compartmen- tal model (top row) and agent based model (bottom row). (A, B) The impact of surveillance every day, 3 days, weekly, or biweekly, on the reproductive number R, calculated as 100 × (R0 −R)/R0, is shown for LODs 103 and 105 and delays of 0, 1, or 2 days (small text below axis). Values of R were estimated from 50 independent simulations of dy- namics (see Methods). (C, D) Relative to no testing (grey bars), surveillance suppresses the total number of infections in both models when testing every day or every three days, but delayed results lead to only partial mitigation of total cases, even for testing every day or 3 days. Error bars indicate inner 95% quantiles of 50 independent simulations each. Surveillance testing to mitigate an ongoing epidemic Based on results from our previous analyses, we considered a less sensitive but rapid test with LOD 105 cp/ml and a zero-day delay in results, and further assumed that 10% of would-be positive samples would be negative due to improper sample collection. We then examined scenarios of testing every 3 days and every 7 days, with either 50% or 75% of individuals participating, starting from a partially mitigated R0 = 1.5. We found that surveillance testing of 75% of individuals every 3 days was sufficient to drive the epidemic toward extinction within 6 weeks and reduce cumulative incidence by 88%, and that other combinations also had successful but less rapid mitigating impacts, particularly when compared with no intervention (Figure 5). Notably, even weekly testing with 50% participation was able to reduce the peak and length of the outbreak, illustrating how even partial surveillance testing using a test with 100X lower molecular sensitivity than PCR can have public health benefits when used frequently (Figure 5). Repeating these simulations using a test with LOD 106 led to similar results (Figure S9). To further generalize these results, we modified our mathematical formula to predict the impacts of per-individual test refusal and per- test sampling-related sensitivity on the reproductive number R (see Supplemental Text S1). 7 7 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint 0 2 4 6 8 weeks after surveillance begins 0% 2% 4% 6% 8% 10% 12% prevalence fully mixed model 12% 38% 27% 58% 100% A 0 2 4 6 8 weeks after surveillance begins 0% 2% 4% 6% prevalence agent based model 13% 37% 28% 54% 100% B no intervention testing regimen begins 50% participation, weekly 50% participation, every 3 days 75% participation, weekly 75% participation, every 3 days Figure 5: Surveillance testing suppresses an ongoing epidemic. Surveillance testing to mitigate an ongoing epidemic Scenarios show the impact of a test with LOD 105, no delay in results, and with 10% of samples assumed to be incorrectly collected (and therefore negative) to reflect decreased sensitivity incurred at sample collection in a mass testing scenario. Annotations show total number of post-intervention infections, as a percentage of the no-intervention scenario, labeled as 100%. See Fig. S9 for identical simulations using a test with LOD 106. Figure 5: Surveillance testing suppresses an ongoing epidemic. Widespread testing and isolation of infected in- dividuals drives prevalence downward for both (A) the fully-mixed compartmental model and (B) the agent based model. Time-series of prevalence, measured as the total number of infectious individuals, are shown for no inter- vention (solid) and surveillance testing scenarios (various dashed; see legend). Surveillance testing began only when prevalence reached 4% (box), and time series are shifted such that testing begins at t = 0. Scenarios show the impact of a test with LOD 105, no delay in results, and with 10% of samples assumed to be incorrectly collected (and therefore negative) to reflect decreased sensitivity incurred at sample collection in a mass testing scenario. Annotations show total number of post-intervention infections, as a percentage of the no-intervention scenario, labeled as 100%. See Fig. S9 for identical simulations using a test with LOD 106. Surveillance testing to mitigate an ongoing epidemic Widespread testing and isolation of infected in- dividuals drives prevalence downward for both (A) the fully-mixed compartmental model and (B) the agent based model. Time-series of prevalence, measured as the total number of infectious individuals, are shown for no inter- vention (solid) and surveillance testing scenarios (various dashed; see legend). Surveillance testing began only when prevalence reached 4% (box), and time series are shifted such that testing begins at t = 0. Scenarios show the impact of a test with LOD 105, no delay in results, and with 10% of samples assumed to be incorrectly collected (and therefore negative) to reflect decreased sensitivity incurred at sample collection in a mass testing scenario. Annotations show total number of post-intervention infections, as a percentage of the no-intervention scenario, labeled as 100%. See Fig. S9 for identical simulations using a test with LOD 106. 0 2 4 6 8 weeks after surveillance begins 0% 2% 4% 6% 8% 10% 12% prevalence fully mixed model 12% 38% 27% 58% 100% A 0 2 4 6 8 weeks after surveillance begins 0% 2% 4% 6% prevalence agent based model 13% 37% 28% 54% 100% B no intervention testing regimen begins 50% participation, weekly 50% participation, every 3 days 75% participation, weekly 75% participation, every 3 days 0 2 4 6 8 weeks after surveillance begins 0% 2% 4% 6% 8% 10% 12% prevalence fully mixed model 12% 38% 27% 58% 100% A % 0 2 4 6 8 weeks after surveillance begins 0% 2% 4% 6% prevalence agent based model 13% 37% 28% 54% 100% B no intervention testing regimen begins 50% participation, weekly 50% participation, every 3 days 75% participation, weekly 75% participation, every 3 days Figure 5: Surveillance testing suppresses an ongoing epidemic. Widespread testing and isol Figure 5: Surveillance testing suppresses an ongoing epidemic. Widespread testing and isolation of infected in- dividuals drives prevalence downward for both (A) the fully-mixed compartmental model and (B) the agent based model. Time-series of prevalence, measured as the total number of infectious individuals, are shown for no inter- vention (solid) and surveillance testing scenarios (various dashed; see legend). Surveillance testing began only when prevalence reached 4% (box), and time series are shifted such that testing begins at t = 0. Viral Loads Viral loads were drawn from a simple viral kinetics model intended to capture (1) a variable latent period, (2) a rapid growth phase from the lower limit of PCR detectability to a peak viral load, (3) a slower decay phase, and (4) prolonged clearance for symptomatic infections vs asymptomatic infections. These dynamics were based on the following observations. Latent periods prior to symptoms have been estimated to be around 5 days [40]. Latent periods prior to detection via virological tests at secondary sites of replication or shedding have been estimated to be up to 4 days [41], corresponding to a latent or eclipse phase observed with other viruses [42]. Viral load appears to peak prior to symptom onset [21], and peaks within 2 days of challenge in a macaque model [43, 44], though it should be noted that macaque challenge doses were high. Viral load decreases monotonically from the time of symptom onset [21, 45, 46, 47, 48], but may be high and detectable 3 or more days before symptom onset [1, 49]. Peak viral loads are difficult to measure due to lack of prospective sampling studies of individuals prior to exposure and infection, but viral loads have been reported in the range of O(104) to O(109) copies per ml [12, 47, 48]. Viral loads appear to become undetectable by PCR within 3 weeks of symptom onset [45, 48, 50], but detectability and timing may differ depending on the degree or presence of symptoms [50, 51]. The majority of studies reviewed by Cevik et al [18] found initial viral loads to be similar between symptomatic and asymptomatic infections [1, 24, 25, 26], but viral clearance was significantly and substantially faster among asymptomatic infections [24, 26, 27, 28, 29]. Finally, we note that the general understanding of viral kinetics may vary depending on the mode of sampling, as demonstrated via a comparison between sputum and swab samples [12]. For a comprehensive review of viral load dynamics, duration of shedding, and infectiousness, see Ref. [18]. To mimic growth and decay, log10 viral loads were specified by a continuous piecewise linear “hinge” func- tion, specified uniquely with three control points: (t0, 3), (tpeak, Vpeak),(tf, 6) (Figure 6A; green squares). The first point represents the time at which an individual’s viral load first crosses 103, with t0 ∼unif[2.5, 3.5], measured in days since exposure. The second point represents the peak viral load. Discussion (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint The difference between clinical and surveillance testing highlights the need for additional tests to be ap- proved and utilized for surveillance. Such tests should not be held to the same degree of sensitivity as clinical tests, in particular if doing so encumbers rapid deployment of faster cheaper SARS-CoV-2 assays. We suggest that the FDA, other agencies, or state governments, encourage the development and use of al- ternative faster and lower cost tests for surveillance purposes, even if they have poorer limits of detection. If the availability of point-of-care or self-administered surveillance tests leads to faster turnaround time or more frequent testing, our results suggest that they would have high epidemiological value. Our modeling suggests that some types of surveillance will subject some individuals to unnecessary quaran- tine days. For instance, the infrequent use of a sensitive test will not only identify (i) those with a low viral load in the beginning of the infection, who must be isolated to limit viral spread, but (ii) those in the recovery period, who still have detectable virus or RNA but are below the infectious threshold [13, 14]. Isolating this second group of patients will have no impact on viral spread but will incur costs of isolation, as would the isolation of individuals who received a false positive test result due to imperfect test specificity. The use of serology, repeat testing 24 or 48 hours apart, or some other test, to distinguish low viral load patients on the upslope of infection from those in the recovery phase could allow for more effective quarantine decisions. Discussion Our results lead us to conclude that surveillance testing of asymptomatic individuals can be used to limit the spread of SARS-CoV-2. However, our findings are subject to a number of limitations. First, the sensi- tivity of a test may depend on factors beyond LOD, including manufacturer variation and improper clinical sampling [33], though the latter may be ameliorated by different approaches to sample collection, such as saliva-based testing [34]. Second, the exact performance differences between testing schemes will depend on whether our model truly captures viral kinetics and infectiousness profiles [21], particularly during the accel- eration phase between exposure and peak viral load. Continued clarification of these within-host dynamics would increase the impact and value of this, and other [31, 32, 35, 36] modeling studies. Finally, we mod- eled participation in surveillance testing regimens (or refusal thereof) as statistically independent between individuals, but health-related behaviors have been shown to be socially [37] and geographically [38, 39] correlated. Clustered refusal of surveillance testing, or refusal to isolate upon testing positive, could present challenging barriers to implementation. Our findings show that the impact of surveillance testing can be expressed as a reduction of the reproductive number R. By mapping a given testing regimen to a reduction in R, the impact of testing regimen can be approximated and generalized without complicated simulations. For instance, one could estimate the max- imum allowable turnaround time delays, or the minimum testing frequency required to bring R below one, based on user-specified and scenario-specific assumptions. To facilitate such generalizations and scenario planning, open-source calculation tools accompany this manuscript. A critical point is that the requirements for surveillance testing are distinct from clinical testing. Clinical diagnoses target symptomatic individuals, need high accuracy and sensitivity, and are not limited by cost. Because they focus on symptomatic individuals, those individuals can isolate such that a diagnosis delay does not lead to additional infections. In contrast, results from the surveillance testing of asymptomatic individuals need to be returned quickly, since even a single day diagnosis delay compromises the surveil- lance program’s effectiveness. Indeed, at least for viruses with infection kinetics similar to SARS-CoV-2, we find that speed of reporting is much more important than sensitivity, although more sensitive tests are nevertheless somewhat more effective. 8 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Viral Loads Peak height was drawn 9 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. h was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: preprint CC-BY-NC-ND 4 0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. peer review) The copyright holder for this preprint this version posted September 8, 2020. . org/10.1101/2020.06.22.20136309 0 2 4 6 8 10 12 14 16 18 20 days since exposure, t 2 3 4 5 6 7 8 9 10 11 log10 viral load (copies/mL) A viral load viral load (discrete time) control points L) A 0 2 4 6 8 10 12 14 16 18 20 days since exposure, t 2 3 4 5 6 7 8 9 10 11 log10 viral load (copies/mL) A viral load viral load (discrete time) control points 0 2 4 6 8 10 12 14 16 18 20 days since exposure, t 2 3 4 5 6 7 8 9 10 11 log10 viral load (copies/mL) symptom onset prolonged clearance B symptomatic modifications Figure 6: Example asymptomatic and symptomatic viral loads with model control points. Examples of model viral loads (lines) and corresponding stochastically drawn control points (squares, circles) are shown for (A) an asymp- tomatic viral load trajectory and (B) a symptomatic viral load trajectory. Because simulations took place in discrete time, black dots show points at which this example viral load would have been sampled. Light grey lines show 20 alternative trajectories in each panel to illustrate the diversity of viral loads drawn from the simple model. Red circles indicate the control points which are modified in symptomatic trajectories to account for symptom onset and prolonged time till clearance. Viral Loads viral load viral load (discrete time) control points days since exposure, t 0 2 4 6 8 10 12 14 16 18 20 days since exposure, t 2 3 4 5 6 7 8 9 10 11 log10 viral load (copies/mL) symptom onset prolonged clearance B symptomatic modifications L) B Figure 6: Example asymptomatic and symptomatic viral loads with model control points. Figure 6: Example asymptomatic and symptomatic viral loads with model control points. Examples of model viral loads (lines) and corresponding stochastically drawn control points (squares, circles) are shown for (A) an asymp- tomatic viral load trajectory and (B) a symptomatic viral load trajectory. Because simulations took place in discrete time, black dots show points at which this example viral load would have been sampled. Light grey lines show 20 alternative trajectories in each panel to illustrate the diversity of viral loads drawn from the simple model. Red circles indicate the control points which are modified in symptomatic trajectories to account for symptom onset and prolonged time till clearance. Figure 6: Example asymptomatic and symptomatic viral loads with model control points. Examples of model viral loads (lines) and corresponding stochastically drawn control points (squares, circles) are shown for (A) an asymp- tomatic viral load trajectory and (B) a symptomatic viral load trajectory. Because simulations took place in discrete time, black dots show points at which this example viral load would have been sampled. Light grey lines show 20 alternative trajectories in each panel to illustrate the diversity of viral loads drawn from the simple model. Red circles indicate the control points which are modified in symptomatic trajectories to account for symptom onset and prolonged time till clearance. Vpeak ∼unif[7, 11], and peak timing was drawn with respect to the start of the exponential growth phase, tpeak −t0 ∼0.5 + gamma(1.5) with a maximum of 3. The third point represents the time at which an individual’s viral load crosses beneath the 106 threshold, at which point viral loads no longer cause active cultures in laboratory experiments [11, 12, 13, 18]. For asymptomatic infections, this point was drawn with respect to peak timing, tf −tpeak ∼unif[4, 9]. For symptomatic infections, a symptom onset time was first drawn with respect to peak timing, tsymptoms −tpeak ∼unif[0, 3], and then the third control point was drawn with respect to symptom onset, tf −tsymptoms ∼unif[4, 9]. Viral Loads (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint Infectiousness Infectiousness F was assumed to be directly related to viral load V in one of three ways. In the main text, each individual’s relative infectiousness was proportional log10 of viral load’s excess beyond 106, i.e. F ∝log10(V ) −6. In the supplementary sensitivity analyses, we investigated two opposing extremes. To capture a more extreme relationship between infectiousness and viral load, we considered F to be directly proportional to viral load’s excess above 106, i.e. F ∝10log10(V )−6 = V × 10−6, and to capture a more extreme relationship, but in the opposing direction, we considered F to simply be a constant when viral load exceeded 106, i.e. F ∝1V >106. We call these three functions log-proportional, proportional, and threshold throughout the text and supplemental materials. We note that a comprehensive review of viral loads, shedding, and infectiousness [18] found that across the surveyed literature, no virus was able to be cultured beyond 9 days post-symptoms. Thus, the choice of the final control point in our symptomatic viral load model (Figure 6B), which corresponds to the latest time at which an individual is infectious, is at most 9 days post-symptom onset. Recently, He et al [21] published an analysis of infectiousness relative to symptom onset which was cor- rected by Bonhoeffer et al (see [21] for details). Among our infectiousness functions, this inferred relation- ship bears the greatest similarity, over time, to the log-proportional infectiousness function, as visualized in Figs. 1 and 3. The proportional and threshold models therefore represent one of many types of sensitivity analysis. Results for those models can be found in Figures S3, S4, and S6. In all simulations, the value of the proportionality constant implied by the infectiousness functions above was chosen to achieve the targeted value of R0 for that simulation, and confirmed via simulation as described below. Viral Loads Thus, symptomatic trajectories are systemat- ically longer, in both duration of infectiousness (see below) and duration of viral shedding, reflecting the documented prolonged clearance and relationship with viral culture experiments (Figure 6B; red circles). In simulations, each viral load’s parameters were drawn independently of others, and the continuous function described here was evaluated at 28 integer time points (Figure 6; black dots) representing a four week span of viral load values. Vpeak ∼unif[7, 11], and peak timing was drawn with respect to the start of the exponential growth phase, tpeak −t0 ∼0.5 + gamma(1.5) with a maximum of 3. The third point represents the time at which an individual’s viral load crosses beneath the 106 threshold, at which point viral loads no longer cause active cultures in laboratory experiments [11, 12, 13, 18]. For asymptomatic infections, this point was drawn with respect to peak timing, tf −tpeak ∼unif[4, 9]. For symptomatic infections, a symptom onset time was first drawn with respect to peak timing, tsymptoms −tpeak ∼unif[0, 3], and then the third control point was drawn with respect to symptom onset, tf −tsymptoms ∼unif[4, 9]. Thus, symptomatic trajectories are systemat- ically longer, in both duration of infectiousness (see below) and duration of viral shedding, reflecting the documented prolonged clearance and relationship with viral culture experiments (Figure 6B; red circles). In simulations, each viral load’s parameters were drawn independently of others, and the continuous function described here was evaluated at 28 integer time points (Figure 6; black dots) representing a four week span of viral load values. 10 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Overview CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint (I →SQ), with f = 0.65 for both models, with f = 0.8 and f = 0.5 explored in sensitivity analyses (Figure S5). Presymptomatic individuals were isolated prior to symptom onset only if they received positive test results. When an individual’s viral load dropped below 103, that individual recovered (I, Q, SQ →R). Details follow. Testing, Isolation, and Sample-to-Answer Turnaround Times All individuals were tested every D days, so that they could be moved into isolation if their viral load exceeded the test’s limit of detection V (t) > L. Each person was deterministically tested exactly every D days , but testing days were drawn uniformly at random such that not all individuals were tested on the same day. To account for delays in returning test results, we included a sample-to-answer turnaround time T, meaning that an individual with a positive test on day t would isolate on day t + T. Transmission, Population Structure, and Mixing Patterns: Fully-mixed model Simulations were initialized with all individuals susceptible, S = N. Each individual was chosen to be symptomatic independently with probability f, and each individual’s first test day (e.g. the day of the week that their weekly test would occur) was chosen uniformly at random between 1 and D. Relative infectiousness was scaled up or down to achieve the specified R0 in the absence of any testing policy, but inclusive of any assumed self-isolation of symptomatics. In each timestep, those individuals who were marked for testing that day were tested, and a counter was initialized to T, specifying the number of days until that individual received their results. Next, individuals whose test results counters were zero were isolated, I →Q. Then, symptomatic individuals whose viral load had declined relative to the previous day were self-isolated, I →SQ. Next, each susceptible individual was spontaneously (externally) infected independently with probability 1/N, S →I. Then, all infected individuals contacted all susceptible individuals, with the probability of transmission based on that day’s viral load V (t) for each person and the particular infectiousness function, described above, S →I. To conclude each time step, individuals’ viral loads and test results counters were advanced, with those whose infectious period had completely passed moved to recovery, I, Q, SQ →R. Overview Two models were used to simulate SARS-CoV-2 dynamics, both based on a typical compartmental frame- work. The first model was a fully-mixed model of N = 20, 000 individuals with all-to-all contact structure, zero initial infections, and a constant 1/N per-person probability of becoming infected from an external source. This model could represent, for instance, a large college campus with high mixing, situated within a larger community with low-level disease prevalence. The second model was an agent-based model of N = 8.4 million agents representing the population and contact structure of New York City, as previously described [30]. Contact patterns were based on a combination of individual-level household contacts drawn from census microdata and age-stratified contact matrices which describe outside of household contacts. This model was initialized with 100 initial infections and no external sources of infection. Both the fully-mixed and agent-based models tracked discrete individuals who were Susceptible (S), In- fected (I), Recovered (R), Isolated (Q), and Self-Isolated (SQ) at each discrete one day timestep. Upon becoming infected (S →I), a viral load trajectory V (t) was drawn which included a latent period, growth, and decay. Each day, an individual’s viral load trajectory was used to determine whether their diagnostic test would be positive if administered, as well as their infectiousness to susceptible individuals. Based on a schedule of testing each person every D days, if an individual happened to be tested on a day when their viral load exceeded the limit of detection L of the test, their positive result would cause them to isolate (I →Q), but with the possibility of a delay in turnaround time. A fraction 1 −f of individuals self-isolate on the day of symptom onset, which occurs 0 to 3 days after peak viral load, to mimic symptom-driven isolation 11 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . Calibration to achieve targeted R0 and estimation of R As a consistency check, each simulation’s R0 was estimated as follows, to ensure that simulations were properly calibrated to their intended values. Note that to vary R0, the proportionality constant in the function that maps viral load to infectiousness need only be adjusted up or down. In a typical SEIR model, this would correspond to changing the infectiousness parameter which governs the rate at which I-to-S contacts cause new infections β. For the fully-mixed, the value of R0 was numerically estimated by running single-generation simulations in which a 50 infected individual were placed in a population of N −50 others. The number of secondary infections from those initially infected was recorded and used to directly estimate R0. For the agent-based model, the value of R0 depends on the distribution of infected agents due to stratification by age and household. We numerically estimate R0 by averaging over the number of secondary infections caused by each agent who was infected in the first 15 days of the simulation (at which point the population is still more than 99.99% susceptible). Estimations of R proceeded exactly as estimations of R0 for both models, except with interventions applied to the the viral loads and therefore the dynamics. Prediction of R without direct simulation is described in Supplemental Text S1. Supplemental Text S1. Transmission, Population Structure, and Mixing Patterns: Agent-based model Household structures, along with the age of each individual, were sampled from census microdata for New York City [52]. The probability for an infectious individual to infect each of their household members each day was determined by scaling the relative infectiousness values to match the estimated secondary attack rate for close household contacts previously reported in case cluster studies [53]. Outside of household transmission was simulated using age-stratified contact matrices, which describe the expected number of daily contacts between an individual in a given age group and those in each other age group. Each infectious individual of age i drew Poisson(Mij) contacts with individuals in age group j, where M is the contact matrix. The contacted individuals were sampled uniformly at random from age group j. We use a contact matrix for the United States estimated by [54]. Each contact resulted in infection, S →I, with probability proportional to the relative infectiousness of the infected individual on that day, scaled to obtain the specified value of R0. To conclude each time step, individuals’ viral loads and test results counters were advanced, with those whose infectious period had completely passed moved to recovery, I, Q →R. Transmission, Population Structure, and Mixing Patterns: Agent-based model The agent-based model added viral kinetics and testing policies (as described above) to an existing model for SARS-CoV-2 transmission in New York City. A full description of the agent-based model is available [30]; here we provide an overview of the relevant transmission dynamics. Simulations were initialized with all individuals susceptible, except for 100 initially infected individuals, S = N −100. As in the fully-mixed model, each individual’s test day was chosen uniformly at random and relative infectiousness was scaled to achieve the specified R0. In each timestep, those individuals who were marked for testing that day were tested, and a counter was initialized to T, specifying the number of days until that individual received their results. Next, individuals whose test results counters were zero were isolated, I →Q. There was no self-isolation in this model (and accordingly, the model did not label individuals as symptomatic or asymptomatic). Then, transmission from infected individuals to susceptible individuals was simulated both within and out- side households. To model within-household transmission, each individual had a set of other individuals 12 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity (which was not certified by peer review) The copyright holder for this pre this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint comprising their household. Household structures, along with the age of each individual, were sampled from census microdata for New York City [52]. The probability for an infectious individual to infect each of their household members each day was determined by scaling the relative infectiousness values to match the estimated secondary attack rate for close household contacts previously reported in case cluster studies [53]. comprising their household. References [1] Melissa M Arons, Kelly M Hatfield, Sujan C Reddy, Anne Kimball, Allison James, Jesica R Jacobs, Joanne Taylor, Kevin Spicer, Ana C Bardossy, Lisa P Oakley, et al. 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Contents Figure S1: Sample simulation trajectories from fully mixed model Figure S2: Epidemiological model outcomes for various test LODs and frequencies Figure S3: Delays in reporting decrease the epidemiological impact of surveillance-driven isolation (pro portional infectiousness) Figure S4: Delays in reporting decrease the epidemiological impact of surveillance-driven isolation (thresh old infectiousness) Figure S5: Robustness of surveillance effectiveness to the fraction of individuals who are symptomatic Figure S6: Epidemiological model outcomes for various test LODs, frequencies, infectiousness functions and with reporting delays Figure S7: Robustness of surveillance effectiveness to epidemiological model parameters Figure S8: Predicted and simulated impact of surveillance on the reproductive number R Figure S9: Surveillance testing suppresses an ongoing epidemic using a test with LOD 106 Text S1: Estimating the impact of surveillance testing on R Figure S1: Sample simulation trajectories from fully mixed model Figure S2: Epidemiological model outcomes for various test LODs and frequencies Figure S3: Delays in reporting decrease the epidemiological impact of surveillance-driven isolation (pro- portional infectiousness) Figure S4: Delays in reporting decrease the epidemiological impact of surveillance-driven isolation (thresh- old infectiousness) Figure S5: Robustness of surveillance effectiveness to the fraction of individuals who are symptomatic Figure S6: Epidemiological model outcomes for various test LODs, frequencies, infectiousness functions, and with reporting delays Figure S7: Robustness of surveillance effectiveness to epidemiological model parameters Figure S8: Predicted and simulated impact of surveillance on the reproductive number R Figure S9: Surveillance testing suppresses an ongoing epidemic using a test with LOD 106 Text S1: Estimating the impact of surveillance testing on R 0 50 100 150 200 250 300 350 Day 0 2000 4000 6000 Count no surveillance A infected test-based isolation symptom-based isolation 0 50 100 150 200 250 300 350 Day 0 200 400 600 800 Count LOD 105, every 7 days C 0 50 100 150 200 250 300 350 Day 0 100 200 300 Count LOD 103, every 7 days B 0 50 100 150 200 250 300 350 Day 0 10 20 30 Count LOD 105, every 3 days D Figure S1: Sample simulation trajectories from fully mixed model. Simulation trajectories show the number of infected individuals in a population of N = 20, 000 with a constant rate of external infection set to 1/N per person per day, i.e. around 1 imported case per day. References [51] Yang Liu, Li-Meng Yan, Lagen Wan, Tian-Xin Xiang, Aiping Le, Jia-Ming Liu, Malik Peiris, Leo LM Poon, and Wei Zhang. Viral dynamics in mild and severe cases of COVID-19. The Lancet Infectious Diseases, 2020. [52] Minnesota Population Center. Integrated public use microdata series, international: Version 7.2 [dataset], 2019. https://doi.org/10.18128/D020.V7.2. [53] Yang Liu, Rosalind Eggo, and Adam Kucharski. Secondary attack rate and superspreading events for SARS-CoV-2. The Lancet, 2020. [54] Kiesha Prem, Alex Cook, and Mark Jit. Projecting social contact matrices in 152 countries using contact surveys and demographic data. PLoS Computational Biology, 13(9):e1005697, 2017. S0 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint Contents CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 total infections 1e4 fully mixed model A LOD 103 LOD 105 LOD 106 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 effective repr. num. R fully mixed model C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0 1 2 3 4 5 total infections 1e6 agent based model B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 effective repr. num. R agent based model D Figure S2: Epidemiological model outcomes for various test LODs and frequencies. The fully mixed model (top row) and agent based model (bottom row) were simulated (Methods) with various test frequencies, ranging from daily to once every 14 days, and with LODs of 103, 105, and 106. Modeling results show mean outcomes from 50 independent simulations at each point, expressed as (A, B) total infections and (C, D) effective reproductive number R, from a baseline of R0 = 2.5. For the fully mixed model, only secondary infections are shown, excluding imported infections. Total population sizes were N = 2 × 104 for the fully mixed model and 8.4 × 106 for the agent based model. Dashed lines indicate R = 1 for reference. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 total infections 1e4 fully mixed model A LOD 103 LOD 105 LOD 106 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 effective repr. Contents num. R fully mixed model C 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 effective repr. num. R fully mixed model C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 total infections 1e4 fully mixed model A LOD 103 LOD 105 LOD 106 1 0.0 0.5 1.0 1.5 effective repr. num. R C days between tests 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 effective repr. num. R agent based model D days between tests 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0 1 2 3 4 5 total infections 1e6 agent based model B effective repr num R D B D 6 8 9 days between tests Figure S2: Epidemiological model outcomes for various test LODs and frequencies. The fully mixed model (top row) and agent based model (bottom row) were simulated (Methods) with various test frequencies, ranging from daily to once every 14 days, and with LODs of 103, 105, and 106. Modeling results show mean outcomes from 50 independent simulations at each point, expressed as (A, B) total infections and (C, D) effective reproductive number R from a baseline of R0 = 2 5 For the fully mixed model only secondary infections are shown excluding imported Figure S2: Epidemiological model outcomes for various test LODs and frequencies. The fully m Figure S2: Epidemiological model outcomes for various test LODs and frequencies. The fully mixed model (top row) and agent based model (bottom row) were simulated (Methods) with various test frequencies, ranging from daily to once every 14 days, and with LODs of 103, 105, and 106. Modeling results show mean outcomes from 50 independent simulations at each point, expressed as (A, B) total infections and (C, D) effective reproductive number R, from a baseline of R0 = 2.5. For the fully mixed model, only secondary infections are shown, excluding imported infections. Total population sizes were N = 2 × 104 for the fully mixed model and 8.4 × 106 for the agent based model. Dashed lines indicate R = 1 for reference. Contents g p g q y ( p row) and agent based model (bottom row) were simulated (Methods) with various test frequencies, ranging from daily to once every 14 days, and with LODs of 103, 105, and 106. Modeling results show mean outcomes from 50 independent simulations at each point, expressed as (A, B) total infections and (C, D) effective reproductive number R, from a baseline of R0 = 2.5. For the fully mixed model, only secondary infections are shown, excluding imported infections. Total population sizes were N = 2 × 104 for the fully mixed model and 8.4 × 106 for the agent based model. Dashed lines indicate R = 1 for reference. daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 A LOD 103 LOD 105 no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 C daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 B dx delay none 1 day 2 days no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 D Figure S3: Delays in reporting decrease the epidemiological impact of surveillance-driven isolation (propor- tional infectiousness). This figure presents results from simulations which were identical to those shown in the main text Figure 4, but in which infectiousness was assumed to be directly proportional to viral load. Compare with thresh- old (binary) infectiousness in Fig. S4 and log-proportional infectiousness in Fig 4. See Methods. Contents Infections (blue), test-based isolation (black), and symptom-based isolation (red) are shown for four scenarios, with R0 = 2.5. (A) No surveillance. (B) Weekly testing at LOD 103. (C) Weekly testing at LOD 105. (D) Testing every 3 days with LOD 105. Note the variation in the vertical axis scales. The model is fully described in Methods. 0 50 100 150 200 250 300 350 Day 0 2000 4000 6000 Count no surveillance A infected test-based isolation symptom-based isolation 0 50 100 150 200 250 300 350 Day 0 200 400 600 800 Count LOD 105, every 7 days C 0 50 100 150 200 250 300 350 Day 0 100 200 300 Count LOD 103, every 7 days B 0 50 100 150 200 250 300 350 Day 0 10 20 30 Count LOD 105, every 3 days D 350 0 50 100 150 200 250 300 350 Day 0 200 400 600 800 Count LOD 105, every 7 days C Figure S1: Sample simulation trajectories from fully mixed model. Simulation trajectories Figure S1: Sample simulation trajectories from fully mixed model. Simulation trajectories show the number of infected individuals in a population of N = 20, 000 with a constant rate of external infection set to 1/N per person per day, i.e. around 1 imported case per day. Infections (blue), test-based isolation (black), and symptom-based isolation (red) are shown for four scenarios, with R0 = 2.5. (A) No surveillance. (B) Weekly testing at LOD 103. (C) Weekly testing at LOD 105. (D) Testing every 3 days with LOD 105. Note the variation in the vertical axis scales. The model is fully described in Methods. Figure S1: Sample simulation trajectories from fully mixed model. Simulation trajectories show the number of infected individuals in a population of N = 20, 000 with a constant rate of external infection set to 1/N per person per day, i.e. around 1 imported case per day. Infections (blue), test-based isolation (black), and symptom-based isolation (red) are shown for four scenarios, with R0 = 2.5. (A) No surveillance. (B) Weekly testing at LOD 103. (C) Weekly testing at LOD 105. (D) Testing every 3 days with LOD 105. Note the variation in the vertical axis scales. The model is fully described in Methods. S1 S1 . Contents daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 A LOD 103 LOD 105 B no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 C D B daily 3 days weekly 14 days daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 B dx delay none 1 day 2 days no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 D Figure S3: Delays in reporting decrease the epidemiological impact of surveillance-driven isolation (propor- tional infectiousness). This figure presents results from simulations which were identical to those shown in the main text Figure 4, but in which infectiousness was assumed to be directly proportional to viral load. Compare with thresh- old (binary) infectiousness in Fig. S4 and log-proportional infectiousness in Fig 4. See Methods. S2 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. ch was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . Contents daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 A LOD 103 LOD 105 no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 C daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 B dx delay none 1 day 2 days no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 D daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 A LOD 103 LOD 105 no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 C D Figure S4: Delays in reporting decrease the epidemiological impact of surveillance-driven isolation (threshold infectiousness). This figure presents results from simulations which were identical to those shown in the main text Figure 4, but in which infectiousness was assumed to be binary, i.e. no infectiousness below 106 and equal infec- tiousness for any viral load above 106. Compare with proportional infectiousness in Fig. S3 and log-proportional infectiousness in Fig 4. See Methods. Contents https://doi.org/10.1101/2020.06.22.20136309 doi: preprint daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 A LOD 103 LOD 105 no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 C daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 B dx delay none 1 day 2 days no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 D Figure S4: Delays in reporting decrease the epidemiological impact of surveillance-driven isolation (threshold infectiousness). This figure presents results from simulations which were identical to those shown in the main text Figure 4, but in which infectiousness was assumed to be binary, i.e. no infectiousness below 106 and equal infec- tiousness for any viral load above 106. Compare with proportional infectiousness in Fig. S3 and log-proportional infectiousness in Fig 4. See Methods. Contents CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 total infections 1e4 fully mixed model logproportional infectiousness A LOD 103 105 106 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 2.0 effective repr. num. R fully mixed model logproportional infectiousness C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0 2 4 6 total infections 1e6 agent based model logproportional infectiousness B dx delay 0 days 1 day 2 days 3 days 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 2.0 effective repr. num. R agent based model logproportional infectiousness D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 total infections 1e4 fully mixed model proportional infectiousness E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 2.0 2.5 effective repr. num. R fully mixed model proportional infectiousness G 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0 2 4 6 total infections 1e6 agent based model proportional infectiousness F 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 2.0 2.5 effective repr. num. R agent based model proportional infectiousness H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 total infections 1e4 fully mixed model threshold infectiousness I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 2.0 effective repr. num. Contents R fully mixed model threshold infectiousness K 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0 2 4 6 total infections 1e6 agent based model threshold infectiousness J 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 2.0 effective repr. num. R agent based model threshold infectiousness L demiological model outcomes for various test LODs, frequencies, infectiousne delays. The fully mixed model and agent based model were simulated (Methods ging from daily to once every 14 days, with LODs of 103, 105, and 106, and w r log-proportional, proportional, and threshold infectiousness functions (see Met ndicate LODs and delays, and in-plot annotations describe various conditions. Mod from 50 independent simulations at each point, expressed as total infections and eff a baseline of R0 = 2.5. For the fully mixed model, only secondary infections are ons. Total population sizes were N = 2 × 104 for the fully mixed model and 8.4 × horizontal line indicates R = 1 for reference 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0 2 4 6 total infections 1e6 agent based model logproportional infectiousness B dx delay 0 days 1 day 2 days 3 days 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 2.0 2.5 effective repr. num. R fully mixed model proportional infectiousness G 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 total infections 1e4 fully mixed model proportional infectiousness E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 2.0 2.5 effective repr. num. Contents daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R fully mixed model A LOD 103 LOD 105 no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model D daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R agent based model G no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections agent based model J daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R fully mixed model B no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model E daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R agent based model H no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections agent based model K daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R fully mixed model C no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model F daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% reduction in R agent based model I no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections agent based model L Figure S5: Robustness of surveillance effectiveness to the fraction of individuals who are symptomatic. (A-F) Results from fully-mixed simulations and (G-L) agent-based simulations using an asymptomatic rate of 50% (top row), 65% (middle row; identical to main text Fig 2), and 80% (bottom row). Figure S5: Robustness of surveillance effectiveness to the fraction of individuals who are symptomatic. (A-F) Results from fully-mixed simulations and (G-L) agent-based simulations using an asymptomatic rate of 50% (top row), 65% (middle row; identical to main text Fig 2), and 80% (bottom row). S3 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . Contents R fully mixed model proportional infectiousness G 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 total infections 1e4 fully mixed model proportional infectiousness E G 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0 2 4 6 total infections 1e6 agent based model proportional infectiousness F 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days between tests 0.0 0.5 1.0 1.5 2.0 2.5 effective repr. num. R agent based model proportional infectiousness H 1 2 3 4 5 6 7 8 9 10 days between tests 0 Figure S6: Epidemiological model outcomes for various test LODs, frequencies, infectiousness functions, and with reporting delays. The fully mixed model and agent based model were simulated (Methods) with various test frequencies, ranging from daily to once every 14 days, with LODs of 103, 105, and 106, and with delays of 0, 1, 2, or 3 days, for log-proportional, proportional, and threshold infectiousness functions (see Methods). Legends in panels A and B indicate LODs and delays, and in-plot annotations describe various conditions. Modeling results show mean outcomes from 50 independent simulations at each point, expressed as total infections and effective reproductive number R, from a baseline of R0 = 2.5. For the fully mixed model, only secondary infections are shown, excluding imported infections. Total population sizes were N = 2 × 104 for the fully mixed model and 8.4 × 106 for the agent based model. A horizontal line indicates R = 1 for reference. S4 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Contents CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint 0.0 0.5 1.0 1.5 2.0 2.5 Predicted R (Eqs. S1 and S2) 0.0 0.5 1.0 1.5 2.0 2.5 Measured R (simulation) agent based model, LOD 103 fully mixed model, LOD 103 agent based model, LOD 105 fully mixed model, LOD 105 Figure S8: Predicted and simulated impact of surveillance on the reproductive number R. Mathematical predic- tions of the reproductive number R (see Equation (S1) in Supplemental Text S1) are scattered against their empirical measurements for the simulations shown in the main text (Figs. 2 and 4). Pearson’s r = 0.998, p < 10−6. 0.0 0.5 1.0 1.5 2.0 2.5 Predicted R (Eqs. S1 and S2) 0.0 0.5 1.0 1.5 2.0 2.5 Measured R (simulation) agent based model, LOD 103 fully mixed model, LOD 103 agent based model, LOD 105 fully mixed model, LOD 105 Figure S8: Predicted and simulated impact of surveillance on the reproductive number R. Mathematical predic- tions of the reproductive number R (see Equation (S1) in Supplemental Text S1) are scattered against their empirical measurements for the simulations shown in the main text (Figs. 2 and 4). Pearson’s r = 0.998, p < 10−6. 0 2 4 6 8 weeks after surveillance begins 0% 2% 4% 6% 8% 10% 12% prevalence fully mixed model 13% 47% 33% 59% 100% A 0 2 4 6 8 weeks after surveillance begins 0% 2% 4% 6% prevalence agent based model 14% 40% 31% 58% 100% B no intervention testing regimen begins 50% participation, weekly 50% participation, every 3 days 75% participation, weekly 75% participation, every 3 days Figure S9: Surveillance testing suppresses an ongoing epidemic using a test with LOD 106. Widespread testing and isolation of infected individuals drives prevalence downward for both (A) the fully-mixed compartmental model and (B) the agent based model. Contents peer review) The copyright holder for this preprint this version posted September 8, 2020. . rg/10.1101/2020.06.22.20136309 no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model A LOD 103 LOD 105 no dx delay 1 day delay 2 day delay no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 B no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 C Figure S7: Robustness of surveillance effectiveness to epidemiological model parameters. (A) Results from the fully-mixed simulation with a tripled rate of external infection, i.e. 3/N per person per day. (B) Results from the fully mixed simulation with R0 doubled, i.e. R0 = 5. (C) Results from the agent-based simulation with R0 doubled, i.e. R0 = 5. 1 A no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model A LOD 103 LOD 105 no dx delay 1 day delay 2 day delay B 1 B no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections fully mixed model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 B C no test daily 3 days weekly 14 days 0% 20% 40% 60% 80% 100% total infections agent based model 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 C Figure S7: Robustness of surveillance effectiveness to epidemiological model parameters. (A) Results from the fully-mixed simulation with a tripled rate of external infection, i.e. 3/N per person per day. (B) Results from the fully mixed simulation with R0 doubled, i.e. R0 = 5. (C) Results from the agent-based simulation with R0 doubled, i.e. R0 = 5. S5 . Contents CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: edRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309 doi: medRxiv preprint Contents Time-series of prevalence, measured as the total number of infectious individuals, are shown for no intervention (solid) and surveillance testing scenarios (various dashed; see legend). Surveillance testing began only when prevalence reached 4% (box), and time series are shifted such that testing begins at t = 0. Scenarios show the impact of a test with LOD 106, no delay in results, and with 10% of samples assumed to be incorrectly collected (and therefore negative) to reflect decreased sensitivity incurred at sample collection in a mass testing scenario. Annotations show total number of post-intervention infections, as a percentage of the no-intervention scenario, labeled as 100%. See Fig. 5 for identical simulations using a test with LOD 105. 0 2 4 6 8 weeks after surveillance begins 0% 2% 4% 6% 8% 10% 12% prevalence fully mixed model 13% 47% 33% 59% 100% A 0 2 4 6 8 weeks after surveillance begins 0% 2% 4% 6% prevalence agent based model 14% 40% 31% 58% 100% B no intervention testing regimen begins 50% participation, weekly 50% participation, every 3 days 75% participation, weekly 75% participation, every 3 days 0 2 4 6 8 weeks after surveillance begins 0% 2% 4% 6% 8% 10% 12% prevalence fully mixed model 13% 47% 33% 59% 100% A % 0 2 4 6 8 weeks after surveillance begins 0% 2% 4% 6% prevalence agent based model 14% 40% 31% 58% 100% B B A Figure S9: Surveillance testing suppresses an ongoing epidemic using a test with LOD 106. Widespread testing and isolation of infected individuals drives prevalence downward for both (A) the fully-mixed compartmental model and (B) the agent based model. Time-series of prevalence, measured as the total number of infectious individuals, are shown for no intervention (solid) and surveillance testing scenarios (various dashed; see legend). Surveillance testing began only when prevalence reached 4% (box), and time series are shifted such that testing begins at t = 0. Scenarios show the impact of a test with LOD 106, no delay in results, and with 10% of samples assumed to be incorrectly collected (and therefore negative) to reflect decreased sensitivity incurred at sample collection in a mass testing scenario. Annotations show total number of post-intervention infections, as a percentage of the no-intervention scenario, labeled as 100%. See Fig. 5 for identical simulations using a test with LOD 105. S6 . S1 Predicting the impact of surveillance testing on R The impact of surveillance on the reproductive number can be estimated by considering the ratio of pop- ulation infectiousness with surveillance testing to population infectiousness with no surveillance testing. However, note that the impact of a surveillance testing policy may depend on two additional factors. First, not all individuals may wish to participate in a testing program. Let the fraction of individuals who participate be given by φ. Second, a test may produce a false negative result unrelated to its limit of detection—for instance due to an improperly collected sample. Let se be the test sensitivity, in the particular sense of the probability of correctly diagnosing an individual as positive when that person’s viral load should, in principle, have provided a sufficiently high RNA concentration to be detectable. Let f0 be the total infectiousness removed with no testing policy, i.e. due to symptom-driven self isola- tion. Let ftest(se) be the fraction of total infectiousness removed with a chosen testing policy, inclusive of symptom-driven self isolation, as well as the test sensitivity se introduced above. Both f0 and ftest(se) can be estimated rapidly via Monte Carlo by drawing trajectories and applying a surveillance policy to them in which a fraction 1 −se positive tests are discarded uniformly at random. In the main text, we found that estimating these values using 10, 000 randomly drawn trajectories was sufficient to produce stable estimates. Under the assumption of statistical independence between an individual’s participation or refusal, viral load, and se, we can approximate the reproductive number as R ≈  φ1 −ftest(se) 1 −f0 + 1 −φ  R0 , (S1) (S1) which simply expresses a weighted combination of removed infectiousness via surveillance testing partic- ipation and no test. Intuitively, note that if there is complete refusal to participate (φ = 0) or an entirely ineffective test (ftest(se) = f0), then R ≈R0, as expected. S7
https://openalex.org/W2976812342
https://link.springer.com/content/pdf/10.1007/s10826-019-01581-8.pdf
English
null
Feasibility Study of the Enhancing Parenting Skills Programme
Journal of child and family studies
2,019
cc-by
10,231
Abstract Objectives This study reports on the feasibility and initial effectiveness of an individually delivered parent programme for parents of young children with behaviour problems. Whilst parenting programmes are known to be effective in reducing behaviour problems, numerous barriers can prevent families from accessing programmes. Individually delivered parent programmes may be more accessible. In the UK, health visitors provide support to all families with a child under 5 years of age and are ideally placed to deliver interventions for child behaviour problems. Methods Fifty-eight parents reporting children with behaviour problems were recruited from four areas to intervention (n = 29) and treatment as usual, wait-list control (n = 29) conditions. Feasibility outcomes included recruitment, retention, programme delivery, and satisfaction. Baseline and six-month post-randomisation follow-up measures were collected in parents’ homes and included parent-report measures of child behaviour, parenting skills, and parental mental health as well as an observation of parenting behaviour during a parent-child play task. Results Significant changes in child behaviour, lax parenting, and parental mental health were found for the whole sample but there were no significant differences between conditions. Recruitment and retention rates were lower than expected questioning the feasibility of delivering the parent programme as it is in existing services. Conclusions This paper provides limited evidence for the feasibility of the Enhancing Parenting Skills programme delivered in existing health services. Further feasibility work, particularly for recruitment and retention, would be needed before conducting a larger study to examine the effectiveness of the programme. Keywords Parenting ●Child behaviour ●RCT ●Feasibility ●Individual treatment Keywords Parenting ●Child behaviour ●RCT ●Feasibility ●Individual treatment Globally, the most common mental disorders in children are behavioural problems, with a worldwide estimated pre- valence of 5.7% (Polanczyk et al. 2015). In the UK, they are the most common reason for referral to Child and Adoles- cent Mental Health Services (CAMHS) and cost society thousands of pounds every year (National Collaborating Centre for Mental Health [NCCMH] 2013; Rivenbark et al. 2018). Numerous risk factors have been identified for the development of child behaviour problems, including indi- vidual factors such as child gender or temperament (Martel 2013; Miner and Clarke-Stewart 2008), and familial/social Globally, the most common mental disorders in children are behavioural problems, with a worldwide estimated pre- valence of 5.7% (Polanczyk et al. 2015). Journal of Child and Family Studies (2020) 29:686–698 https://doi.org/10.1007/s10826-019-01581-8 Journal of Child and Family Studies (2020) 29:686–698 https://doi.org/10.1007/s10826-019-01581-8 ORIGINAL PAPER Margiad E. Williams 1 ●Zoe Hoare2 ●Dawn A. Owen1 ●Judy Hutchings1 Margiad E. Williams 1 ●Zoe Hoare2 ●Dawn A. Owen1 ●Judy Hutchings1 Published online: 25 September 2019 © The Author(s) 2019 * Margiad E. Williams margiad.williams@bangor.ac.uk 1 Centre for Evidence Based Early Intervention, Nantlle Building, Normal Site, Bangor University, Bangor LL57 2PZ, UK 2 North Wales Organisation for Randomised Trials in Health and Social Care, Bangor University, Bangor LL57 2PZ, UK * Margiad E. Williams margiad.williams@bangor.ac.uk 1 Centre for Evidence Based Early Intervention, Nantlle Building, Normal Site, Bangor University, Bangor LL57 2PZ, UK 2 North Wales Organisation for Randomised Trials in Health and Social Care, Bangor University, Bangor LL57 2PZ, UK Abstract The majority of parents are not seen by their health visitor after the 27-month visit unless there is an identified need. Vulnerable families receive additional support from their health visitor including for issues such as sleep, behaviour, and feeding. For all health visitors, approximately 9% of their caseloads would require additional support at some point in the child’s life, up until the age of 5 years (Munday 2018). A trusting parent-practitioner relationship is vital for working with vulnerable families, and the close one-to-one contact through home visits that health visitors provide places them in a good position to develop a meaningful understanding of family needs (Whittaker 2014). Health visitors are a valued source of advice for parents who especially value their knowledge of parenting, child devel- opment and behaviour (Brook and Salmon 2017; Hogg et al. 2013). Their knowledge of child development and access to all families of young children makes them ideally placed to deliver parenting interventions (Cowley et al. 2013; Myors et al. 2014). A survey of health visitor skills showed that they typically use some evidence-based beha- vioural techniques as part of their work with families, but most were non-specific and not in the form of a structured intervention (Williams and Hutchings 2018). Many health visitors report growing caseloads of children with behaviour difficulties, and spend a lot of time dealing with these cases (Wilson et al. 2008), suggesting that a structured interven- tion may be a useful tool for working with families. Parenting programmes can be delivered in several formats including group-based, individual, and self-administered. However, there are practical and psychological obstacles that often prevent families from accessing parenting pro- grammes (e.g., financial costs, transportation, stigma, work schedules) (Duppong-Hurley et al. 2016; Heath et al. 2018; Lavigne et al. 2010; Sayal et al. 2010). Parents’ perceptions of barriers associated with parenting programmes, demands imposed by the intervention, relevance, and their relation- ship with the provider can also have an effect (Kazdin 1997). Individually delivered parenting programmes over- come some of the practical barriers because they can be delivered in the family home, making them potentially more accessible, and by a professional with which the parent already has a good relationship (Kazdin 1997; Lundahl et al. 2006; Reyno and McGrath 2006). Individually administered parenting programmes have stronger effects in reducing child behaviour problems compared to group- based programmes, especially for vulnerable parents (Lun- dahl et al. 2006). Abstract In the UK, they are the most common reason for referral to Child and Adoles- cent Mental Health Services (CAMHS) and cost society thousands of pounds every year (National Collaborating Centre for Mental Health [NCCMH] 2013; Rivenbark et al. 2018). Numerous risk factors have been identified for the development of child behaviour problems, including indi- vidual factors such as child gender or temperament (Martel 2013; Miner and Clarke-Stewart 2008), and familial/social factors such as poverty (Shaw and Shelleby 2014). How- ever, despite there being a range of factors correlated with increased risk for children, it is the extent to which these risk factors compromise parenting that predicts child behaviour problems (Gach et al. 2018; Hoeve et al. 2009). The most effective interventions to address child beha- viour problems are parenting programmes that teach appropriate behaviour management skills based on social learning theory principles (Furlong et al. 2012; NCCMH 2013). Many evaluations have shown significant improve- ments in child behaviour, parenting skills, and parental mental health (Barlow and Coren 2018). Changes in par- enting have also been shown to mediate changes in child behaviour (Gach et al. 2018). Parenting programmes typi- cally teach parents positive strategies (e.g., praise, rewards, clear instructions and household rules) to encourage posi- tive child behaviours, and behaviour management strategies such as ignoring to reduce the incidence of child behaviour problems. Parents are also taught the importance of spending time playing and/or in special time activities with Journal of Child and Family Studies (2020) 29:686–698 687 their children to encourage the development of strong parent-child relationships. Video clips and modelling are used in many programmes to demonstrate the behavioural skills being taught, and parents are given the opportunity to practice implementing these skills through role-play or in home-based practice with their children. Some, particularly the more effective programmes, use homework tasks designed to promote skill development at home (Kaminski et al. 2008). A number of effective programme components have been identified including positive interactions with their child, active rehearsal of skills, teaching of principles not techniques, modelling, and time-out (e.g., Barth and Liggett-Creel 2014; Kaminski et al. 2008). visits that health visitors are required to complete with all families, including an ante-natal visit, new baby review at two weeks, six- to eight-week assessment, 12-month assessment, and a 27-month assessment. Abstract This may be due to the inclusion of live coaching of skills with parents and children, a component which has been shown to be effective in teaching beha- vioural techniques (Kaminski et al. 2008). Vulnerable families are also more likely to have complex needs and may benefit more from individually focussed interventions (Hutchings and Williams 2019). Due to the heavy demand on health visitors, the Enhancing Parenting Skills (EPaS) programme was devel- oped with the goal of training health visitors in behavioural management skills to utilise in their work with families of children with disruptive behaviours. The model was developed based on social learning theory principles with the intention of providing individualised support to families. Individualised support is the cornerstone of effective work with families experiencing significant difficulties with their children. The EPaS programme is conceptualised as a pro- cess consisting of three phases: a standardised assessment; a structured case analysis formulation process to facilitate the identification of the specific problem behaviours, their functions, and the necessary replacement behaviours; and an intervention phase designed to support parents in implementing evidence-based behaviour change strategies. Parenting programmes are typically delivered by pro- fessionally trained staff including clinical psychologists, health visitors/public health nurses, and social workers. Health visitors in the UK are trained nurses with specialist training in public health promotion, including a specific focus on the factors associated with positive child devel- opment. They provide a universal service to all families with children under the age of 5 years, known as the Healthy Child Programme, and targeted services for those in need (e.g., vulnerable families) (Cowley et al. 2007; Department of Health 2009). The recommended caseload size for each health visitor is approximately 250 families but caseloads are often much larger (Munday 2018). As part of a universal service, there are a minimum of five mandated The EPaS programme was developed from an intensive coaching intervention involving video-feedback that was delivered and evaluated with families of CAMHS referred children with severe behavioural problems. Findings showed significant improvements in child behaviour, par- enting, and parental mental health (Hutchings et al. 2002) Journal of Child and Family Studies (2020) 29:686–698 688 the EPaS training due to lack of time, personal issues, or job change. Abstract They were asked to identify two families of children aged between 30 and 60 months who were reporting having a child with significant behavioural difficulties to take part in the project. Health visitors used the Eyberg Child Behaviour Inventory (ECBI; Eyberg et al. 1980) to identify children from their caseloads who were scoring above the clinical cut-off for behaviour problems on either the Intensity (≥131) or Problem scale (≥15). Of the 37 health visitors who completed the EPaS training, 29 managed to identify two families for the project. All health visitors were female (n = 29; 100%) and had a wide range of experience (Median = 3.00 years; range = 2 months – 28 years). and were maintained at a four-year follow-up (Hutchings et al. 2004). The main limitations were that it targeted CAMHS referred school-aged children with severe beha- vioural problems, was resource intensive, and therefore not accessible to many families. To meet the needs of more families, the programme was adapted for home delivery by health visitors with pre-school children and named the EPaS programme (Lane and Hutchings 2002). A small-scale study reported significant improvements in child behaviour and parental mental health, and health visitors reported increased confidence in and frequency of use of behavioural techniques (Lane and Hutchings 2002). However, the training delivered to the health visitors was very intensive, with sessions delivered weekly over a 12-week period. Due to this, a further study adapted the programme for wider dissemination in the form of a two-day training for a variety of staff working with parents (Hutchings and Williams 2013). Data from a small sample showed significant reductions in child behaviour and dysfunctional parenting and improvements in parental mental well-being (Hutchings and Williams 2013). A criticism from staff on the training was that two days was too short to cover the skills needed to deliver the programme. A lack of knowledge of some staff regarding child development was another concern. ( y g y ) Eighty-seven families were approached to take part in the study. Of these, 11 (12.6%) were not eligible due to scoring below the cut-off on the ECBI; eight (9.2%) were not contacted due to their health visitor withdrawing from the study; six (6.9%) were not randomised due to their health visitor being unable to identify a second family for the study; and four (4.6%) were not interested in participating in the trial. Abstract Fifty-eight families were recruited and randomised to either the intervention or a treatment as usual (TAU) waitlist control condition (see CONSORT Fig. 1). Mean age of the children was 40.52 months (SD = 8.78) with over 70% being male. The mean score for child behaviour problems was 165.34 on the ECBI Intensity subscale, indicating significant levels above the clinical cut-off (131). All but one of the primary carers were female (n = 57; 98.3%) and 53 (91.4%) were reporting living in poverty. Two-thirds of the sample were reporting three or more risk factors associated with socio-economic disadvantage (see Table 1). There were no significant differences between families in the intervention and families in the TAU control in terms of baseline characteristics. No power calculation was conducted. The present trial was designed to address the limitations of the previous studies and incorporate participant feedback. First, the trial reverted to the initial EPaS trial strategy of training health visitors due to the variability of child development knowledge seen in the participants of the Hutchings and Williams (2013) study and the inaccessi- bility of the Hutchings et al. (2002) intervention. Second, the training was revised into a three-day course due to the intensive nature of the training in the Lane and Hutchings (2002) trial and the criticism on the shortness of the training in the Hutchings and Williams (2013) study. The main aims of the present trial were to explore feasibility questions of the new EPaS training model in existing services and examine initial evidence for effectiveness. The key ques- tions were around the feasibility of target population recruitment, intervention delivery, retention in the research study, and acceptability of the intervention to parents. Health visitor satisfaction with the training is reported elsewhere (see Williams and Hutchings 2018). Initial effectiveness was explored by comparing outcomes for families in the EPaS programme to a control condition. Randomisation After collection of baseline measures, families were randomly assigned to either the intervention or the TAU control condition (ratio 1:1). Randomisation was within health visitor so that each health visitor had one intervention and one TAU control family. Randomisation occurred using an online programme with random permuted blocks (www.randomization.com). Data collection Forty-nine health visitors initially consented to partici- pate, however 12 (24.5%) withdrew before commencing Ethical approval was granted by Bangor University Ethics Committee in May 2014 (application number: 2014-12886) 689 Journal of Child and Family Studies (2020) 29:686–698 Journal of Child and Family Studies (2020) 29:686–698 Fig. 1 CONSORT diagram of participants Fig. 1 CONSORT diagram of participants and the North West Wales Research Ethics Committee in July 2014 (application number: 14/WA/0187). The study was registered with ISRCTN (ISRCTN06867279). Families were initially approached by their health visitor to examine eligibility using the ECBI questionnaire. The families approached were from the health visitors’ own caseloads and they would have had previous contact with the family. Eligible families were informed of the study and asked if they would be interested in taking part (see Fig. 1 for flow of participants through the study). Interested families completed a note of interest which was sent to the research team. The parent would then be contacted within one week to arrange a home visit. During the home visit, the study was explained in detail and any questions from parents were answered. If the parent was happy to continue, written informed consent was obtained. Only once informed con- sent was obtained did the parent complete the baseline and the North West Wales Research Ethics Committee in July 2014 (application number: 14/WA/0187). The study was registered with ISRCTN (ISRCTN06867279). Families were initially approached by their health visitor to examine eligibility using the ECBI questionnaire. The families approached were from the health visitors’ own caseloads and they would have had previous contact with the family. Eligible families were informed of the study and asked if they would be interested in taking part (see Fig. 1 for flow of participants through the study). Interested families completed a note of interest which was sent to the research team. The parent would then be contacted within one week to arrange a home visit. During the home visit, the study was explained in detail and any questions from parents were answered. If the parent was happy to continue, written informed consent was obtained. Only once informed con- sent was obtained did the parent complete the baseline measures and observation. Home visits to collect data were conducted at baseline and six months later for follow-up measures. Parents received a children’s book at each time- point as a thank you for providing data. Data collection Four families (7%) completed the parent-child observation in Welsh while the rest were completed in English. Eligible families were informed of the study and asked if they would be interested in taking part (see Fig. 1 for flow of participants through the study). Interested families completed a note of interest which was sent to the research team. The parent would then be contacted within one week to arrange a home visit. During the home visit, the study was explained in detail and any questions from parents were answered. If the parent was happy to continue, written informed consent was obtained. Only once informed con- sent was obtained did the parent complete the baseline Intervention Sessions are normally conducted on a weekly basis but it was up to the health visitors to arrange appointments to implement the sessions. standardised assessment tools including parent-report questionnaires, interview schedules, and observation tools to collect information about the family, their current circumstances, specific child problem behaviours, and parents’ goals. The second phase consists of teaching the implementer how to produce a case analysis using the information collected in the assessment sessions. It involves using the information to develop an under- standing of the problem, its history and current function, the assets available in the situation that will support change, and some potential short- and longer-term goals. The case analysis is shared with the family and an inter- vention contract is negotiated. The third phase of the programme introduces strategies that parents could use to achieve their short- and longer-term goals. Health visitors coach parents in the use of the strategies during home visits. Intervention strategies focus on improving parent- child relationships and teaching replacement behaviours (e.g., praise and rewards, ignoring unwanted behaviours, redirecting, consequences, time-out). that clarify whether the intervention strategies are being effective. These records and homework assignments are reviewed by the health visitors at the start of each session. It is not a requirement of the EPaS programme to com- plete all three phases of the programme, since the first phase may be sufficient on its own for some families. However, there are recommended number of sessions for each phase that implementers can use as a guide: three assessment sessions, one case analysis session, and between six and eight intervention sessions. The number of sessions com- pleted depends on the complexity of the problem(s) being targeted. Sessions are normally conducted on a weekly basis but it was up to the health visitors to arrange appointments to implement the sessions. Table 1 Participant baseline characteristics Intervention All health visitors received three days of training, each day approximately one month apart. The training was delivered by the last author, an experienced clinician who developed the programme. All intervention resources were provided including a detailed manual and assessment tools (see Hutchings and Williams 2019). The programme has three phases. The first phase consists of assessment procedures that are completed with the parents. These entail Journal of Child and Family Studies (2020) 29:686–698 690 Health visitor characteristics All (N = 29) Age, years: M (SD) 41.76 (8.86) Gender, female: n (%) 29 (100.0) HV experience, years: Median (range) 3.00 (0–28) All (N = 58) Intervention (n = 29) Control (n = 29) Child characteristics Child gender, male: n (%) 42 (72.4) 20 (69.0) 22 (75.9) Child age, months: M (SD) 40.52 (8.78) 39.52 (9.09) 41.52 (8.51) Child behaviour, ECBI: M (SD) 165.34 (28.68) 167.62 (30.07) 163.07 (27.56) Parent characteristics Parent gender, female: n (%) 57 (98.3) 29 (100.0) 28 (96.6) Parent age, years: M (SD) 30.55 (8.90) 29.07 (8.02) 32.03 (9.61) Age birth 1st child, years: M (SD) 21.80 (5.59) 21.45 (5.12) 22.19 (6.13) Low educationa: n (%) 45 (77.6) 23 (79.3) 22 (75.9) Living in povertyb: n (%) 53 (91.4) 27 (93.1) 26 (90.0) Single parent: n (%) 19 (32.8) 12 (41.4) 7 (24.1) Unemployment: n (%) 29 (50.0) 16 (55.2) 13 (44.8) Depressionc: n (%) 35 (60.3) 17 (58.6) 18 (62.1) High risk indexd: n (%) 39 (67.2) 20 (69.0) 19 (65.5) HV health visitor, ECBI Eyberg Child Behavior Inventory Intensity subscale aLeft education before or at 16 years bIncome less than 60% median based on Office for National Statistics (2014) cPresence of depression indicated by scoring borderline or above on BDI dThree or more risk factors Table 1 Participant baseline characteristics that clarify whether the intervention strategies are being effective. These records and homework assignments are reviewed by the health visitors at the start of each session. It is not a requirement of the EPaS programme to com- plete all three phases of the programme, since the first phase may be sufficient on its own for some families. However, there are recommended number of sessions for each phase that implementers can use as a guide: three assessment sessions, one case analysis session, and between six and eight intervention sessions. The number of sessions com- pleted depends on the complexity of the problem(s) being targeted. Parent-child observation An observation of parent-child interaction was conducted using categories from the Dyadic Parent-Child Interaction Coding System (Eyberg and Robinson 1981). This measure uses a frequency count of observed behaviours over a 30-minute time period. Parents were asked to play with their child as they normally would. The categories used included: Observed positive parenting, observed social-emotional coaching, and observed negative parenting. All parent- child observations were live coded by one of two trained coders who were unaware of which condition parents had been allocated. Inter-rater reliability was examined for over 20% of observations at each time point (baseline = 24.1%; follow-up = 29.3%). For the current study, overall intra- class correlations (ICC) for the categories were as follows: Observed positive parenting ICC = 0.950; Observed social- emotional coaching ICC = 0.961; Observed negative par- enting ICC = 0.933. Beck Depression Inventory II (BDI-II) baseline family demographics. The questionnaire covers key socioeconomic circumstances (e.g., education, marital status, income, employment status). Education was dichotomised to represent low (left school before or at 16 years of age) or high (left school after 16 years of age); Living in poverty was dichotomised to represent low (income less than 60% median) or high (income above 60% median) based on figures from Office for National Statistics (2014). A socio-economic risk index was also calculated based on categories from Hutchings (1996) and Rutter and Quinton (1977). These were: unemployment, single parent, teenage parent (<20 years of age) at birth of first child, low education, living in poverty, and symptoms of depression indicated by scoring borderline or above on the Beck Depression Inventory II (Beck et al. 1996). High risk is indicated by the presence of three or more risk factors. The BDI-II is a 21-item, parent-reported measure of depressive symptomatology (Beck et al. 1996). Higher scores indicate more symptoms of depression. The internal consistency for the present study was good (baseline α = 0.92 and follow-up α = 0.95). Parent satisfaction A questionnaire was developed to measure parent satisfac- tion with the programme. Questions asked about pro- gramme content (7 questions), overall process (2 questions), and health visitor (1 question). Responses were on a five- point Likert scale from Very unhelpful to Very helpful. Parents were also asked if they would recommend the programme to other parents. Higher scores represent more satisfaction. This questionnaire was collected by health visitors at their final session with the parent. Conners Abbreviated Parent-Teacher Questionnaire (Abbreviated Conners) The Abbreviated Conners is a 10-item inventory used to measure symptoms of child hyperactive behaviour (Conners 1994). Higher scores indicate more behaviour problems. The internal consistency for the present study was good (Baseline α = 0.85 and follow-up α = 0.87). Data Analyses The PS is a 30-item, parent-reported inventory used to assess dysfunctional parenting practices (Arnold et al. 1993). As well as a total score, there are three subscales: Laxness, Over-reactivity, and Verbosity. Higher scores indicate higher dysfunctional parenting. The internal con- sistency for the total score and each subscale were as fol- lows: total (baseline α = 0.87 and follow-up α = 0.92); laxness (baseline α = 0.89 and follow-up α = 0.93); over- reactivity (baseline α = 0.82 and follow-up α = 0.85); verbosity (baseline α = 0.52 and follow-up α = 0.67). Because of the low internal consistency for the verbosity subscale, it was not used in the analyses. Eyberg Child Behaviour Inventory The ECBI is a 36-item, parent-reported inventory designed to assess the frequency and intensity of behavioural pro- blems in children aged 2 to 16 years. The questionnaire consists of two subscales with clinical cut-offs: Intensity (≥131) and Problem (≥15). Higher scores indicate more behaviour problems. The internal consistency across the two time points in the present study was good (Intensity: baseline α = 0.88 and follow-up α = 0.95; Problem: base- line α = 0.81 and follow-up α = 0.93). Sociodemographic measures A questionnaire was developed for health visitors asking the following demographic questions: age, gender, and number of years working as a health visitor. Parents are involved throughout the EPaS process; they are asked to keep records throughout the process including information about their child during the assessment phase. They are also asked to undertake homework assignments A revised version of the Personal Data and Health Questionnaire (Hutchings 1996) was used to collect Journal of Child and Family Studies (2020) 29:686–698 691 Beck Depression Inventory II (BDI-II) Results Families in the intervention condition were asked to com- plete a satisfaction questionnaire about the intervention at the final session with their health visitor. Fifteen families (52%) completed this, 13 of whom had completed all three phases of EPaS and two who had completed the assessment and case analysis phases. Satisfaction levels were high with 96% rating different aspects of the intervention as helpful (range 80–100%). Ninety-three per cent would recommend the programme to other parents. Feasibility outcomes Follow-up retention rates are reported as percentages. Any differences in demographic characteristics and baseline scores between families lost to follow-up and those who remained in the study were explored using independent t- tests and chi-square tests. Programme delivery was explored by examining the percentage of parents that received each of the three EPaS phases and mean number of sessions received. Mean percentages from the parent satisfaction questionnaire are reported. Journal of Child and Family Studies (2020) 29:686–698 692 Initial effectiveness Of the 29 families in the intervention condition, 27 (93%) of the families completed the assessment phase, 18 (62%) completed the assessment and case analysis phase and 15 (51.7%) completed all three phases of the EPaS programme. No data were available for two families. The mean number of home visits for the intervention families was 6.91 (SD = 2.75, range = 2–12). Of the 29 families in the intervention condition, 27 (93%) of the families completed the assessment phase, 18 (62%) completed the assessment and case analysis phase and 15 (51.7%) completed all three phases of the EPaS programme. All data analyses were completed using SPSS version 24. Variable residuals were examined using P-P plots to detect any violations in normality. Four variables, namely BDI-II, observed positive parenting, observed social-emotional coaching, and observed negative parenting were found to violate normality assumptions, and were therefore normal- ised using square-root transformations. The square-rooted values were used in the analyses. To account for missing data at follow-up, analyses were conducted using last observation carried forward. Repeated measures ANOVA models were conducted with time as the within-group variable and condition as the between-group variable. Effect sizes were calculated by dividing the model estimate with the pooled standard deviation at baseline. Interpretation was based on Cohen’s d values (Cohen 1988). No data were available for two families. The mean number of home visits for the intervention families was 6.91 (SD = 2.75, range = 2–12). Because the control group was TAU, some families (n = 7; 24%) also received visits from their health visitor for support for their child’s behavioural challenges (M = 1.26, SD = 2.90, range = 0–13). Five received generic beha- vioural support, one was referred to the Triple-P pro- gramme, and one received an intensive support package delivered by a family support worker alongside the health visitor. Study Retention Twenty-two families (38%) were lost to the six-month follow-up, ten (34%) intervention and 12 (41%) TAU families (see Fig. 1). For the ten intervention families, nine could not be contacted at follow-up and one had moved. For the 12 TAU families, nine withdrew because of reported improvements in their child’s behaviour when contacted by researchers at follow-up, two had moved, and one withdrew because of a long-term illness. There were no significant differences between those lost to follow-up and those not lost in terms of demographic characteristics or baseline data (p > 0.05). Initial Effectiveness BDI-II Beck Depression Inventory II aObserved outcomes Table 2 Descriptive statistics for intervention and control Table 2 Descriptive statistics for intervention and control Journal of Child and Family Studies (2020) 29:686–698 693 Table 3 Results of repeated measures ANOVA models Outcomes Estimate (95% CI) Effect size (95% CI) ECBI intensity Condition −3.59 (−18.99, 11.82) −0.13 (−0.66, 0.41) Time −6.83 (−14.02, 0.37) −0.24 (−0.49, 0.01) ECBI problem Condition −1.55 (−4.70, 1.60) −0.28 (−0.84, 0.29) Time −2.24 (−3.95, −0.53)* −0.40 (−0.70, −0.09)* Abbreviated conners Condition −1.35 (−4.11, 1.41) −0.26 (−0.78, 0.27) Time −1.64 (−2.79, −0.48)* −0.31 (−0.53, −0.09)* PS laxness Condition −0.08 (−0.70, 0.54) −0.06 (−0.56, 0.43) Time −0.21 (−0.38, −0.05)* −0.17 (−0.30, −0.04)* PS overreactive Condition −0.40 (−0.86, 0.07) −0.43 (−0.93, 0.08) Time −0.04 (−0.19, 0.12) −0.04 (−0.21, 0.13) PS total Condition −0.20 (−0.60, 0.20) −0.25 (−0.75, 0.25) Time −0.12 (−0.25, 0.01) −0.15 (−0.32, 0.01) BDI-II Condition −0.30 (−1.01, 0.42) −0.22 (−0.73, 0.30) Time −0.30 (−0.59, −0.01)* −0.22 (−0.42, −0.01)* Observed positive parent Condition −0.17 (−0.98, 0.64) −0.10 (−0.58, 0.38) Time −0.21 (−0.63, 0.21) −0.12 (−0.37, 0.12) Observed SE coaching Condition 0.01 (−1.47, 1.49) 0.003 (−0.50, 0.51) Time −0.49 (−0.98, 0.00) −0.17 (−0.33, 0.00) Observed negative parent Condition −0.22 (−0.98, 0.55) −0.14 (−0.61, 0.34) Time −0.13 (−0.41, 0.16) −0.08 (−0.25, 0.10) ECBI Eyberg Child Behavior Inventory, Conners abbreviated conners, PS Parenting Scale, BDI-II Beck Depression Inventory II EPaS programme in its current form within existing health visiting services is questionable and requires further examination. There was limited evidence of programme effectiveness; however, the results should be interpreted in context due to a number of challenges. The results of the analyses indicated that child behaviour problems, lax parenting, and parental depression sig- nificantly reduced over time for the whole sample. How- ever, there was no significant difference between families in the intervention and TAU control conditions at follow-up. The significant whole group results should be interpreted with caution since they may be due to regression to the mean (Barnett et al. 2004). There was some evidence of a stronger effect on several key measures for families in the intervention condition based on small effect sizes; however, the differences were not significant. There is increased variability in the scores at follow-up, reflected by larger standard deviations, suggesting a large range of change in scores. This may be why some of the reductions in scores seen in the intervention condition were not significant changes. Initial Effectiveness Descriptive statistics are displayed in Table 2. Results from the repeated measures ANOVA models can be seen in TAU, WL Control Intervention Baseline Follow-up Baseline Follow-up n M (SD) n M (SD) n M (SD) n M (SD) ECBI intensity 29 163.07 (27.56) 17 169.82 (38.08) 29 167.62 (30.07) 19 144.53 (40.53) ECBI problem 29 20.31 (5.29) 17 20.12 (7.96) 29 20.31 (6.01) 18 14.22 (9.81) Conners 29 21.47 (4.97) 17 21.00 (5.43) 29 20.90 (5.60) 19 17.79 (6.70) PS laxness 29 3.21 (1.23) 17 3.11 (1.30) 29 3.23 (1.32) 19 2.86 (1.39) PS overreactive 29 2.84 (0.76) 17 2.75 (1.01) 29 2.47 (1.04) 19 2.27 (0.85) PS total 29 3.24 (0.71) 17 3.22 (0.94) 29 3.14 (0.89) 19 2.84 (0.86) BDI-II 29 21.01 (11.64) 17 19.68 (13.80) 29 19.05 (11.36) 19 15.21 (13.95) Positive parenta 29 26.93 (23.12) 16 17.75 (17.05) 29 20.21 (13.64) 19 22.11 (14.17) SE coachinga 29 104.90 (52.28) 16 85.88 (56.53) 29 108.41 (74.20) 19 91.53 (14.17) Negative parenta 29 19.45 (17.66) 16 16.25 (12.01) 29 16.38 (11.01) 19 12.74 (7.83) TAU treatment as usual, WL waitlist, ECBI Eyberg Child Behavior Inventory, Conners abbreviated conners, PS Parenting Scale. Initial Effectiveness The lack of differences between conditions may also be due to the TAU nature of the control condition or the flawed design whereby each health visitor identified two families with whom to work, one randomised into the intervention and the other to the control condition. This means that parents in the control condition still had access to their health visitor if needed. The design was imple- mented due to restrictions in time and resources and in order to try to maximise the recruitment of families. It also meant that the training only had to be delivered to one set of health visitors who could then implement the programme with their families. Records kept by health visitors to monitor visits showed that seven families from the TAU control condition did receive support from their health visitor dur- ing the intervention phase. It is not possible to know whe- ther the health visitors used some of the skills taught in the EPaS training with their control families. Another possibi- lity to the lack of between-group differences is that the families in each condition were not equivalent at follow-up. As previously mentioned, there were no significant differ- ences between conditions in terms of demographics and baseline characteristics; however, the CONSORT diagram shows that nine families in the control condition dropped out because of child behaviour improvements. This was not the case for those who dropped out of the intervention condition, indicating differential attrition. D li f h bl i h 93% f Table 3. There was a significant main effect of time for ECBI Problem, Abbreviated Conners, PS laxness, and BDI- II indicating a reduction in scores from baseline to follow- up with small effect sizes. There was no significant effect of condition with all confidence intervals crossing zero. However, there were small effect sizes favouring the intervention condition for ECBI Problem, Abbreviated Conners, PS over-reactivity, PS total, and BDI-II. Delivery of the programme was acceptable with 93% of families completing the assessment phase. It is not a requirement of the EPaS programme that parents must complete all three phases. It is possible that the assessment phase alone may have been enough to elicit changes for some families, as found in the Family Check-Up interven- tion (FCU; Dishion et al. 2008; Shaw et al. 2006) in which Discussion This study explored the feasibility of delivering an indivi- dually administered parenting programme within existing health visiting services. The feasibility of delivering the Journal of Child and Family Studies (2020) 29:686–698 694 families undergo three sessions of assessment based on motivational interviewing techniques and are offered addi- tional follow-up sessions if needed. The FCU programme has shown significant results for child behaviour problems (Dishion et al. 2008) with maintenance effects up to 7.5 years (Dishion et al. 2014), but some parents do not opt for the follow-up sessions, indicating that the assessment is sufficient for them to recognise more effective parenting strategies. This may have been why some parents in the present study did not complete all three phases of the EPaS programme. to implement the intervention. Seventy-four percent of families identified met the eligibility criteria and 93.3% consented to take part (Hutchings, Bywater and Daley 2007). This is in contrast to the current trial whereby only 87 families were identified, 87% met the eligibility criteria, and 67% consented to participate. The lower numbers of iden- tified families may be partly due to the creation of govern- ment schemes such as the Welsh Government Flying Start programme, specifically targeting young children aged 2 and 3 years with additional support including parent groups, language and play sessions, and enhanced health visiting support (Welsh Government 2016). Parents reported high rates of satisfaction with the EPaS programme suggesting that the programme is acceptable to families of young children with behaviour problems. An earlier paper on the outcomes for health visitors also showed high rates of programme acceptability (Williams and Hutchings 2018). However, the results should be interpreted with caution. In the current study, parent satis- faction was collected using a self-report questionnaire dur- ing the final EPaS session with their health visitor. The ratings may be subject to participant response bias since parents may have felt pressured to give positive responses to please their health visitor. Also, only 52% of the inter- vention families returned the satisfaction questionnaire. Qualitative interviews with an independent researcher would have been more useful since they would provide a richer source of data and reduce the risk of bias. Retention at the six-month follow-up was poor with only 62% of families providing data. Discussion There were no significant differences between families lost at follow-up and those who remained in the study, and drop-outs were relatively evenly distributed across the conditions (10 intervention and 12 control families). Other studies have found a number of predictors associated with lower retention. These include demographic characteristics such as low socioeconomic status, unemployment, low education, single parent status, younger maternal age, and ethnic minority status (e.g., Fernandez and Eyberg 2009; Reyno and McGrath 2006; Robinson et al. 2016). The families in the current study showed high rates of poverty, unemployment, and low education with two-thirds of the sample reporting three or more socioeconomic risk factors. When taking into account the level of disadvantage in the sample, the attrition rate is similar to other studies of individually delivered parenting programmes (e.g., 39% in Fernandez and Eyberg 2009; 49% in Werba et al. 2006). Challenges A survey of their skills showed that they used evidence-based techniques as part of their work Journal of Child and Family Studies (2020) 29:686–698 695 with families (see Williams and Hutchings 2018); however, the rates were very variable with some using many techni- ques and others using very few. This means that more experienced health visitors may be better equipped to deliver the intervention. There are also issues around health visitor competence to consider. This was the first time that the health visitors had delivered the EPaS programme. Many RCTs ensure that facilitators have delivered the programme at least once before implementing as part of research (Hutchings, Bywater and Daley 2007), however this was not possible for the current trial. There are also questions around health visitor inexperience, as described above, meaning that some may not have had the skills to deliver the intervention with fidelity. There were no eligibility criteria based on the health visitors’ experience. It is also possible that health visitors’ prior learning and behaviour/expectations may have affected the way they delivered the intervention which, in turn, may have affected the results. Nevertheless, overall, families in both the intervention and control conditions showed significant improvements in child behaviour, lax parenting, and parental mental health. (Lavigne et al. 2010). Individually delivered programmes are more effective for hard to engage, vulnerable parents who often have complex problems that require individually focussed interventions (Lundahl et al. 2006). In the UK, health visitors provide parenting support as part of their universal access to families of children under 5 years of age (Myors et al. 2014). They are good at identifying families of children with behaviour problems (Hutchings et al. 2007), but with recent changes to their role, they may not have the time or relevant training to deliver social learning theory- based programmes. The EPaS programmes contains components of effective interventions (e.g., live coaching, social learning theory principles, homework assignments; Kaminski et al. 2008). Previous evaluations of the EPaS programme reported positive outcomes (Hutchings et al. 2002, 2004; Hutchings and Williams 2013; Lane and Hutchings 2002), but the programme required adaptations to the training format. The results of the current study are promising, with good rates of acceptability for parents and health visitors (see Williams and Hutchings 2018), but limited evidence of effectiveness. Challenges It is clear that the roles of health visitors have significantly changed since the earlier trial (Lane and Hutchings 2002), with less time available for attending training, conducting research related activities, and systematic support for families. This suggests that health visitors may not be the best choice as facilitators. The original version of the EPaS programme was developed for CAMHS-based workers working with children with severe behaviour problems (Hutchings et al. 2002). It was later developed for use by health visitors due to the limited accessibility to families when delivered through CAMHS. However, health visitor time restraints and lack of clinical supervision in the current study meant that they struggled to identify and engage families in the intervention. Given the risks of poor long- term outcomes for children with early onset behaviour problems, this suggests the importance of targeting a dif- ferent facilitator population (e.g., CAMHS-based primary care workers) for the programme to be delivered as intended. Implementation fidelity is the degree to which an intervention is delivered as intended by the developer. It encompasses not only adherence to the intervention manual but also facilitator competence in terms of com- ponents of the intervention and clinical skills (McMahon and Pasalich 2018). The implementation of a programme can have significant effects on outcomes (Kaminski et al. 2008; McMahon and Pasalich 2018), therefore monitoring implementation fidelity is important. For the current study, all health visitors received a detailed intervention manual and three days of training. The training sessions were delivered one month apart which enabled some supervision as part of the training process; however, clinical supervision sessions on individual cases were not available. The lack of clinical supervision is particularly problematic since it is an important aspect of any inter- vention delivery (Flay et al. 2005) and may have affected health visitors’ abilities to effectively apply the inter- vention. Clinical supervision had been intended and agreed upon by the collaborating services involved in the project; local clinical psychologists were going to provide supervision within their locality; however, due to sche- duling difficulties, this did not happen (with the exception of one site). This is a significant limitation, particularly given the high levels of need within the families recruited, and has implications for the interpretation of the results. Challenges Recruitment for the trial was more difficult than expected. Recruitment difficulties in RCTs are not unusual and are well documented, with an estimated 60% of RCTs failing to reach recruitment targets (McDonald et al. 2006; Robinson et al. 2016; Watson and Torgerson 2006). The planned sample size was 60 health visitors and 120 families; how- ever, only 29 health visitors and 58 families participated in the study. Preliminary meetings with health visiting man- agers during study inception were positive and managers reported that the planned study would be feasible. After commencement of the study, the reorganisation of tier 1 services in the health service, including the role of health visitors, was announced and implemented. This may have affected health visitors’ abilities to dedicate time to partici- pating in the research study. Difficulties in identifying eli- gible families for the study were also unexpected although those identified were reporting, on average, very significant problems with their children. In a similar trial conducted by Hutchings et al. (2007) health visitors identified over 200 families, with young children displaying clinical levels of behaviour problems. That trial targeted a similar population from similar areas as the current trial, used similar eligibility criteria, but only used health visitors to identify families not Another factor that may have influenced retention and recruitment could be health visitor inexperience. The med- ian time working as a health visitor was three years. This was very different from the Lane and Hutchings (2002) evaluation of the EPaS programme in which health visitors reported means of between 9 and 11 years’ experience. The relationship between health visitors and their clients is of utmost importance, especially when working with vulner- able families, in order to develop an understanding of the family circumstances and when introducing intervention programmes (Whittaker 2014). Vulnerable families are much harder to engage in intervention programmes (Lavigne et al. 2010; Reyno and McGrath 2006). Because many of the health visitors were relatively inexperienced, it is possible that they may not have developed the skills needed to form strong, trusting relationships with parents on their caseloads which may have impacted on parents’ engagement with the intervention. The health visitors also lacked experience in delivering evidence-based behavioural interventions since this was the first time they had delivered the EPaS programme. Limitations and Future Research Directions This study has a number of limitations. First, the sample size was very small and the attrition rate at follow-up was high with 38% of families lost to follow-up. Second, data were not collected on any services used by the families in the TAU control condition. It is possible that control family access to other services may have contributed to the lack of significant differences between conditions. Third, there may have been a knowledge contamination effect due to the fact that health visitors were trained in the EPaS programme but had access to the control families as part of TAU. Fourth, Childhood behaviour difficulties are a significant pro- blem not only to families but also society. Without social learning theory-based intervention, these problems can lead to long-term poor outcomes for children (NCCMH 2013). Group based parenting programmes are effective but not always accessible, especially for vulnerable families 696 Journal of Child and Family Studies (2020) 29:686–698 number: 2014-12886, and the North West Wales Research Ethics Committee in July 2014, application number: 14/WA/0187. All pro- cedures performed in the study were in accordance with the APA ethical standards in the treatment of the study sample. number: 2014-12886, and the North West Wales Research Ethics Committee in July 2014, application number: 14/WA/0187. All pro- cedures performed in the study were in accordance with the APA ethical standards in the treatment of the study sample. the randomization did not result in equivalent groups, leading to differential attrition which may have affected the between-group results. Also, due to scheduling difficulties, clinical supervision for the participating health visitors by primary care clinical psychologists did not happen. Informed Consent Informed written consent was obtained from all individual participants included in the study. The results of this feasibility trial suggest some future directions. First, an exploration of different recruitment strategies is warranted to try to maximise the number of families being identified. Lack of time was a significant barrier for health visitors in participating in the trial and also identifying families from their caseloads. It is possible that utilising other services, such as the Welsh Government- funded Flying Start Initiative, may be a better way of identifying parents. Second, further feasibility work around retention strategies would also be useful because of the high attrition level at follow-up. Robinson et al. References Arnold, D. S., O’Leary, S. G., Wolff, L. S., & Acker, M. M. (1993). The Parenting Scale: a measure of dysfunctional parenting in discipline situations. Psychological Assessment, 5, 137–144. https://doi.org/10.1037/1040-3590.5.2.137. Barlow, J., & Coren, E. (2018). The effectiveness of parenting programs: a review of Campbell reviews. Research on Social Work Practice, 28, 99–102. https://doi.org/10.1177/1049731517725184. Barnett, A. G., Van Der Pols, J. C., & Dobson, A. J. (2004). Regression to the mean: what it is and how to deal with it. International Journal of Epidemiology, 34, 215–220. https://doi. org/10.1093/ije/dyh299. Barth, R. P., & Liggett-Creel, K. (2014). Common components of parenting programs for children birth to eight years of age involved with child welfare services. Children and Youth Ser- vices Review, 40, 6–12. https://doi.org/10.1016/j.childyouth. 2014.02.004. Beck, A. T., Steer, R. A., & Brown, G. K. (1996). Manual for the Beck Depression Inventory-II. San Antonio: Psychological Corporation. Acknowledgements The authors thank the health visitors and families who took part in the study. Brook, J., & Salmon, D. (2017). A qualitative study exploring parental perspectives and involvement in health visiting services during the Health Visitor Implementation Plan in the South West of England. Health and Social Care in the Community, 25, 349–356. https://doi.org/10.1111/hsc.12308. Authors’ Contributions MEW: designed and executed the study, conducted data analyses, and wrote the paper. ZH: assisted with data analyses and collaborated with writing of the study. DAO: assisted with executing the study (data collection) and collaborated in the writing of the final manuscript. JH: designed and assisted in executing the study, collaborated in the writing and editing of the final manuscript. Cohen, J. (1988). Statistical power analysis for the behavioural sci- ences. Hillsdale: Erlbaum. Conners, C. K. (1994). The Conners rating scales: use in clinical assessment, treatment planning and research. In M. E. Maruish (Ed.), Use of psychological testing for treatment planning and outcome assessment (pp. 467–497). Hillsdale: Erlbaum. Funding This work was funded directly by an anonymous donation and coordinated through the Development and Alumna Relations Office, Bangor University. The funder had no involvement in the conduct of the research and/or preparation of the article. Cowley, S., Caan, W., Dowling, S., & Weir, H. (2007). What do health visitors do? A national survey of activities and service organisation. Public Health, 121, 869–879. https://doi.org/10. 1016/j.puhe.2007.03.016. Cowley, S., Whittaker, K., Grigulis, A., Malone, M., Donetto, S., Wood, H., & Maben, J. (2013). Why health visiting? References A review of the literature about key health visitor interventions, processes and outcomes for children and families (Report No. 016 0058). London: National Nursing Research Unit. Limitations and Future Research Directions (2015) identify a number of retention strategies used in RCTs, some of which were not used in this study. For example, financial incen- tives are commonly used, with over half the studies iden- tified in the Robinson et al. (2015) review utilising them. The current study utilised a non-financial incentive in the form of a children’s book for research participation. It is possible that the use of a financial incentive would have been more effective at retaining parents in the research at follow-up, especially since the level of disadvantage was high. Finally, health visitors struggled with time restrictions within their roles and there were questions around compe- tency to deliver the intervention with high fidelity. There- fore, future research should explore whether other staff (e.g., CAMHS-based primary care workers) would be better placed to deliver the EPaS programme. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://crea tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Compliance with Ethical Standards Conflict of Interest M.E.W., Z.H., and D.A.O. declare that they have no competing interests. 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Bloqueio auriculo-ventricular total congênito em indivíduo com fibroplastose endocardiaca e persistência do conduto arterial: Diagnóstico in útero
Revista do Instituto Adolfo Lutz
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BLOQUEIO AURíCULO-VENTRICULAR TOTAL CONGÊNITO EM INDIVíDUO COM FIBROPLASTOSE ENDOCARDíACA E PERSISTÊNCIA DO CONDUTO ARTERIAL Diagnóstico in utero * MARCOS FABIO LION EVANDRO PIMENTA ** DE CAMPOS *** A fibroelastose endocardíaca ocorre associada a cardiopatias congênitas, ou surge como entidade isolada, discutindo-se, neste caso, a etiologia congênita ou adquirida. Publicou-se muito sôbre o assunto, ainda que sejam raros os trabalhos onde há associação do processo de fibroelastose endocardíaca com a persistência do conduto arterial. De outro lado, são inúmeros os casos divulgados de bloqueio aurfculo-ventricular total na primeira infância, com a hipótese de que êste distúrbio do ritmo seja consecutivo a transtornos ocorridos durante a gestação. Entretanto, os casos em que há suspeita de bloqueio aurículo-ventricular total durante a gestação, pela baixa freqüência das pulsações fetais cardíacas e confirmação, após o nascimento, pelo eleetrocardiograma, são mais raros. Em nosso paciente, suspeitou-se da existência do bloqueio aurfculo-ventrlcular total durante a gestação, e a criança era portaTrabalho do Serviço de Cardiologia do Departamento de Medicina (Serviço do Prof. Luiz V. Décourt) da Faculdade de Medicina da Universidade de São Paulo; do Departamento de Anatomia Patolôgíca (Serviço do Prof. Constantino Mignone) da Faculdade de Medicina da Universidade de São Paulo e da Diretoria de Patologia do Instituto Adolfo Lutz. Publicado em Arch. Inst. Cardo Mex. 30: 1960 . .,.'" Do Serviço de Cardiologia (Prof. Luiz V. Décourt). Ex-residente interno do LN.C., México, D.F. ,.* Do Departamento de Anatomia Patológica da Faculdade de Medicina da USP (Serviço do Prof. Constantino Mignoni) e Diretor da Diretoria de Patologia do Instituto Adolfo Lutz. Patologista agregado ao Serviço do Prof. Luíz Décourt. Recebido para publicação em 24 de julho de 1961. Rev. Inst, Adolfo Lutz, 1961, V. 21. 18 REVISTA DO INSTITUTO ADOLFO LUTZ dora de fibroelastose endocardíaca congênita e persistência do conduto arterial. A raridade dessa associação animou-nos a publicar o caso. Em 1928, AYLWARDpublicou um caso de bloqueio aurtculo-ventricular total, com freqüência ventrícular de 48 pulsações por minuto, e diagnóstico feito durante a gestação. O paciente tinha irmão, também, com bloqueio aurículo-ventricular total, o que é particularidade interessante. Em 1930, LEECH publicou caso de bloqueio aurículo-ventricular total, associado a persistência do conduto arterial, com diagnóstico feito quando o paciente tinha cinco anos e meio de idade. Neste caso, supunha-se bloqueio aurfculo-ventrícular total congênito, não havendo verificação antes do nascimento. Quanto à persistência do conduto arterial, não houve comprovação anátomo-patológíca. SANKEY, em 1948, encontrou na literatura 10 casos de bloqueio aurículo-ventrlcular total, cujo diagnóstico poderia ser previsto antes do parto, visto a menor freqüência ventrícular, após o nascimento, ser de 40 pulsações por minuto. A êstes 10 casos, juntou um de sua experiência. Em nenhum dêles, há menção à presença de conduto arterial persistente ou de fibroelastose endocardíaca. Em 1950, STADLERet alii publicaram caso de bloqueio aurículoventrícular total, cuia freqüência ventrícular mais baixa foi de 42 pulsações por minuto. Fêz-se o diagnóstico de bloqueio, durante a gestação. A necrópsía revelou hipertrofia de ambos os ventrículos, dilatação da auríeula direita e do ventrfculo direito, e fibroelastose endocardíaea, KELLY & ANDERSEN, em 1956, fizeram revisão bibliográfica sôbre a fibroelastose endocardíaca e publicaram 2 casos desta enfermidade, com grande comprometimento do ventrículo direito e do ventrículo esquerdo, associado a bloqueio auriculo-ventrlcular total. A freqüência cardíaca mais baixa encontrada foi de 28 por minuto. Também, em 1956, SANTOROestudou caso de fibroelastose endo cardíaca, com envolvimento de todo o coração, e bloqueio aurículoventr icular total, suspeitando-se do diagnóstico durante a vida intra-uterina, Recentemente, em 1957, DEVITT & PINTO publicaram caso semelhante. OBSERVAÇõES Antecedentes familiares: DO PACIENTE mãe de 35 anos de idade; teve febre reumática aos 25 anos, de curta duração, sem seqüelas cardíacas. Nessa ocasião fêz amígdalectomia. O pai, há 9 anos, teve tuberculose BLOQUEIO AURÍCULO-VENTRICULAR •.• ------~~-~---~--~-- 19 pulmonar, seguida de cura. Primeira gestação. Examinada periodicamente, durante a gestação, até o 5.° mês. Nesta ocasião apresentava auscultação normal do foco fetal. Na véspera do parto, que chegou a término, verificou-se a freqüência de pulsações do foco fetal, sendo de 40 por minuto, em ritmo regular. O parto foi normal. A mãe não sofreu infecção ou traumatismo durante a gravidez. Histórico da doença atual: paciente do sexo feminino, raça branca, pesando 1.920 g ao nascer. Chorou imediatamente após o nascimento e não apresentou cianose. No exame clínico, não se encontrou nada de anormal, com exceção dos dados do aparelho cardiovascular. Verificou-se bradicardía acentuada, com freqüência cardíaca de cêrca de 25 pulsações por minuto. Não apresentou frêmitos ou sôpros e os ruídos cardíacos eram normais, ainda que intensos, durante algumas revoluções cardíacas. As artérias dos membros superiores e inferiores eram palpáveis e o pulso normal. Não se tomou a pressão arterial. Não se verificaram sinais de insuficiência cardíaca. O electrocardíograma, nesta ocasião, mostrou bloqueio aurículo ventrícular total e hípertrof'ia ventricular esquerda. Em conseqüência da bradicardia, colocou-se a menina em incubadora com oxigênio. No terceiro dia de vida, uma chapa do tórax mostrou cardíomegalia acentuada, sem alterações pleuropulmonares, ou da parede costal. No quarto dia de evolução, escutou-se sôpro sistólico, em todo o precórdio. No 5.° dia de vida, apareceu sôpro diastólico, audível também, em tôda a região precordíal, Os componentes sistólico e diastólico do sôpro separavam-se pelo segundo ruído, não havendo, portanto, o caráter de sôpro contínuo. No 6.° dia, além de acentuada bradícardia persistente e da presença dos sopros, verificou-se a aparição de extrassístoles freqüentes. O electrocardíograma, nesta ocasião, mostrou bloqueio aurículo-ventricular total e hípertrofía ventrícular esquerda, com extrassístoles ventriculares freqüentes. Nova radiografia do tórax, aos 12 dias, mostrou aumento da área cardíaca. Aos 15 dias de vida, desapareceram as extrassístoles e o ritmo se regularizou. Desde então, a freqüência ventrícular variou de 27 a 35 pulsações por minuto. Durante tôda a evolução da doença da paciente, houve pouco aumento de pêso, variando de 1.920 a 2.120 g. Do 20.0 dia em diante, apresentou ligeira diarréia e discreta icterícia. No 35.° dia, continuava a diarréia, acentuando-se, ligei- 20 REVISTA DO INSTITUTO ADOLFO LUTZ ramente, a icterícia. Diagnosticou-se desidratação; em palpações, verificou-se o fígado a 3 dedos do bordo costal direito, e o baço a um dedo do bordo costal esquerdo. No 36.° dia, apareceu dispnéía intensa, coríza e tiragem acentuada. A menina faleceu, com o pêso de 1.970 g. Exames complementares: Hematôcrito (2.° dia) - 55% H emocitotôaico (20.° dia) Erítrócitos : 4.900.000/mm:1• Hemoglobina : 16,2 g/% = 101 %. Leucôcitos : 6.000/mm3, com contagem diferencial normal para a idade. Hemocitolôçico (35.° dia) Hemoglobína : 11,3 g % Leucócitos: 12.300/mm3• 71 % Radiografia do tó(3.° dia) : cardíomegalia acentuada, principalmente no ventr ículo esquerdo. Campos pulmonares normais (figura 1). J'aX Electrocardiograrna (1.° dia) - Bloqueio aurículo-ventricular total, com freqüência aurícular de 125 por minuto e freqüência ventricular de 25 por minuto. O foco da origem do ritmo ventricular encontrava-se situado antes da bifurcação do Feixe de Hiss. QR8; 0,7 sego 8AP a F'ig. 1 - Radiografia do tórax, póstero-anterior. 80°, dirigido para trás. Terceiro dia de vida Ondas P difásicas, aumentada em DI, diminuída em VI, V2 e V3, com depressões em V4 e V5, e com ápice em D2, D3 e VF; duração de 0,055 sego e voltagem de 3,2 mm em D2. 8AQR8 a 30°, dirigido para trás. Complexo 1'8 de VI a V5, ondas 8 com depressões em V4 e V5. Onda "R" pura em V6. 8AT a 40°, orientado para frente. Ondas T positivas de VI a V6. + + + BLOQUEIO AURÍCULO- VENTRICULAR ooo 21 Conciueõo: Bloqueio aurículo-ventricular total. Ritmo ídioventricular com origem acima da bifurcação do feixe de Hiss. Hipertrofia ventrícular esquerda (fig. 2). A Figo 2 - Electrocardiograma. B Primeiro dia de vida Electrocardiograma (6.0 dia) - Em comparação com as gravações anteriores, nota-se o aparecimento de complexos ventriculares extensos, empastados, prematuros, com provável foco de origem no ventriculo esquerdo (fig. 3). Figo 3 -0- Electrocardiograma. Sexto dia de vida 22 REVISTA DO INSTITUTO ADOLFO LUTZ Electrocardíograma (35.° dia). Em comparações com as gravações anteriores, observa-se o desaparecimento das extrassístoles. Aumento da freqüência auricular a 170 por minuto. AP passou de + 80° a + 60°. Diminuição da voltagem da onda P em VI e V2. Aparecimento dos complexos rSr' em V6 (transtornos da condução íntr a-veritr cular de estímulo?) (fig. 4). Terapêutica: A criança recebeu ácido pangâmíco (vitamina B 15), na dose de 10 mg/24 horas, íntramuscular, do 3.° ao 15.° dia. AlimenFig. 4 - Electrocardiograma. Trigésimo quinze dia tação feita com leite de vida em pó. Exame amátomo-paioíáçieo: í- Coração de forma globular, com volume aumentado, principalmente no ventrículo esquerdo; 27 g de pêso (normal, cêrca de 20 g). Ventrículo esquerdo: endoeárdío ligeiramente fibroso em tôda a extensão. Músculos papílares, ligeiramente hipertrófícos e lisos. Colunas carnosas lisas. Miocárdio pálido com ligeiras áreas levemente amarelas. Epicárdio liso e brilhante. Capacidade muito aumentada. Aurícula esquerda: dade pouco aumentada. endocárdio, ligeiramente Discreta hipertrofia. fibroso. Capaci- Ventrículo direito: endocárdio, discretamente fibroso. Colunas carnosas e músculos papílares aumentados e lisos. Capacidade aumentada. Aurícula direita: endocárdío mentada. Discreta hípertrofía. normal. Capacidade pouco au- 23 BLOQUEIO AURÍCULO-VENTRICULAR... Espessura do míocárdío no ventrículo média 1,5 mm; ápex 1,5 mm. direito: base 2 mm; parte Ventrículo esquerdo: via de saída 33 mm, via de entrada 33 mm. Ventrículo direito: via de saída 42 mm, via de entrada 25 mm. Válvula pulmonar: 19 mm. vula tricúspíde : 40 mm. Válvula aórtica : 18 mm. Vál- Vasos da base: ligeira dilatação da porção inicial da aorta, Conduto arterial persistente, de 5 mm medindo 2,5 em de diâmetro. de cumprimento e diâmetro exterior de 2 mm (figs. 5, 6, 7 e 8). Fig. 5 - Na parte inferior, ventrículo esquerdo dilatado e htpertrófíco. Na parte superior, aurícula esquerda dilatada. Ausência de comunicação ínter-aurícular. V. E.: ventrículo esquerdo. A. E.: aurícula esquerda 24 REVISTA DO INSTITUTO ADOLFO LUTZ Fig. 6 - Ventrículo esquerdo e porção inicial da aorta. Fibroelastose endocardíaca, Colunas carnosas lisas. ventrículo esquerdo hipertrófico. Ausência de comunicação ínter-ventrícular Fig. 7 - Ventrículo direito artéria pulmonar. Veutrículo tado. Endocárdio de aspecto nicação ínter-ventrícular. V. D.: ventrículo direito. aberto e porção inicial da direito hipertrófico e dilafibroso. Ausência de comuOrifício pulmonar normal. A. P.: artéria pulmonar BLO'lUEIO AURÍCULO-VENTRICULAR ... 25 Conclusão: hipertrofía e dilatação globais do coração, com predomínio da dilatação, principalmente do ventrículo esquerdo. Persistência do conduto arterial. Fibroelastose endocardíaca, com exceção da aurícula direita. Exame histopatológico: Fizeram-se cortes do coração nas áreas correspondentes ao nódulo de Keith-Flack, ao nódulo de Fíg. 8 Ventrículo direito aberto, mostrando evidente hipertrofia. Na parte superior da figura a aorta com ramos iniciais. Assinalado com estilete, o conduto arterial persistente. V. D.: ventríoulo direito. Ao: aorta. C. A.: conduto arterial Aschoff-Tawara, ao feixe de Hiss e ramificações. Colheram-se fragmentos dos dois ventrículos e de ambas as aurículas. Colorações: com hema toxílina-eosina, o miocárdío : com Weigert, as fibras elásticas; e com van Gieson, o tecido conjuntivo. 26 REVISTA DO INSTITUTO ADOLFO LUTZ A dissecção macroscópíca do sistema condutor do coração, feita depois de fixação com auxílio de lupa, ofereceu dificuldades em virtude da difícil identificação do tecido. As colorações dos cortes das regiões do sistema condutor não oferecem meios para verificação, sob o ponto de vista hístológico. A'Üexame hístológico, o tecido endocardíaco mostrou-se espêsso, fàcilmente visível pela hematoxilina-eosina, As colorações específicas de Weigert e de van Gieson demonstraram ser o espessamento produto da presença de fibras conjuntivas maior quantidade, na parede do ventrículo áreas. Êsse espessamento mostrou ser 10 coração normal do mesmo pêso e de igual e de fibras elásticas, em esquerdo, que em outras vêzes maior que o de um idade. No coração nor- mal, observou-se, somente, uma delgada capa de tecido conjuntivo, com poucas fibras elásticas. As amostras aproveitadas para comparação foram colhidas da face anterior do ventrículo esquerdo. Verificou-se fàcilmente êsse espessamento nas outras regiões, não sendo, entretanto, tão acentuado como no ventrículo esquerdo. O tecido endocardíaco das colunas carnosas e músculos papilares demonstrou acentuado espessamento A (figs. 9 elO). B Plg, 9 - a) Corte da parede ventricular esquerda, face anterior. Espessamento do endocárdio. Fibras elásticas e conjuntívas grandemente aumentadas (fibroelastose endocardíaca) . b) Corte da parede ventrlcular esquerda, face anterior. Indivíduo normal da mesma idade e com o mesmo pêso. Endocárdio de espessura normal. Aum, oco 10 x obj. 10. Col. Weigert - van Gieson 27 BLOQUEIO AURÍCULO- VENTRICULAR ... A B Fig. 10 - a) Corte do ventrículo direito. Espessamento do endocárdlo, Fibras elásticas e conjuntivas aumentadas. Fibroelastose endocardíaca. b) Corte do míocárdío do ventrículo direito, de indivíduo normal, da mesma idade e com o mesmo pêso. Endocárdio com espessura normal. Aum, oco10 x obj. 10. Col. Weigert - van Gíeson Concluímos que se trata, anâtomopatolõgicamente, de coração com fibroelastose endocardíaca, localizada, principalmente, no ventríeulo esquerdo. COMENTÁRIOS O caso é sumamente raro, pois não encontramos semelhante na literatura. As observações seguintes merecem menção particular: a) suspeitou-se de bloqueio aurículo-ventrícular total, durante a gestação. A freqüência cardíaca que, na véspera do parto, era de 40 pulsações por minuto, demonstrou ser a mesma, pelo electrocardiograma, no primeiro dia de vida; b) a freqüência ventricular foi, no primeiro sações por minuto, portanto, mais baixa trada em crianças da mesma idade. casos publicados referem freqüência de ções por minuto; c) interpretou-se, inicialmente, o sôpro sistólico, audível em todo o precórdio, que apareceu no 4.0 dia da evolução, como devido à cardíopatía congênita, talvez do tipo de comunicação intercavitária, por ser a associação mais comum, proveniente da insuficiência mitral funcional ou, ainda, da estenose relativa do orifício aórtico, resultante da bradícardía e da acentuada hipertrofía e dilatação do dia, de 25 puldo que a enconA maioria dos 27 a 40 pulsa- 28 REVISTA DO INSTITUTO ADOLFO LUTZ ventrículo esquerdo. O aparecimento do sõpro diastólico fêz-nos pensar na possibilidade de conduto arterial persistente, o que foi confirmado na necrópsia, A inaudíbilídade dos sopros sistólico e díastólico, nos primeiros dias, seria provável conseqüência do equilíbrio das pressões dos círculos aórtíco e pulmonar. Com a eventual queda da pressão sistólica pulmonar, conseqüência da diminuição normal da resistência no território pulmonar, apareceu o sôpro sistólico e mais tarde, com o desequilíbrio tensional durante a diástole, apareceu o sôpro diastólíco. A ausência de cianose, durante tôda a evolução da enfermidade, falou em favor de um curtocircuito artérío-venoso pelo conduto arterial; d) as radiografias do tórax mostraram sempre acentuada cardiomegalia, aparentemente a expensas de tôdas as câmaras, mas com indiscutível predomínio do ventrfculo esquerdo, o que poderia relacionar-se com o fato de ser aqui a fibroelastose mais acentuada. A avaliação da magnitude da circulação pulmonar foi difícil, devido à cardíomegalia extensa; e) o electrocardiograma mostrou, desde o princípio, bloqueio aurículo-ventrícular total, com freqüência aurícular, pràticamente normal, para a idade; freqüência ventr.ícular extremamente baixa e franca hipertrofia ventricular esquerda; f) pela evolução da paciente, verificou-se muita dificuldade para ganhar pêso. Faleceu com desidratação e icterícia. O exame anátomo-patológico do fígado e das vias biliares revelou, unicamente, trombos biliares intra-hepátícos ; g) apesar da falta de convicção sôbre a ação específica do ácido pangâmíco, administrou-se vitamina Bli3 à paciente, como última tentativa para melhorar as condições do míocárdío ; h) no exame anátomo-patológico, chamaram a atenção a extensa cardíomegalia, o conduto arterial persistente e a fíbroelastose endocardíaca em ambos ventrículos e aurícula esquerda, com predomínio do ventrículo esquer- BLOQUEIO A URÍCULO- VENTRICULAR ... 29 do. Não se verificou nenhum outro defeito congênito do coração. O volume do coração era 4 vêzes maior que o normal, para pacientes da mesma idade e do mesmo pêso. Havia hípertrofía do miocárdio, com espessamento 2 vêzes maior que o normal. Apesar disso, notava-se dilatação de tôdas as cavidades cardíacas. O exame não ofereceu meios para identificação do sistema condutor, visto o coração haver sido dissecado após fixação, o que impediu ulterior impregnação dêsse órgão pelo lugol. RESUMO Os autores descrevem a associação rara de varres defeitos congênitos do coração: bloqueio aurículo-ventrícular total, fibroelastose endocardíaca e persistência do conduto arterial. Suspeitou-se de bloqueio auriculo-ventricular total, durante a gestação, pela baixa freqüência cardíaca do foco fetal. Confirmou-se o bloqueio, no primeiro dia de vida, pelo electrocardiograma. A freqüência ventricular, nesta ocasião, foi extremamente baixa, 25 pulsações por minuto. Confirmou-se a fíbroelastose endocardíaca pelo exame neeróptíco macro e microscópico, havendo envolvimento dos dois ventrículos e da aurícula esquerda, sendo mais intenso no ventrículo esquerdo. O tamanho do coração era cêrca de 4 vêzes o tamanho normal, com ligeiro aumento de pêso. O mioeárdio do ventrículo esquerdo era 2 vêzes mais espêsso, e o endocárdio cêrca de 10 vêzes mais espêsso que o normal. O conduto arterial era patente. A criança, prematura, viveu 36 dias e faleceu com quadro de desidratação. SUMMARY The authors present the case of arare association of several congenítal heart malformations: a complete A-V block, an endoeardial fibroelastosis and a patent ductus arteriosus. The complete atríoventricular block had been suspected duríng pregnancy by the low heart rate of the fetus. It was confirmed on the first day of life with electrocardiogram, The heart rate was extrcmely low, with a ventrícular 25 beats per minute. rate of REVISTADOINSTITUTOADOLFOLUTZ 30 Endocardial fibroelastosis was conf'irmed by the macro and mícroscopícal examination. Both ventricles and the left auricle were involved. It was more marked on the left ventricle. The size of the heart was four times the normal size; its weight was lightly íncreased. The myocardium of the left ventricle was twice as thick as normal and its endocardium ten times as thick as normal. The ductus arteríosus was patent. The child was bom prematurely as a result of extreme dehydration. and lived 36 days. She died SOMMAIRE Les auteurs présentent une rare association de plusieurs anomalies congénitales du coeur : bloc aurtculo-ventrículaíre total, fíbro-élastose endocardíaque et persistance du canal artéríel. Le bloc aurlculo-ventriculaire total avait été suspecté pendant Ia grossesse, en raison de Ia basse fréquence des pulsatíons du foetus. Le premier jour aprês Ia naissance, on a eu confirmation du bloc aurlculo-ventrículaíre par l'électrocardiograrnme. Il faut souligner Ia fréquence ventriculaire extrêmement basse, de 25 battements par minute. La fibro-élastose endocardiaque fut confirmée à Ia nécropsie, .par des examens macro et microscopiques. Elle existait dans les deu x ventricules et dans l'oreillette gauche. La taille du coeur était prês de 4 fois celle d'un coeur normal, avec une légêre augmentatíon du poids. Le myocarde du ventrícule gauche était deux fois plus gros et son endocarde prês de 10 fois plus épaís que le normal. Le canal artéríel était persístant, L'enfant, du sexe fémínín, né prematurément, vécut 36 jours et mourut au cours d'un état de déshydratation. BIBLIOGRAFIA AYLWARD, R. D. - 1928 - Congenital heart bloek. Brit. M. J. 1: 943. DEVITT,R. E. & C. J. PINTO - 1957 - Congenital heart block due to endocardíal fibroelastosis interpreted as foetal distress. J. Obsto & Gynaec. Brit. Emp. 64: 885-7. KELLY, J. & D. H. ANDERSEN - 1956 - Congenital endocardial fibroelastosis. lI. A clínícal and pathological investigation of those cases without associated cardiac malformations íncludíng report of two familiar instances. Pediatrics 18: 539-55. BLOQUEIOAURÍCULO-VENTRICULAR... 31 LEECH,C. B. - 1930 - Congenital complete heart block. Report of a case wíth an associated patent ductus arteríosus. Am. J. Diseases Children 39: 131-140. SANKEY,A. O. et aZii - 1948 - Congenítal heart símulating report of two cases. Brit. M. J. 2 (4579): 676··7. SANTORO, E. V. - 1956 - Congenital endocardial block. Report of case. Arch, Pediat. 73: 94-98. foetal dístress, fibroelastosis A and total heart STADLER, H. E., C. A. REID & H. P. FRIEDMAN - 1950 - Prenatal fibroelastosis ("fetal endocardítís") manifested clinical1y by total heart block. J. Pediat, 36: 370-5.
https://openalex.org/W4390344664
https://www.qeios.com/read/MINY0N/pdf
English
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Review of: "In-Vitro Antibacterial Activity of some Ganoderma Species: A Review"
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2,023
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Qeios, CC-BY 4.0 · Review, December 28, 2023 Qeios ID: MINY0N · https://doi.org/10.32388/MINY0N Review of: "In-Vitro Antibacterial Activity of some Ganoderma Species: A Review" Unnikrishnan Sneha1 Unnikrishnan Sneha1 B.S. Abdur Rahman Crescent Institute of Science and Technology Potential competing interests: No potential competing interests to declare. The article is an extensive review of the in-vitro antibacterial activity of some Ganoderma species. It is well-written with recent references. Qeios ID: MINY0N · https://doi.org/10.32388/MINY0N 1/1
https://openalex.org/W2041623144
https://europepmc.org/articles/pmc3305035?pdf=render
English
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T2-Imaging of the ischemic area-at-risk predicts recovery of cardiac function after acute ST-elevation myocardial infarction
Journal of cardiovascular magnetic resonance
2,012
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T2-Imaging of the ischemic area-at-risk predicts recovery of cardiac function after acute ST- elevation myocardial infarction mean LDL was 118.7 ± 33.0, and the mean HgA1c was 6.7 ± 1.8. The baseline mean LVEF was 42.1 ± 6.6%, and increased to 45.1 ± 9.8%, a mean increase of 3.0%. The mean percentage T2 enhancement was 41.2 ± 17.4%, and the mean burden of LGE was 23.5 ± 14.0%, giving a percentage myocardial salvage of 17.7%. The degree of myocardial salvage correlated moderately with an improvement of LVEF from the acute to recovery CMR, with a Spearman correlation coefficient of 0.59 (p= 0.020). Bourque et al. Journal of Cardiovascular Magnetic Resonance 2012, 14(Suppl 1):P26 http://www.jcmr-online.com/content/14/S1/P26 Bourque et al. Journal of Cardiovascular Magnetic Resonance 2012, 14(Suppl 1):P26 http://www.jcmr-online.com/content/14/S1/P26 Bourque et al. Journal of Cardiovascular Magnetic Resonance 2012, 14(Suppl 1):P26 http://www.jcmr-online.com/content/14/S1/P26 Background T2-weighted edema imaging identifies the ischemic area at risk during acute myocardial infarction. Myocardial salvage occurs in the area at risk without late gadoli- nium enhancement (LGE). The degree of myocardial salvage is prognostically important, but its effect on recovery of left ventricular (LV) function is not known. The purpose of this study was to determine if the degree of myocardial salvage predicts recovery of func- tion after an acute ST-elevation myocardial infarction (STEMI). Author details 1 1Radiology, University of Virginia Health System, Charlottesville, VA, USA. 2Medicine, University of Virginia Health System, Charlottesville, VA, USA. Methods The percentage of myocardial salvage moderately pre- dicts recovery of function after an acute STEMI. Future analysis will focus on identifying additional predictors on post-infarction cardiac magnetic resonance imaging. We assessed patients with no known prior CAD post- STEMI. Imaging was performed within 72 hours of the acute event and was then repeated 6-12 weeks post- infarction. The degree of myocardial salvage was obtained by subtracting the percentage LGE from the T2 area at risk. The degree of salvage was compared to the percentage improvement in LVEF by nonparametric Spearman rank correlation coefficient analysis. POSTER PRESENTATION Open Access Results Twenty-three patients were recruited for the study. One patient refused a second study, 2 had technical difficul- ties, and 5 had a baseline LV ejection fraction (EF) of ≥50%, leaving a final study population of 15 subjects. The mean age of the sample was 59.3 ± 11.7 years; 86.7% were male and 93.3% were Caucasian. Hyperten- sion, hyperlipidemia, and tobacco use was present in 40.0%, 73.3%, and 46.7%, respectively. The mean BMI was 30.8 ± 4.1, and 9/15 (60%) had a BMI ≥30. The Published: 1 February 2012 doi:10.1186/1532-429X-14-S1-P26 Cite this article as: Bourque et al.: T2-Imaging of the ischemic area-at- risk predicts recovery of cardiac function after acute ST-elevation myocardial infarction. Journal of Cardiovascular Magnetic Resonance 2012 14(Suppl 1):P26. 1Radiology, University of Virginia Health System, Charlottesville, VA, USA Full list of author information is available at the end of the article © 2012 Bourque et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
https://openalex.org/W2996939874
https://molecularneurodegeneration.biomedcentral.com/track/pdf/10.1186/s13024-019-0353-1
English
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Longitudinal and nonlinear relations of dietary and Serum cholesterol in midlife with cognitive decline: results from EMCOA study
Molecular neurodegeneration
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(2019) 14:51 (2019) 14:51 An et al. Molecular Neurodegeneration (2019) 14:51 https://doi.org/10.1186/s13024-019-0353-1 An et al. Molecular Neurodegeneration ( https://doi.org/10.1186/s13024-019-0353-1 Open Access Longitudinal and nonlinear relations of dietary and Serum cholesterol in midlife with cognitive decline: results from EMCOA study Yu An1, Xiaona Zhang1, Ying Wang1, Yushan Wang1, Wen Liu1, Tao Wang1, Zhongsheng Qin2 and Rong Xiao1* Abstract Background: Previous studies regarding the cholesterol-cognition relationship in midlife have generated conflicting results. We thus investigated whether dietary and blood cholesterol were associated with cognitive decline. Methods: Participants were drawn from a large cohort study entitled the Effects and Mechanism Investigation of Cholesterol and Oxysterol on Alzheimer’s disease (EMCOA) study. We included 2514 participants who completed a selection of comprehensive cognitive tests and were followed for an average of 2.3 years. Blood concentrations of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) were assessed and dietary intakes were investigated by food frequency questionnaire (FFQ) at baseline. Apolipoprotein E (APOE) was genotyped by Kompetitive Allele Specific PCR (KASP) sequencing. Non-high- density lipoprotein cholesterol (Non-HDL-C) and LDL-C/HDL-C ratio were calculated. The longitudinal effects of dietary and blood cholesterol on risk of global cognitive decline (decrease in Montreal Cognitive Assessment (MoCA) > 2 points) were examined using Cox proportional hazards models. The nonlinear associations with global and domain-specific cognitive decline was evaluated with mixed effect linear models. Results: In Cox proportional hazards models, neither cholesterol nor egg intake was associated with a higher risk of accelerated global cognitive decline. In contrast, the higher serum concentrations of TC, LDL-C, non-HDL-C and LDL-C/HDL-C ratio were positively associated with accelerated global cognitive decline regardless of being evaluated continuously or categorically while higher HDL-C was positively associated with accelerated global cognitive decline only when being evaluated categorically (all P < 0.05). In mixed effect linear models, quadratic and longitudinal relations of dietary cholesterol and egg intakes to global cognition, processing speed and executive function were observed. Moreover, there were inverted U-shaped relations of HDL-C, with processing speed and executive function but U-shaped relations of HDL-C and LDL-C/HDL-C ratio with verbal memory. Adverse linear associations of higher LDL-C and LDL-C/HDL-C ratio with multiple cognitive comes were also revealed. Additionally adjusting for APOE genotype did not modify cholesterol-cognition associations. Dietary and serum cholesterol had variable associations with global and domain-specific cognitive decline across educational groups. Conclusion: Differential associations between dietary/serum cholesterol and cognitive decline across different domains of function were observed in a particular population of middle-aged and elderly Chinese. Interventions to improve cognitive reserve regarding dietary instruction and lipid management should be tailored according to specific target. Conclusion: Differential associations between dietary/serum cholesterol and cognitive decline across different domains of function were observed in a particular population of middle-aged and elderly Chinese. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: xiaor22@ccmu.edu.cn 1School of Public Health, Capital Medical University, No.10 Xitoutiao, You An Men Wai, Beijing 100069, China Full list of author information is available at the end of the article Abstract d d d l d h ld b * Correspondence: xiaor22@ccmu.edu.cn 1School of Public Health, Capital Medical University, No.10 Xitoutiao, You An Men Wai, Beijing 100069, China Full list of author information is available at the end of the article * Correspondence: xiaor22@ccmu.edu.cn 1School of Public Health, Capital Medical University, No.10 Xitoutiao, You An Men Wai, Beijing 100069, China Full list of author information is available at the end of the article Materials and methods Participants p Participants from the EMCOA study, a multicenter pro- spective study of community-dwelling volunteers initiated by Capital Medical University in 2014, returned to the re- spective research center in three locations approximately every 2 years. This study was registered at Chinese Clinical Trial Registry as ChiCTR-OOC-17011882. Beginning in 2014, participants between 50 to 70 years old were admin- istered face-to-face interviews with the collection of socio- demographic information (e.g. age, sex and education years), medical history of chronic diseases, neuropsycho- logical testing and dietary survey. Fasting venous blood samples were collected from the antecubital vein after a 12-h fast during all the interviews, following standardized protocols for storage of blood samples. The exclusion cri- teria for the original study included suffering from severe diseases or conditions known to affect cognitive function (e.g. depression, malignant tumors, a history of traumatic brain injury, cerebral infarction or cerebrovascular disease, long-term frequency intake of drugs and medication or dietary supplement to improve cognitive function). Finally, longitudinal data from 2514 middle-aged and elderly par- ticipants entered the study and were used for this analysis (Fig. 1). Because the EMCOA used continuous enrollment procedures, participants have different numbers of visits and follow times are also variable with a median time of 2.3 years. The medical Ethics Committee of Capital Med- ical University (No. 2013SY35) approved the study proto- col and written informed consents were obtained from all subjects. An updating meta-analysis of 17 studies indicated diver- gent cholesterol–cognition associations [5]. When measured in midlife, higher serum cholesterol levels were associated with an increased risk of late-life cognitive decline, AD and other dementia. However, this risk relationship has not been extended to late life. Prior studies of increased late-life serum cholesterol and subsequent risk of incident cognitive dysfunction report either null results or protective associa- tions [6]. There is evidence that decreased cholesterol levels may be a manifestation of underlying dementia-related neuropathology [7]. Therefore, a non-linear pattern of both high and low serum cholesterol is related to increased risk of cognitive decline or AD have been noted. Wendell et al. have observed non-linear longitudinal [8] and cross-sectional [9] associations between serum choles- terol levels and cognitive function in Baltimore Longi- tudinal Study of Aging. Our group have also reported such similar sex-specific, non-linear, cross-sectional associa- tions [10]. Background An extensive yet conflicting research has documented longi- tudinal associations between serum cholesterol and pro- spective cognitive decline [1]. Meanwhile, a sparse prior literature has identified no associations between cholesterol/ egg intakes and incident dementia or Alzheimer’s disease (AD) [2]. However, a recent article published in JAMA has concluded that higher consumption of cholesterol and eggs was significantly associated with higher risk of incident car- diovascular disease (CVD) in a dose-response manner [3]. Since CVD are known to predict the risk of dementia [4], the role of dietary and serum cholesterol in cognitive func- tion and AD is not as clear cut. Materials and methods Participants Despite that, there was a lack of cohort data and hence we do not know if non-linear patterns were replicated in longitudinal settings. With respect to dietary cholesterol, Vincent et al. [11] have indicated from meta-regression ana- lyses that there is a positive, nonlinear relation between the changes in LDL-C and dietary cholesterol, suggesting a complex network of interrelationships between dietary chol- esterol, serum cholesterol, which may obscure the role of dietary cholesterol in cognitive function. Similar to serum cholesterol levels, associations of dietary cholesterol and cognitive impairment, AD or dementia are mixed, albeit lim- ited [2, 12], suggesting a need for nonlinear examination. The present study thus aimed to augment the current un- derstanding of both serum and dietary cholesterol-cognition non-linear associations in our longitudinal settings—the Effects and Mechanism investigation of Cholesterol and © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 19 (2019) 14:51 An et al. Molecular Neurodegeneration (2019) 14:51 An et al. Molecular Neurodegeneration (Continued from previous page) Trial registration: EMCOA, ChiCTR-OOC-17011882, Registered 5th, July 2017-Retrospectively registered, http:// www.medresman.org/uc/project/projectedit.aspx?proj=2610 Trial registration: EMCOA, ChiCTR-OOC-17011882, Registered 5th, July 2017-Retrospectively registered, http:// www.medresman.org/uc/project/projectedit.aspx?proj=2610 Keywords: Cholesterol, Cognitive decline, Nonlinear Oxysterol on Alzheimer’s disease (EMCOA) study [13]. While the study serves as an extension of our group’s prior cross-sectional examination, to our knowledge, no prior study has directly addressed both serum and dietary choles- terol nonlinearly. We aimed to examine potential quadratic relations of multiple serum cholesterol levels (TC, TG, HDL-C and LDL-C), cholesterol and egg intake to global and domain-specific cognitive decline, which may be more sensitive and helpful to elucidate the impacts of cholesterol on brain integrity and function. Oxysterol on Alzheimer’s disease (EMCOA) study [13]. While the study serves as an extension of our group’s prior cross-sectional examination, to our knowledge, no prior study has directly addressed both serum and dietary choles- terol nonlinearly. We aimed to examine potential quadratic relations of multiple serum cholesterol levels (TC, TG, HDL-C and LDL-C), cholesterol and egg intake to global and domain-specific cognitive decline, which may be more sensitive and helpful to elucidate the impacts of cholesterol on brain integrity and function. Trial registration: EMCOA, ChiCTR-OOC-17011882, Registered 5th, July 2017-Retrospectively registered, http:// www.medresman.org/uc/project/projectedit.aspx?proj=2610 Cognitive tests At each study visit, standard cognitive tests were adminis- tered by trained study personnel in a standard order in a quiet room. The Montreal Cognitive Assessment (MoCA) Page 3 of 19 (2019) 14:51 An et al. Molecular Neurodegeneration (2019) 14:51 An et al. Molecular Neurodegeneration Fig. 1 Study flow chart the values of dietary nutrients were energy adjusted ac- cording to the regression–residual method [19]. [14] were used for global cognitive evaluation. Symbol Digit Modalities Test (SDMT) [15] was used to assess processing speed. The Auditory Verbal Learning Test (AVLT) [16] in- cluding 5 trials of recall of 12-word list measured immedi- ate recall (AVLT-IR), short recall (AVLT-SR) and long recall (AVLT-LR) of memory. Logical Memory Test (LMT) [17] and Digit Span Forwards (DSF) [18] of Wechsler Memory Scale—Revised, Chinese version (WMS-RC) were used to measure attention and execu- tive function respectively. [14] were used for global cognitive evaluation. Symbol Digit Modalities Test (SDMT) [15] was used to assess processing speed. The Auditory Verbal Learning Test (AVLT) [16] in- cluding 5 trials of recall of 12-word list measured immedi- ate recall (AVLT-IR), short recall (AVLT-SR) and long recall (AVLT-LR) of memory. Logical Memory Test (LMT) [17] and Digit Span Forwards (DSF) [18] of Wechsler Memory Scale—Revised, Chinese version (WMS-RC) were used to measure attention and execu- tive function respectively. Covariates Sociodemographic information included age at study base- line, gender, education (in years). Lifestyle factors included smoking status (determined by self-report and dichoto- mized as current smoker or not). Risk factors for cognitive decline included body mass index (the ratio of weight to squared height, BMI), diabetes (fasting glucose≥7.0 mmol/L or antidiabetic medication), hypertension (measured blood pressure > 140/90 mmHg or antihypertensive medication), coronary heart disease (CHD) and per se use of a lipid- lowering medication (yes or no). Dietary assessment Detailed dietary information at baseline was collected using food frequency questionnaire (FFQ) that asked about habitual intake of foods over the past year. Nutri- ents and energy intake were derived by multiplying the nutrients and energy content of each food of the specific portion size by the frequency of consumption as stated on the FFQ and then summed over all food items from the China Food Composition Database [12]. Consump- tion frequencies of food items were converted into esti- mated number per day using the middle value (eg 3–4 times per week = 0.5 times per day). Estimated daily total energy (in kJ/d), eggs (in g/d), cholesterol (in mg/d), carbohydrate, fat, saturated fatty acid (SFA), polyunsat- urated fatty acid (PUFA), monounsaturated fatty acid (MUFA) and protein (in g/d) intakes were derived. All Statistical analysis y Statistical analyses were performed using STATA version 13.0 (STATA, College Station, TX). Prior to analysis, the normality of data distribution was checked. Continuous variables were expressed as medians (interquartile ranges, IQR) when non-normally distributed or the mean ± stand- ard deviation (SD) when normally distributed. Analysis of variance (ANOVA) or the Kruskal-Wallis rank test was used for continuous variables as appropriate. The energy- adjusted values according to the residual method for all of the nutrients and eggs were calculated. The differences in frequencies of the categorical variables were evaluated using chi-square test or Fisher’s exact test. Dietary intakes of cholesterol and eggs were categorized into quartiles. The serum levels of cholesterol were categorized into two or three groups according to reference value or range. Cox proportional hazards regression models were used to esti- mate hazards ratios (HRs) for accelerated cognitive decline (a decrease in MoCA > 2 points between follow-up and baseline [22]) in baseline cholesterol and egg intakes as well as serum cholesterol. Longitudinal associations of dietary and serum cholesterol with global and domain-specific cognitive decline were estimated using linear mixed-effect models. We included quadratic terms in mixed-effect models when exploring the nonlinearity of the association between continuous baseline dietary cholesterol and egg intake as well as serum cholesterol concentrations and subsequent cognitive change. Each cognitive test was en- tered as a single outcome variable in separate mixed-effects regression models. During a median follow-up of 2.3 years, 546 partici- pants (21.7%) were defined as accelerated cognitive de- cline. In multivariable Cox proportional hazards regression model 1 adjusted for AD risk factors (Table 2), neither cholesterol nor egg intake was associated with risk of accelerated cognitive decline regardless of being evaluated continuously (cholesterol: HR: 1.0002; 95% CI: 0.9995–1.0009; P = 0.590; egg: HR:1.002; 95% CI: 0.999– 1.006; P = 0.128) or in quartiles (cholesterol: HR for highest compared with lowest quartiles: 1.18; 95% CI: 0.89–1.58; P = 0.256; egg: HR for highest compared with lowest quartiles: 1.04; 95% CI: 0.81–1.33; P = 0.786). Laboratory analysis l d Centralized measurements of baseline fasting serum choles- terol levels were measured by enzymatic method (TC and TG) or direct method (HDL-C and LDL-C) using Automatic Biochemistry Analyzer (Olympus AU480, Japan) and com- mercially available diagnostic kits (Intec Products, Xiamen, China) in Beijing. Non-HDL-C was calculated by subtracting HDL-C from TC. The desirable concentrations of TC, TG and LDL-C are respectively less than 5.20 mmol/L, 1.70 mmol/L and 3.12 mmol/L. The reference range of HDL-C concentration and LDL-C/HDL-C ratio are respectively Page 4 of 19 Page 4 of 19 An et al. Molecular Neurodegeneration An et al. Molecular Neurodegeneration (2019) 14 1.04–1.7 mmol/L and 1.31–3.19. Less than 3.4 mmol/L of non-HDL-C concentration is considered normal [20]. 1.04–1.7 mmol/L and 1.31–3.19. Less than 3.4 mmol/L of non-HDL-C concentration is considered normal [20]. and intakes of energy, protein, carbohydrates, fat, choles- terol, SFA, PUFA and MUFA. Model 2 was adjusted as for model 1 and mutually for number of APOE ε4 alleles to test specially whether APOE genotype of the subjects have an influence on the identified relationship between cholesterol and various cognitive functions. Moreover, further subanalysis by four educational group, Elemen- tary school (≤6 years of education), Junior middle school (7–9 years of education), Senior middle school (10–12 years of education) and College and above (at least college or university; ≥13 years of education) [13], were performed taking generally lower educational back- ground into consideration. A two-sided P < 0.05 were considered statistically significant. Results Of 2514 participants, 54.0% were women. The median age was 59 years and the median education years was 9 years. Median cholesterol and egg intakes were respect- ively 282.83 mg/d and 45.21 g/d. Compared with those with a lower cholesterol intake, participants with a higher cholesterol intake were less likely to be women, have hypertension and lipid-lowering medication per se use, have lower energy and carbohydrates intakes, but more educated, more likely to drink and have diabetes (all P < 0.05, Table 1). They also had higher protein, fat, SFA, PUFA, MUFA and egg intakes but lower serum TG concentrations (P < 0.05). In regard to baseline cognitive performance, participants with higher cholesterol intake performed significantly better than that with lower in- take (all P < 0.05) except for AVLT-SR and AVLT-LR. No differences were observed with other covariates. Par- ticularly, no significant differences emerged with respect to APOE ε4 allele, the distribution of which with higher ε2 and lower ε4 differed greatly from European and American populations [23]. APOE genotyping Identification and measurement of the APOE genotype with none ε4 (ε2/ε3, ε2/ε2, ε3/ε3), one ε4 (ε3/ε4, ε2/ε4) and two ε4 (ε4/ε4) in this study were achieved through the KASP genotyping assay by BioMiao Biological Tech- nology, Beijing, China. In brief, leukocyte total genomic DNA was extracted from 400 μL of peripheral blood samples by using the Whole Blood DNA Extraction Kit (QIAamp® DNA Blood Mini Kit). DNA samples were then randomly placed on batches of 96-well plate. Geno- typing of APOE was performed according to the manu- facturer’s instructions [21]. For the purpose of quality control, 5% of samples were repeated and non-template controls were set in each plate. Statistical analysis With respect to serum cholesterol levels, additional serum concentrations of TC, LDL-C, non-HDL-C and LDL-C/HDL-C ratio were significantly associated with accelerated global cognitive decline when being evalu- ated continuously (HR for TC: 1.15, 95% CI: 1.06–1.26, P = 0.002; HR for LDL-C:1.26, 95% CI: 1.14–1.40, P < Covariates in the models were selected based on estab- lished and previously published risk factors for AD or associations with exposures in the current analysis. Model 1 included age, sex, years of education, BMI, smoking and drinking status, diabetes, hypertension and CHD history, per se use of lipid-lowering medication An et al. Molecular Neurodegeneration (2019) 14:51 An et al. Statistical analysis Their effect size changed only modestly without loss of significance if APOE genotype was included in the model. Therefore, the number of APOE ε4 risk alleles did not modify the association of either cholesterol intake or serum choles- terol levels with risk of global cognitive decline (Table 2, Fig. 2). 0.001; HR for non-HDL-C: 1.15, 95%CI: 1.05–1.27, P = 0.004; HR for LDL-C/HDL-C ratio: 1.20, 95%CI: 1.07– 1.34, P = 0.002). After being dichotomized, serum choles- terol was associated with an HR of 1.26 (95% CI: 1.04– 1.53, P = 0.020) for TC higher than 5.20 mmol/L, 1.37 (95%CI: 1.00–1.87, P = 0.048) for HDL-C higher than 1.70 mmol/L, 1.60 (95% CI: 1.34–1.92, P < 0.001) for LDL-C higher than 3.12 mmol/L, 1.54 (95% CI: 1.29– 1.84, P < 0.001) for non-HDL-C higher than 3.40 mmol/ L and 1.54 (95% CI: 1.15–2.06, P = 0.003) for LDL-C/ HDL-C ratio higher than 3.19 with significant adverse impact on global cognitive decline (Fig. 2). Their effect size changed only modestly without loss of significance if APOE genotype was included in the model. Therefore, the number of APOE ε4 risk alleles did not modify the association of either cholesterol intake or serum choles- terol levels with risk of global cognitive decline (Table 2, Fig. 2). risk of cognitive decline in Junior middle school group but higher risk in College and above group whereas egg intake was significantly associated with higher risk of cognitive decline in both Senior middle school group and College and above group. g g p Findings from mixed-effects linear regression analyses for dietary cholesterol and egg intake were shown in Table 5. Regarding nonlinear effects, significant longitu- dinal, quadratic effects of dietary cholesterol were identi- fied for MoCA (β = −0.00000142, P = 0.023, Fig. 4a) and SDMT (β = −0.00000713, P = 0.001, Fig. 4b) and egg intake for DSF (β = −0.000022, P = 0.008, Fig. 4d). Besides, posi- tive linear associations of dietary cholesterol were identified for DSF (β = 0.005, P = 0.048, Fig. 4c). Table 6 demon- strated nonlinear or linear longitudinal associations of serum cholesterol with cognitive outcomes. No quadratic associations of TC, TG and Non-HDL-C with cognitive decline were observed. When it comes to HDL-C, mixed- effect linear models revealed significant U-shaped effects of HDL-C on AVLT-LR (β = 0.514, P = 0.045, Fig. 5a). Statistical analysis Molecular Neurodegeneration (2019) 14:51 An et al. Molecular Neurodegeneration Page 6 of 19 Table 1 Baseline characteristics according to cholesterol intakes in 2514 participants in the EMCOA (Continued) Cholesterol intake quartile, mg/d P value Q1(< 188) Q2 (188–283) Q3 (283–385) Q4 (> 385) Baseline Cognitive Performance MoCA 24 (22, 26) 25 (22, 26) 25 (22, 27) 26 (24, 27) < 0.001* AVLT-IR 14 (11, 18) 14 (11, 18) 15 (12, 18) 15 (12, 19) 0.004* AVLT-SR 5 (3, 7) 5 (3, 7) 5 (3, 7) 5 (4, 7) 0.154 AVLT-LR 4 (2, 6) 4 (2, 6) 4 (2, 6) 4 (3, 6) 0.087 SDMT 32 (25, 40) 34 (28, 41) 34 (26, 42) 35 (29, 44) < 0.001* DSF 8 (7, 8) 8 (7, 9) 8 (7, 9) 8 (7, 9) < 0.001* LMT 9.5 (5.5, 14.0) 10.0 (6.0, 14.0) 11.0 (6.5, 15.0) 11.0 (7.5, 15.0) < 0.001* Abbreviations: MoCA Montreal Cognitive Assessment, AVLT-IR auditory verbal learning test-immediate recall, AVLT-SR auditory verbal learning test-short recall, AVLT-LR auditory verbal learning test-long recall, SDMT symbol digit modalities test, LMT logical memory test, DSF digit span forwards, DSB digit span backwards, BMI body mass index, CHD coronary heart disease, TC total cholesterol, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, TG triglycerides, Non-HDL-C non-high-density lipoprotein cholesterol, APOE apolipoprotein E Data shown as median (interquartile range) were compared between 4 groups using Kruskal-Wallis rank test; Data shown as n (%) were compared between 4 groups using the chi-square test or Fisher’s exact test †All dietary nutrients and egg intake are energy adjusted according to the regression–residual method ∗P < 0.05 eristics according to cholesterol intakes in 2514 participants in the EMCOA (Continued) 0.001; HR for non-HDL-C: 1.15, 95%CI: 1.05–1.27, P = 0.004; HR for LDL-C/HDL-C ratio: 1.20, 95%CI: 1.07– 1.34, P = 0.002). After being dichotomized, serum choles- terol was associated with an HR of 1.26 (95% CI: 1.04– 1.53, P = 0.020) for TC higher than 5.20 mmol/L, 1.37 (95%CI: 1.00–1.87, P = 0.048) for HDL-C higher than 1.70 mmol/L, 1.60 (95% CI: 1.34–1.92, P < 0.001) for LDL-C higher than 3.12 mmol/L, 1.54 (95% CI: 1.29– 1.84, P < 0.001) for non-HDL-C higher than 3.40 mmol/ L and 1.54 (95% CI: 1.15–2.06, P = 0.003) for LDL-C/ HDL-C ratio higher than 3.19 with significant adverse impact on global cognitive decline (Fig. 2). Statistical analysis Molecular Neurodegeneration Page 5 of 19 Table 1 Baseline characteristics according to cholesterol intakes in 2514 participants in the EMCOA Cholesterol intake quartile, mg/d P value Q1(< 188) Q2 (188–283) Q3 (283–385) Q4 (> 385) Demographic characteristics Age 58 (56, 62) 59 (55, 62) 59 (55, 62) 59 (55, 62) 0.707 Women, n(%) 349 (55.57%) 362 (57.37%) 355 (56.71%) 291 (46.26%) < 0.001* Education years 9 (9, 12) 9 (9, 12) 12 (9, 12) 12 (9, 12) 0.001* BMI (kg/m2) 24.6 (22.9, 26.7) 24.6 (22.6, 26.6) 24.4 (22.6, 26.4) 24.5 (22.6, 26.5) 0.371 Lifestyle Current smoker, n(%) 161 (25.64%) 151 (23.93%) 131 (20.93%) 154 (24.48%) 0.242 Current drinker, n(%) 143 (22.77%) 140 (22.19%) 151 (24.12%) 195 (31.00%) 0.001* Medical History Diabetes, n(%) 69 (10.99%) 73 (11.57%) 96 (15.34%) 140 (22.26%) < 0.001* Hypertension, n(%) 235 (37.42%) 212 (33.60%) 195 (31.15%) 187 (29.73%) 0.022* CHD, n(%) 79 (12.58%) 63 (9.98%) 52 (8.31%) 55 (8.74%) 0.051 vLipid-lowering medication per se use, n(%) 80 (12.74%) 74 (11.73%) 52 (8.31%) 57 (9.06%) 0.030* APOE genotype with 0/1/2 ɛ4 risk alleles 0.473 0 (ε2/ε3, ε2/ε2, ε3/ε3) 525 (83.60%) 517 (81.93%) 515 (82.27%) 527 (83.78%) 1 (ε3/ε4, ε2/ε4) 98 (15.61%) 109 (17.27%) 102 (16.29%) 100 (15.90%) 2 (ε4/ε4) 5 (0.80%) 5 (0.79%) 9 (1.44%) 2 (0.32%) Dietary intakes† Energy, kJ/d 7165 (5732, 9169) 6477 (5005, 8524) 6927 (5691, 8135) 6823 (5159, 9034) < 0.001* Carbohydrates, g/d 271.88 (231.75, 310.47) 245.47 (214.74, 278.66) 244.60 (213.94, 274.34) 224.99 (197.91, 251.91) < 0.001* Protein, g/d 59.40 (52.85, 66.49) 63.22 (57.85, 69.18) 65.24 (59.68, 71.10) 71.69 (65.79, 80.71) < 0.001* Fat, g/d 59.29 (44.18, 74.88) 67.37 (54.51, 79.38) 66.69 (55.59, 79.09) 70.34 (59.43, 80.46) < 0.001* SFA, g/d 15.83 (12.50, 18.71) 18.63 (15.71, 21.42) 19.08 (16.56, 21.93) 21.87 (19.55, 24.87) < 0.001* PUFA, g/d 25.27 (16.77, 33.38) 27.88 (20.90, 34.62) 26.16 (19.34, 33.59) 23.69 (16.41, 31.20) < 0.001* MUFA, g/d 20.98 (15.27, 25.35) 24.16 (19.39, 28.39) 24.03 (20.06, 28.55) 27.68 (23.47, 32.52) < 0.001* Eggs, g/d 12.01 (4.01, 20.75) 29.97 (23.47, 41.12) 58.00 (49.11, 61.11) 62.66 (57.16, 66.61) < 0.001* Serum Cholesterol TC, mmol/L 4.51 (3.79, 5.19) 4.50 (3.80, 5.23) 4.51 (3.74, 5.25) 4.67 (3.98, 5.34) 0.054 TC > 5.20 mmol/L, n(%) 150 (23.92%) 160 (25.36%) 170 (27.16%) 177 (28.27%) 0.308 TG, mol/L 1.50 (1.09, 2.08) 1.59 (1.11, 2.13) 1.40 (1.06, 2.00) 1.36 (0.99, 2.00) < 0.001* TG > 1.70 mmol/L, n(%) 247 (39.39%) 277 (43.90%) 219 (34.98%) 224 (35.78%) 0.004* HDL-C, mmol/L 1.24 (1.05, 1.48) 1.26 (1.10, 1.46) 1.30 (1.10, 1.50) 1.29 (1.10, 1.50) 0.06 HDL-C < 1.04 mmol/L, n(%) 146 (25.13%) 113 (19.25%) 111 (19.68%) 115 (20.35%) 0.052 HDL-C > 1.70 mmol/L, n(%) 46 (9.56%) 44 (8.49%) 62 (12.04%) 61 (11.94%) 0.166 LDL-C, mmol/L 2.77 (2.20, 3.31) 2.79 (2.20, 3.35) 2.72 (2.12, 3.31) 2.82 (2.30, 3.39) 0.438 LDL-C > 3.12 mmol/L, n(%) 206 (32.85%) 207 (32.81%) 209 (33.39%) 221 (35.30%) 0.762 Non-HDL, mmol/L 3.24 (2.62, 3.81) 3.20 (2.59, 3.92) 3.22 (2.58, 3.85) 3.35 (2.71, 3.93) 0.100 Non-HDL ≥3.40, n(%) 270 (43.06%) 262 (41.52%) 255 (40.73%) 296 (47.28%) 0.088 LDL-C/HDL-C 2.22 (1.68, 2.73) 2.20 (1.73, 2.71) 2.11 (1.72, 2.60) 2.18 (1.71, 2.71) 0.325 LDL-C/HDL-C ≤1.31, n(%) 50 (8.85%) 53 (9.20%) 63 (10.92%) 54 (9.52%) 0.654 LDL-C/HDL-C ≥3.19, n(%) 62 (10.75%) 55 (9.52%) 49 (8.70%) 59 (10.31%) 0.667 An et al. Statistical analysis Molecular Neurodegeneration Table 2 Risk of accelerated cognitive decline in dietary and serum cholesterol levels in 2514 participants in the EMCOA Variables Model 1 Model 2 HR (95% CI) P value HR (95% CI) P value Dietary cholesterol, mg/d 1.0002 (0.9995–1.0009) 0.59 1.0002 (0.9995–1.0009) 0.564 Q1(< 188) Ref Ref Q2 (188–283) 0.93 (0.71–1.20) 0.558 0.93 (0.71–1.20) 0.569 Q3 (283–385) 1.08 (0.83–1.40) 0.571 1.10 (0.84–1.43) 0.488 Q4 (> 385) 1.18 (0.89–1.58) 0.256 1.19 (0.89–1.60) 0.231 Egg intake, g/d 1.002 (0.999–1.006) 0.128 1.003 (0.999–1.006) 0.111 Q1(< 21) Ref Ref Q2 (21–45) 0.78 (0.61–1.01) 0.058 0.79 (0.61–1.02) 0.068 Q3 (45–60) 1.21 (0.95–1.55) 0.125 1.22 (0.96–1.56) 0.108 Q4 (> 60) 1.04 (0.81–1.33) 0.786 1.05 (0.82–1.35) 0.703 Serum cholesterol, mmol/L TC, mmol/L 1.15 (1.06–1.26) 0.002* 1.15 (1.05–1.26) 0.002* TC ≤5.20 mmol/L Ref Ref TC > 5.20 mmol/L 1.26 (1.04–1.53) 0.020* 1.26 (1.03–1.53) 0.022* TG, mol/L 0.96 (0.89–1.05) 0.382 0.96 (0.89–1.05) 0.377 TG ≤1.70 mmol/L Ref Ref TG > 1.70 mmol/L 1.02 (0.86–1.22) 0.798 1.02 (0.85–1.22) 0.818 HDL-C, mmol/L 1.31 (0.97–1.79) 0.083 1.31 (0.96–1.78) 0.088 1.70 ≥HDL-C ≥1.04 mmol/L Ref Ref HDL-C < 1.04 mmol/L 0.83 (0.66–1.05) 0.114 0.83 (0.66–1.05) 0.127 HDL-C > 1.70 mmol/L 1.37 (1.00–1.87) 0.048* 1.39 (1.02–1.90) 0.040* LDL-C, mmol/L 1.26 (1.14–1.40) < 0.001* 1.26 (1.14–1.40) < 0.001* LDL-C ≤3.12 mmol/L Ref Ref LDL-C > 3.12 mmol/L 1.60 (1.34–1.92) < 0.001* 1.60 (1.34–1.92) < 0.001* Non-HDL-C, mmol/L 1.15 (1.05–1.27) 0.004* 1.15 (1.05–1.27) 0.004* Non-HDL-C < 3.4 mmol/L Ref Ref Non-HDL-C ≥3.4 mmol/L 1.54 (1.29–1.84) < 0.001* 1.54 (1.29–1.84) < 0.001* LDL-C/HDL-C 1.20 (1.07–1.34) 0.002* 1.20 (1.07–1.34) 0.002* 1.31 < LDL-C/HDL-C < 3.19 Ref Ref LDL-C/HDL-C ≤1.31 1.10 (0.82–1.49) 0.521 1.11 (0.82–1.50) 0.506 LDL-C/HDL-C ≥3.19 1.54 (1.15–2.06) 0.003* 1.55 (1.16–2.07) 0.003* Abbreviations: TC total cholesterol, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, TG triglycerides, Non-HDL-C non-high- density lipoprotein cholesterol, HR hazards ratio Values were obtained from Cox proportional hazards regression models cognitive decline in dietary and serum cholesterol levels in 2514 participants in the EMCOA remain significant in Senior middle school group. More- over, only inverted U-shaped effects of HDL-C, LDL-C and ratio of LDL-C/HDL-C for MoCA, AVLT-SR and SDMT were observed in College and above group. No significant associations were demonstrated in Elemen- tary school group. Subgroup analysis by educational groups differed greatly from general analysis when it comes to nonlinear effects (Tables 7 and 8). Statistical analysis Simi- larly, U-shaped patterns of ratio of LDL-C/HDL-C were also identified for AVLT-SR (β = 0.054, P = 0.032, Fig. 5e) and AVLT-LR (β = 0.054, P = 0.032, Fig. 5f). Besides, inverted U-shaped effects of HDL-C for SDMT (β = − 3.046, P = 0.004, Fig. 5b) and DSF (β = −0.342, P = 0.006, Fig. 5c) were also identified, such that participants per- formed better at midrange HDL-C than at high and low levels. In Sion, the models also revealed adverse linear longitudinal effects of LDL-C for LMT (β = −1.099, P = 0.028, Fig. 5g) and ratio of LDL-C/HDL-C for AVLT-IR (β = −0.547, P = 0.047, Fig. 5d). Further adjustment of number of APOE ε4 risk alleles did not modify these associations. No significant effects arose for the remain- der of serum cholesterol and cognitive tests. Table 3 demonstrated significant differences among different educational groups regarding to cholesterol and egg intakes (P = 0.0001), serum TG (P = 0.0145), HDL-C (P = 0.0413) and ratio of LDL-C/HDL-C (P = 0.0089). Generally, subjects with more years of education had more dietary cholesterol and egg intake and higher ratio of LDL-C/HDL-C. Subanalysis were thus performed and the whole population was stratified by different educa- tional groups (Table 4, Fig. 3). Table 4 showed that higher serum cholesterol levels still increased risk of accelerated global cognitive decline generally but had some differences across different educational groups. Contrary to that, associations of dietary cholesterol and egg intake with cognitive decline in subanalysis differed from that in general analysis when being evaluated con- tinuously. Dietary cholesterol had a trend toward lower An et al. Molecular Neurodegeneration (2019) 14:51 Page 7 of 19 An et al. Statistical analysis U-shaped effects of serum mul- tiple cholesterol measurements (TC, TG, HDL-C, LDL- C and Non-HDL-C) for all the cognitive performance except DSF were identified in Junior middle school group whereas only U-shaped effects of HDL-C and ra- tio of LDL-C/HDL-C for AVLT-SR and AVLT-LR To summarize the effects most clearly, plots (Fig. 4 and 5) were generated using the predicted cognitive test An et al. Molecular Neurodegeneration (2019) 14:51 An et al. Molecular Neurodegeneration Page 8 of 19 Fig. 2 Forest plot for Cox proportional hazards model1 and model 2. HR: hazards ratio; CI: confidence interval. Model 1 was adjusted for sex, age, education years, BMI, smoking and drinking status, diabetes, hypertension and coronary artery disease history and per se use of lipid-lowering medication and intakes of energy, protein, carbohydrates, fat, SFA, PUFA, and MUFA. Model 2 was adjusted as for model 1 and for number of APOE ε4 alleles Fig. 2 Forest plot for Cox proportional hazards model1 and model 2. HR: hazards ratio; CI: confidence interval. Model 1 was adjusted for sex, age, education years, BMI, smoking and drinking status, diabetes, hypertension and coronary artery disease history and per se use of lipid-lowering medication and intakes of energy, protein, carbohydrates, fat, SFA, PUFA, and MUFA. Model 2 was adjusted as for model 1 and for number of APOE ε4 alleles Fig. 2 Forest plot for Cox proportional hazards model1 and model 2. HR: hazards ratio; CI: confidence interval. Model 1 was adjusted for sex, age, education years, BMI, smoking and drinking status, diabetes, hypertension and coronary artery disease history and per se use of lipid-lowering medication and intakes of energy, protein, carbohydrates, fat, SFA, PUFA, and MUFA. Model 2 was adjusted as for model 1 and for number of APOE ε4 alleles was adversely associated with immediate recall of ver- bal memory and attention decline. scores associated with dietary cholesterol, egg intake and serum concentrations of cholesterol. Each graph depicted the significant quadratic or linear, longitudinal relation- ship between cognitive performance and cholesterol levels. In general, the plots showed that both lower and higher cholesterol/egg intakes were associated with poorer cognitive performance of global cognition, processing speed and executive function; serum concentrations of HDL-C within reference range was associated with better processing speed and executive function. Statistical analysis Add- itionally, short and long recall of verbal memory was performed best at high and low levels of HDL-C and LDL-C/HDL-C ratio than at midrange. Last but not least, higher ratio of LDL-C/HDL-C and LDL-C levels density lipoprotein cholesterol Elementary school: ≤6 years of education; Junior middle school: 7–9 years of education; Senior middle school: 10–12 years of education; College and above: at least college or university; ≥13 years of education) ∗P < 0 05 Discussion In this prospective study of 2514 community-dwelling participants initially with normal cognitive performance in middle-aged and elderly, we showed that higher levels of multiple cholesterol measurements were associated with higher risk of accelerated global cognitive decline. Moreover, we identified nonlinear or linear associations of dietary and serum cholesterol with domain-specific cognitive decline. Distribution of APOE ε4 risk alleles in our Asian population did not modify their associations. Subanalysis by educational group further demonstrated Table 3 Comparison of dietary and serum cholesterol levels by different educational groups Variables Elementary school (n = 418) Junior middle school (n = 925) Senior middle school (n = 764) College and above (n = 407) P value Dietary cholesterol, mg/d 255.5 (160.3, 367.0) 262.2 (176.5, 370.2) 296.0 (207.8, 392.7) 327.7 (212.8, 412.2) 0.0001* Egg intake, g/d 39.6 (18.3, 59.5) 35.8 (18.3, 59.0) 48.4 (23.7, 60.4) 52.8 (25.8, 60.9) 0.0001* TC, mmol/L 4.61 (3.90, 5.20) 4.51 (3.80, 5.28) 4.53 (3.78, 5.20) 4.62 (3.83, 5.30) 0.518 TG, mol/L 1.40 (1.00, 1.90) 1.48 (1.02, 2.05) 1.49 (1.10, 2.15) 1.45 (1.07, 2.10) 0.0145* HDL-C, mmol/L 1.30 (1.10, 1.50) 1.27 (1.10, 1.50) 1.24 (1.09, 1.43) 1.26 (1.10, 1.47) 0.0413* LDL-C, mmol/L 2.80 (2.24, 3.30) 2.76 (2.13, 3.30) 2.76 (2.20, 3.39) 2.86 (2.27, 3.44) 0.0962 Non-HDL-C, mmol/L 3.29 (2.70, 3.83) 3.22 (2.61, 3.85) 3.25 (2.56, 3.88) 3.26 (2.72, 3.95) 0.5925 LDL-C/HDL-C 2.13 (1.72, 2.57) 2.15 (1.67, 2.67) 2.22 (1.73, 2.75) 2.25 (1.75, 2.77) 0.0089* Abbreviations: TC total cholesterol, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, TG triglycerides, Non-HDL-C non-high- density lipoprotein cholesterol Elementary school: ≤6 years of education; Junior middle school: 7–9 years of education; Senior middle school: 10–12 years of education; College and above: at least college or university; ≥13 years of education) ∗P < 0.05 Table 3 Comparison of dietary and serum cholesterol levels by different educational groups Page 9 of 19 An et al. Molecular Neurodegeneration (2019) 14:51 An et al. Discussion Molecular Neurodegeneration Table 4 Subanalysis for risk of accelerated cognitive decline in dietary and serum cholesterol levels by different educational groups Variables Elementary school (n = 418) Junior middle school (n = 925) Senior middle school (n = 764) College and above (n = 407) HR (95% CI) P value HR (95% CI) P value HR (95% CI) P value HR (95% CI) P value Dietary cholesterol, mg/d 1.002 (0.9999–1.0034) 0.063 0.999 (0.998–0.99998) 0.046* 1.001 (0.999–1.003) 0.077 1.002 (1.000–1.003) 0.041* Q1(< 188) Ref Ref Ref Ref Q2 (188–283) 1.16 (0.60–2.24) 0.666 1.12 (0.75–1.67) 0.57 0.67 (0.38–1.16) 0.153 0.66 (0.31–1.40) 0.276 Q3 (283–385) 1.21 (0.60–2.43) 0.596 0.77 (0.49–1.22) 0.269 1.30 (0.78–2.16) 0.309 1.72 (0.90–3.27) 0.098 Q4 (> 385) 1.97 (0.95–4.11) 0.07 0.90 (0.54–1.48) 0.667 1.47 (0.85–2.56) 0.171 1.49 (0.72–3.11) 0.287 Egg intake, g/d 1.006 (0.998–1.014) 0.119 0.996 (0.991–1.001) 0.155 1.008 (1.002–1.014) 0.013* 1.010 (1.001–1.018) 0.020* Q1(< 21) Ref Ref Ref Ref Q2 (21–45) 0.92 (0.47–1.80) 0.817 0.71 (0.48–1.04) 0.076 0.86 (0.50–1.46) 0.572 0.72 (0.37–1.41) 0.337 Q3 (45–60) 1.72 (0.90–3.28) 0.103 0.87 (0.57–1.33) 0.527 1.50 (0.90–2.47) 0.116 1.02 (0.55–1.87) 0.96 Q4 (> 60) 1.18 (0.61–2.26) 0.623 0.73 (0.48–1.10) 0.128 1.58 (0.95–2.62) 0.08 1.79 (0.97–3.29) 0.062 Serum cholesterol, mmol/L TC, mmol/L 1.42 (1.10–1.82) 0.006* 1.06 (0.91–1.25) 0.433 1.08 (0.93–1.26) 0.313 1.258 (1.002–1.578) 0.048* TC ≤5.20 mmol/L Ref Ref Ref Ref TC > 5.20 mmol/L 2.00 (1.25–3.21) 0.004* 0.97 (0.69–1.37) 0.868 1.10 (0.75–1.62) 0.638 1.82 (1.12–2.94) 0.015* TG, mol/L 0.86 (0.68–1.08) 0.183 0.97 (0.85–1.10) 0.622 0.96 (0.82–1.13) 0.643 0.97 (0.80–1.19) 0.783 TG ≤1.70 mmol/L Ref Ref Ref Ref TG > 1.70 mmol/L 0.94 (0.58–1.50) 0.786 0.95 (0.70–1.28) 0.742 1.13 (0.80–1.58) 0.491 0.91 (0.58–1.42) 0.669 HDL-C, mmol/L 1.88 (0.95–3.74) 0.071 0.99 (0.60–1.64) 0.977 1.13 (0.61–2.09) 0.705 1.73 (0.78–3.82) 0.177 1.70 ≥HDL-C ≥1.04 mmol/L Ref Ref Ref Ref HDL-C < 1.04 mmol/L 1.28 (0.71–2.32) 0.414 0.98 (0.68–1.42) 0.922 0.62 (0.39–1.002) 0.051 0.67 (0.38–1.19) 0.169 HDL-C > 1.70 mmol/L 2.29 (1.23–4.27) 0.009* 1.25 (0.75–2.08) 0.399 0.91 (0.43–1.94) 0.806 1.14 (0.47–2.76) 0.763 LDL-C, mmol/L 1.56 (1.16–2.10) 0.003* 1.18 (0.98–1.42) 0.076 1.23 (1.03–1.45) 0.020* 1.32 (1.02–1.70) 0.032* LDL-C ≤3.12 mmol/L Ref Ref Ref Ref LDL-C > 3.12 mmol/L 2.71 (1.71–4.29) < 0.001* 1.34 (0.98–1.83) 0.07 1.57 (1.12–2.21) 0.009* 1.81 (1.16–2.82) 0.008* Non-HDL-C, mmol/L 1.40 (1.06–1.85) 0.019* 1.08 (0.91–1.27) 0.394 1.09 (0.92–1.28) 0.325 1.25 (0.97–1.59) 0.081 Non-HDL-C < 3.4 mmol/L Ref Ref Ref Ref Non-HDL-C ≥3.4 mmol/L 2.06 (1.29–3.31) 0.003* 1.28 (0.95–1.73) 0.098 1.55 (1.11–2.16) 0.010* 1.79 (1.16–2.77) 0.009* LDL-C/HDL-C 1.30 (0.94–1.80) 0.108 1.23 (0.99–1.53) 0.065 1.18 (0.98–1.42) 0.077 1.13 (0.85–1.52) 0.405 1.31 < LDL-C/HDL-C < 3.19 Ref Ref Ref Ref LDL-C/HDL-C ≤1.31 1.43 (0.66–3.09) 0.363 1.15 (0.73–1.80) 0.544 0.84 (0.42–1.71) 0.636 1.25 (0.57–2.73) 0.583 LDL-C/HDL-C ≥3.19 1.89 (0.73–4.89) 0.19 2.02 (1.20–3.42) 0.008* 1.36 (0.83–2.21) 0.219 1.20 (0.62–2.30) 0.589 Abbreviations: TC total cholesterol, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, TG triglycerides, Non-HDL-C non-high-density lipoprotein cholesterol, HR hazards ratio Values were obtained from Cox proportional hazards regression models adjusted for sex, age, BMI, smoking and drinking status, diabetes, hypertension and coronary artery disease history and per se use of lipid-lowering medication and intakes of energy, protein, carbohydrates, fat, SFA, PUFA, MUFA and number of APOE ε4 alleles Elementary school: ≤6 years of education; Junior middle school: 7–9 years of education; Senior middle school: 10–12 years of education; College and above: at least college or university; ≥13 years of education) ∗P < 0.05 An et al. Discussion Molecular Neurodegeneration (2019) 14:51 An et al. Molecular Neurodegeneration Page 10 of 19 Fig. 3 Forest plot of subanalysis for Elementary school (a), Junior middle school (b), Senior middle school (c), College and above (d) in Cox proportional hazards. HR: hazards ratio; CI: confidence interval Fig. 3 Forest plot of subanalysis for Elementary school (a), Junior middle school (b), Senior middle school (c), College and above (d) in Cox proportional hazards. HR: hazards ratio; CI: confidence interval investigate the impacts of dietary and serum cholesterol in middle-aged and elderly Chinese. education-specific associations between cholesterol and cognition. This is, to our knowledge, the first report of nonlinear relations of both dietary and serum concentra- tions of cholesterol to longitudinal changes in cognitive performance. There are only two longitudinal human studies concern- ing the impact of dietary cholesterol on cognitive dysfunc- tion but neither the risk of incident AD or dementia in Kuopio Ischaemic Heart Disease Risk Factor Study (KIHD) [2] nor cognitive decline in the Chicago Health and Aging Project (CHAP) [25] was associated with dietary cholesterol intake. In line with the results from aforementioned cohort studies, our longitudinal findings also report a non- significant association between dietary cholesterol/egg con- sumption and accelerated global cognitive decline with or without APOE adjustment. However, our previous studies have shown a beneficial association of dietary cholesterol Since the 2015–2020 Dietary Guidelines for Americans issued 2 seemingly contradictory statements concerned with dietary cholesterol [24], the worldwide controversy of dietary cholesterol has intensified primarily due to sparse data from human studies as well as contradictory conclusions resulting from between-study heterogeneity. It may not be applicable to follow the American dietary guidelines without regard to native conditions. Under the circumstances, the EMCOA study was conducted to Page 11 of 19 An et al. Molecular Neurodegeneration (2019) 14:51 An et al. Discussion Molecular Neurodegeneration Table 5 Results of mixed-effects regression models predicting cognitive test performance from dietary cholesterol and egg intake Cognitive tests Dietary cholesterol2 Dietary cholesterol Egg intake2 Egg intake β P value β P value β P value β P value Model 1 MoCA −0.00000142 0.023* 0.0020 0.001* −0.0000197 0.331 0.0046 0.135 AVLT-IR −0.00000124 0.19 0.0018 0.052 −0.0000157 0.605 0.0030 0.508 AVLT-SR −0.000000754 0.122 0.0006 0.223 −0.0000111 0.478 0.0003 0.895 AVLT-LR −0.000000834 0.121 0.0006 0.28 −0.00000582 0.737 −0.0005 0.836 SDMT −0.00000713 0.001* 0.0066 0.003* −0.0001176 0.097 0.0145 0.172 DSF −0.00000047 0.072 0.0005 0.048* −0.000022 0.008* 0.0044 0.001* LMT −0.00000158 0.201 0.0023 0.064 −0.0000214 0.588 0.0073 0.225 Model 2 MoCA −0.00000142 0.023* 0.0020 0.001* −0.0000198 0.328 0.0046 0.133 AVLT-IR −0.00000124 0.19 0.0018 0.053 −0.000016 0.598 0.0031 0.504 AVLT-SR −0.000000751 0.122 0.0006 0.231 −0.0000113 0.469 0.0003 0.893 AVLT-LR −0.000000831 0.122 0.0006 0.288 −0.00000602 0.728 −0.0005 0.836 SDMT −0.0000071 0.001* 0.0066 0.003* −0.0001177 0.096 0.0145 0.174 DSF −0.000000475 0.069 0.0005 0.045* −0.0000221 0.008* 0.0044 < 0.001* LMT −0.00000157 0.205 0.0023 0.067 −0.0000215 0.587 0.0072 0.229 Abbreviations: MoCA Montreal Cognitive Assessment, AVLT-IR auditory verbal learning test-immediate recall, AVLT-SR auditory verbal learning test-short recall, AVLT-LR auditory verbal learning test-long recall, SDMT symbol digit modalities test, DSF digit span forwards, LMT logical memory test β: unstandardized regression coefficients were obtained from mixed-effects regression models Model 1 was adjusted for sex, age, education years, BMI, smoking and drinking status, diabetes, hypertension and coronary artery disease history and per se use of lipid-lowering medication and intakes of energy, protein, carbohydrates, fat, SFA, PUFA, and MUFA Model 2 was adjusted as for model 1 and for number of APOE ε4 alleles ∗P < 0.05 studies, including our cross-sectional studies [10], began to investigate nonlinear associations. The current study thus aimed to augment the understanding of nonlinear lon- gitudinal cholesterol–cognition associations and served as an extension of nonlinear examination of non-HDL-C and ratio of LDL-C/HDL-C. with mild cognitive impairment (MCI) in cross-sectional settings [12, 26]. On the contrary, another two cross- sectional population-based study including participants with parallel age of ours from Netherlands [27] and Ireland [28] demonstrated higher dietary cholesterol intake was sig- nificantly associated with impaired cognitive performance. In both our studies, significant associations of HDL-C higher than 1.70 mmol/L with increased accelerated global cognitive decline and nonlinear relations of HDL-C with multiple domain-specific cognitive decline were observed. Discussion Model 2 was adjusted as for sex, age, education years, BMI, smoking and drinking status, diabetes, hypertension and coronary artery disease history and per se use of lipid-lowering medication and intakes of energy, protein, carbohydrates, fat, SFA, PUFA, MUFA and number of APOE ε4 alleles Fig. 4 Significant effects of dietary cholesterol for MoCA (a), SDMT (b), D SF (c) and egg intake for DSF(d) in mixed-effect linear model 2. MoCA: digit span forwards. Model 2 was adjusted as for sex, age, education Fig. 4 Significant effects of dietary cholesterol for MoCA (a), SDMT (b), DSF (c) and egg intake for DSF(d) in mixed-effect linear model 2. MoCA: Montreal Cognitive Assessment; SDMT: symbol digit modalities test; DSF: digit span forwards. Model 2 was adjusted as for sex, age, education years, BMI, smoking and drinking status, diabetes, hypertension and coronary artery disease history and per se use of lipid-lowering medication and intakes of energy, protein, carbohydrates, fat, SFA, PUFA, MUFA and number of APOE ε4 alleles sectional study has reported higher level of LDL-C may be considered as a potential protective factor against cognition decline [40]. Such evidence needs future repli- cation but may have important clinical implications when taking that lower TC and LDL-C may be corres- pondingly detrimental owing to poor nutritional status and harmful effects on brain among the elderly. conceivable that studies that examine associations be- tween HDL-C and cognitive change may inevitably pro- duce conflicting results, which may be obscured by a highly heterogeneous particle size of HDL. Further re- search is needed to clarify the association between lipo- protein particle characteristics of HDL, such as particle diameter and concentration, and cognitive changes. In contrast to HDL-C, elevated TC and LDL-C at baseline was associated with greater cognitive decline re- gardless of being evaluated continuously or categorically, which were partially in support of specific adverse linear associations between LDL-C and attention decline. The Cox model findings for TC and LDL-C are consistent with the prospective study by Ma et al. [39]. It has been recognized that higher TC and LDL-C was cognitively detrimental due to correlated CVD risk among middle- aged and elderly individuals. Discussion In contrast, longitudinal and cross-sectional studies from Wendell et al [8, 9] reported nonsignificant quadratic as- sociations between HDL-C and cognitive performance. Moreover, another two large cohort studies from America [33] and France [34] also found HDL-C was not associated with 20-year cognitive decline or risk of incident dementia or its subtypes. Nevertheless, higher HDL-C is reported to be associated with better cognitive function in the Maine- Syracuse Study [35] and lower dementia risk in the Japan Public Health Centre-based prospective (JPHC) Study [36]. Owing to these controversial studies, the trend has turned to subclasses of HDL [37]. Ohtani et al. [38] have found significantly increased small-sized HDL particle levels but not HDL-C levels in MCI group compared with control group, suggesting potential associations between HDL subclasses and development of MCI. It is therefore As indicated by Smith and Refsum [29], the associa- tions between the nutrient status and outcome usually follows a sigmoidal curve, which illustrates that add- itional nutrient intake is beneficial at low status but could be harmful at high intake; and it will have no ef- fect at the plateau. Therefore, the ostensibly conflicting results across prior studies may not be truly contradict- ory. Higher consumption may correspond to the de- scending part of the curve while lower consumption may fell on the ascending part of the curve. Taking into consideration that dietary cholesterol intake increased dramatically in both Americans [30] and Chinese [31] and eggs were a major source of dietary cholesterol, a more cautious approach to dietary cholesterol and egg intake should be considered even though American and Chinese Dietary Guidelines dropped the recommenda- tion on the cholesterol intake limit [32]. In regard to serum concentrations of cholesterol and cognitive changes, a large amount of research has dem- onstrated conflicting results and thus a small number of An et al. Molecular Neurodegeneration (2019) 14:51 Page 12 of 19 An et al. Molecular Neurodegeneration (20 Page 12 of 19 (2019) 14:51 Fig. 4 Significant effects of dietary cholesterol for MoCA (a), SDMT (b), DSF (c) and egg intake for DSF(d) in mixed-effect linear model 2. MoCA: Montreal Cognitive Assessment; SDMT: symbol digit modalities test; DSF: digit span forwards. Discussion However, a recent cross- The longitudinal associations of non-HDL-C, ratio of LDL-C/HDL-C with cognitive decline in cognitively healthy participants have been rarely examined and is still poorly understood, though both of them have been regarded as good predictors for CVD risk [41, 42]. As the sum of all the atherogenic lipoprotein particles other than the HDLs [20], higher serum levels of non-HDL-C was reported to be in- dependent risk factors of cognitive impairment in patients with acute ischemic stroke [43] and MCI in patients with type 2 diabetes [44]. LDL-C/HDL-C ratio help to provide Page 13 of 19 An et al. Molecular Neurodegeneration (2019) 14:51 An et al. Discussion Molecular Neurodegeneration Table 6 Results of mixed-effects regression models predicting cognitive test performance from serum cholesterol Cognitive tests TC2 TC TG2 TG HDL-C2 HDL-C LDL-C2 LDL-C Non-HDL-C2 Non-HDL-C LDL-C/HDL-C2 LDL-C/HDL-C β β β β β β β β β β β β Model 1 MoCA 0.024 −0.210 −0.002 −0.020 0.208 −0.234 0.020 −0.179 0.007 −0.067 0.021 −0.269 AVLT-IR 0.016 −0.157 0.000 −0.012 0.353 −0.677 0.094 −0.748 0.018 −0.159 0.050 −0.547* AVLT-SR 0.014 −0.163 0.005 −0.034 0.270 −0.619 0.038 −0.337 0.018 −0.169 0.054* −0.415* AVLT-LR 0.021 −0.213 0.002 0.007 0.514* −1.362 0.038 −0.337 0.025 −0.200 0.06* −0.401* SDMT −0.096 0.655 0.008 −0.130 −3.046* 8.662* −0.248 1.181 −0.161 0.797 −0.147 0.311 DSF −0.009 0.091 −0.002 0.010 −0.342* 1.008* −0.015 0.096 −0.023 0.167 −0.019 0.084 LMT 0.071 −0.848 −0.011 0.171 0.661 −2.015 0.133 −1.099* 0.053 −0.544 0.023 −0.457 Model 2 MoCA 0.024 −0.216 −0.003 −0.018 0.209 −0.240 0.020 −0.183 0.008 −0.070 0.021 −0.269 AVLT-IR 0.018 −0.178 0.000 −0.009 0.358 −0.699 0.097 −0.767 0.020 −0.169 0.051 −0.553* AVLT-SR 0.016 −0.183 0.005 −0.032 0.275 −0.640 0.041 −0.356 0.019 −0.180 0.055* −0.423* AVLT-LR 0.022 −0.230 0.002 0.009 0.518* −1.380 0.048 −0.375 0.026 −0.209 0.061* −0.409* vSDMT −0.094 0.620 0.008 −0.130 −3.036* 8.627* −0.243 1.141 −0.160 0.780 −0.143 0.282 DSF −0.009 0.092 −0.002 0.011 −0.343* 1.008* −0.015 0.098 −0.023 0.167 −0.020 0.088 LMT 0.073 −0.868 −0.011 0.170 0.667 −2.036 0.136 −1.123* 0.054 −0.554 0.026 −0.474 Abbreviations: MoCA Montreal Cognitive Assessment, AVLT-IR auditory verbal learning test-immediate recall, AVLT-SR auditory verbal learning test-short recall, AVLT-LR auditory verbal learning test-long recall, SDMT symbol digit modalities test, DSF digit span forwards, LMT logical memory test, TC total cholesterol, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, TG triglycerides, Non-HDL-C non- high-density lipoprotein cholesterol β: unstandardized regression coefficients were obtained from mixed-effects regression models Model 1 was adjusted for sex, age, education years, BMI, smoking and drinking status, diabetes, hypertension and coronary artery disease history and per se use of lipid-lowering medication and intakes of energy, protein, carbohydrates, fat, SFA, PUFA, and MUFA Model 2 was adjusted as for model 1 and for number of APOE ε4 alleles An et al. Molecular Neurodegeneration (2019) 14:51 Page 14 of 19 An et al. Molecular Neurodegeneration (2019) 14:51 (2019) 14:51 An et al. Molecular Neurodegeneration Page 14 of 19 Fig. Discussion 5 Significant effects of HDL-C for AVLT-LR (a), SDMT (b) and DSF (c), LDL-C/HDL-C ratio for AVLT-IR (d), AVLT-SR (e) and AVLT-LR (f) and LDL- C for LMT (g) in mixed-effect linear model 2. AVLT-IR: auditory verbal learning test-immediate recall; AVLT-SR: auditory verbal learning test- immediate recall; AVLT-LR: auditory verbal learning test-long recall; LMT: Logical Memory Test; SDMT: symbol digit modalities test; DSF: digit span forwards. TC: total cholesterol; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol. Model 2 was adjusted as for sex, age, education years, BMI, smoking and drinking status, diabetes, hypertension and coronary artery disease history and per se use of lipid- lowering medication and intakes of energy, protein, carbohydrates, fat, SFA, PUFA, MUFA and number of APOE ε4 alleles. Fig. 5 Significant effects of HDL-C for AVLT-LR (a), SDMT (b) and DSF (c), LDL-C/HDL-C ratio for AVLT-IR (d), AVLT-SR (e) and AVLT-LR (f) and LDL- C for LMT (g) in mixed-effect linear model 2. AVLT-IR: auditory verbal learning test-immediate recall; AVLT-SR: auditory verbal learning test- immediate recall; AVLT-LR: auditory verbal learning test-long recall; LMT: Logical Memory Test; SDMT: symbol digit modalities test; DSF: digit span forwards. TC: total cholesterol; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol. Model 2 was adjusted as for sex, age, education years, BMI, smoking and drinking status, diabetes, hypertension and coronary artery disease history and per se use of lipid- lowering medication and intakes of energy, protein, carbohydrates, fat, SFA, PUFA, MUFA and number of APOE ε4 alleles. Fig. 5 Significant effects of HDL-C for AVLT-LR (a), SDMT (b) and DSF (c), LDL-C/HDL-C ratio for AVLT-IR (d), AVLT-SR (e) and AVLT-LR (f) and LDL- C for LMT (g) in mixed-effect linear model 2. AVLT-IR: auditory verbal learning test-immediate recall; AVLT-SR: auditory verbal learning test- immediate recall; AVLT-LR: auditory verbal learning test-long recall; LMT: Logical Memory Test; SDMT: symbol digit modalities test; DSF: digit span forwards. TC: total cholesterol; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol. Model 2 was adjusted as for sex, age, education years, BMI, smoking and drinking status, diabetes, hypertension and coronary artery disease history and per se use of lipid- lowering medication and intakes of energy, protein, carbohydrates, fat, SFA, PUFA, MUFA and number of APOE ε4 alleles. Page 15 of 19 An et al. Molecular Neurodegeneration (2019) 14:51 An et al. Discussion Molecular Neurodegeneration Table 7 Subanalysis for results of mixed-effects regression models predicting cognitive test performance from dietary cholesterol and egg intake by different educational groups Cognitive tests Dietary cholesterol2 Dietary cholesterol Egg intake2 Egg intake β P value β P value β P value β P value Elementary school (n = 418) MoCA 0.00000347 0.257 −0.004239 0.102 0.0000902 0.202 −0.020563 0.048* AVLT-IR −0.00000105 0.746 −0.001233 0.654 0.0000432 0.56 −0.017803 0.107 AVLT-SR 0.00000115 0.501 −0.002667 0.067 0.0000348 0.375 −0.013017 0.026* AVLT-LR 0.000000766 0.693 −0.002141 0.193 0.0000178 0.689 −0.011021 0.095 SDMT 0.00000511 0.519 −0.001092 0.87 0.0001744 0.342 −0.009431 0.726 DSF 0.000000705 0.537 −0.000568 0.557 0.00000138 0.958 −0.001005 0.796 LMT 0.00000044 0.933 −0.002083 0.641 0.0000577 0.624 −0.017358 0.328 Junior middle school (n = 925) MoCA −0.00000223 0.064 0.002945 0.011* −0.0000345 0.167 0.0077031 0.087 AVLT-IR −0.00000349 0.062 0.004374 0.015* −0.0000191 0.622 0.0079525 0.255 AVLT-SR −0.00000199 0.038* 0.001977 0.032* −0.0000191 0.336 0.0032397 0.366 AVLT-LR −0.00000158 0.136 0.001501 0.138 −0.00000175 0.936 0.0001187 0.976 SDMT −0.0000127 0.002* 0.010674 0.005* −0.0001801 0.030* 0.0240724 0.108 DSF −0.000000446 0.378 0.000669 0.167 −0.000023 0.028* 0.0051033 0.007* LMT −0.00000132 0.553 0.003593 0.091 −0.0000423 0.357 0.013091 0.114 Senior middle school (n = 764) MoCA −0.00000155 0.075 0.002437 0.013* −0.00000089 0.988 0.0045697 0.483 AVLT-IR −0.00000141 0.336 0.002893 0.078 0.0000611 0.521 −0.001338 0.902 AVLT-SR −0.00000127 0.091 0.000801 0.342 −0.00000153 0.975 −0.001089 0.845 AVLT-LR −0.00000132 0.112 0.000674 0.47 −0.0000122 0.822 0.0005769 0.926 SDMT −0.0000018 0.606 0.002899 0.459 0.00000761 0.973 0.0012421 0.962 DSF −0.000000936 0.011* 0.001025 0.013* −0.0000315 0.184 0.0068822 0.011* LMT −0.00000268 0.183 0.002725 0.226 0.0000091 0.944 0.0061439 0.679 College and above (n = 407) MoCA −0.0000011 0.511 0.001583 0.309 −0.0001057 0.052 0.0134549 0.053 AVLT-IR −0.00000213 0.544 0.001039 0.75 −0.0002722 0.016* 0.0243167 0.092 AVLT-SR 0.000000363 0.839 0.000177 0.915 −0.0000809 0.16 0.0097477 0.186 AVLT-LR 0.000000472 0.811 0.000178 0.923 −0.0000889 0.162 0.0101086 0.214 SDMT 0.00000371 0.662 −0.005496 0.486 −0.0000766 0.778 −0.012119 0.728 DSF −0.000000251 0.782 −0.000251 0.766 −0.0000345 0.241 0.0033226 0.378 LMT 0.00000291 0.47 −0.001744 0.639 −0.0001277 0.332 0.0196316 0.242 Abbreviations: MoCA Montreal Cognitive Assessment, AVLT-IR auditory verbal learning test-immediate recall, AVLT-SR auditory verbal learning test-short recall, AVLT-LR auditory verbal learning test-long recall, SDMT symbol digit modalities test, DSF digit span forwards, LMT logical memory test β: unstandardized regression coefficients were obtained from mixed-effects regression models adjusted for sex, age, BMI, smoking and drinking status, diabetes, hypertension and coronary artery disease history and per se use of lipid-lowering medication, number of APOE ε4 alleles and intakes of energy, protein, carbohydrates, fat, SFA, PUFA, and MUFA Elementary school: ≤6 years of education; Junior middle school: 7–9 years of education; Senior middle school: 10–12 years of education; College and above: at least college or university; ≥13 years of education) ∗P < 0.05 Table 7 Subanalysis for results of mixed-effects regression models predicting cognitive test performance from dietary cholesterol and egg intake by different educational groups an estimate of how much cholesterol is removed by HDL and delivered to plaques via LDL. Discussion We demonstrated in Cox models that both of non-HDL-C and ratio of LDL-C/HDL- C could act as readily available methods for estimating risk of accelerated global cognitive decline in middle-aged and elderly Chinese. Besides, we also revealed quadratic and linear effects of LDL-C/HDL-C ratio with verbal memory, suggesting disordered cholesterol transport among athero- genic lipoprotein particles may be particularly detrimental to verbal memory. Measurement of non-HDL-C and ratio of LDL-C/HDL-C can be calculated from a usual lipid panel and consequently is simple and inexpensive. Where linear effects of LDL-C/HDL-C ratio with verbal memory, suggesting disordered cholesterol transport among athero- genic lipoprotein particles may be particularly detrimental to verbal memory. Measurement of non-HDL-C and ratio of LDL-C/HDL-C can be calculated from a usual lipid panel and consequently is simple and inexpensive. Where An et al. Molecular Neurodegeneration (2019) 14:51 An et al. Discussion Molecular Neurodegeneration Page 16 of 19 (2019) 14:51 Table 8 Subanalysis for results of mixed-effects regression models predicting cognitive test performance from serum cholesterol b different educational groups Cognitive tests TC2 TC TG2 TG HDL-C2 HDL-C LDL-C2 LDL-C Non-HDL-C2 Non-HDL-C LDL-C/HDL-C2 LDL-C/HDL β β β β β β β β β β β β Elementary school (n = 418) MoCA −0.128 1.102 −0.072 0.366 1.411 −3.634 −0.208 1.016 −0.191 1.103 −0.099 0.232 AVLT-IR −0.027 0.205 0.037 −0.287 0.071 0.002 −0.029 −0.025 −0.079 0.445 −0.010 −0.225 AVLT-SR −0.055 0.503 0.011 −0.036 −0.525 1.604 −0.053 0.266 −0.100 0.646 −0.030 0.042 AVLT-LR −0.061 0.599 −0.018 0.115 −0.129 0.462 −0.102 0.630 −0.124 0.861 −0.044 0.208 SDMT 0.093 −1.779 −0.210 0.802 −1.413 4.817 0.072 −1.138 0.026 −1.357 0.038 −1.229 DSF −0.030 0.254 −0.010 0.106 −0.127 0.271 −0.088 0.468 −0.065 0.419 −0.106 0.513 LMT 0.259 −2.713 −0.110 1.212 2.315 −7.679 0.265 −2.009 0.052 −0.485 0.043 −0.343 Junior middle school (n = 925) MoCA 0.122* −1.150* −0.013 0.082 −0.108 0.598 0.149 −0.876 0.124* −0.869* 0.021 −0.241 AVLT-IR 0.164* −1.536* −0.010 0.149 0.936 −2.163 0.225 −1.515* 0.186* −1.306* 0.055 −0.646 AVLT-SR 0.083* −0.824* −0.001 0.058 0.762* −2.188* 0.143* −0.948* 0.101* −0.730* 0.155 −0.832* AVLT-LR 0.067 −0.669 −0.008 0.164 0.935* −2.849* 0.149* −0.958* 0.095* −0.673* 0.177 −0.846 SDMT −0.011 −0.501 0.058* −0.833* −2.383 6.464 −0.167 0.455 −0.009 −0.586 −0.418 1.403 DSF 0.010 −0.068 −0.004 0.005 −0.354 1.059 0.047 −0.223 −0.015 0.126 −0.014 0.081 LMT 0.104 −1.216 −0.017 0.148 0.927 −3.183 0.297* −1.925* 0.108 −0.940 −0.040 0.021 Senior middle school (n = 764) MoCA 0.008 0.019 0.006 −0.037 0.080 −0.183 0.029 −0.194 −0.036 0.364 0.026 −0.153 AVLT-IR 0.022 −0.195 0.004 0.040 1.178 −3.704 0.139 −1.063 −0.010 0.121 0.059 −0.432 AVLT-SR 0.037 −0.389 0.011 −0.109 1.560* −4.367* 0.038 −0.366 0.021 −0.184 0.062* −0.436* AVLT-LR 0.061 −0.585 0.008 −0.001 1.712* −4.820* 0.062 −0.494 0.052 −0.348 0.069* −0.441 SDMT −0.133 1.337 −0.051 0.909 −1.483 3.480 −0.086 0.292 −0.303 2.237 −0.094 0.391 DSF 0.001 −0.010 −0.001 −0.005 −0.192 0.654 −0.008 0.000 −0.018 0.121 −0.002 −0.071 LMT 0.076 −0.858 −0.005 0.159 0.926 −2.362 0.142 −1.264 0.067 −0.624 0.095 −0.944 College and above (n = 407) MoCA −0.026 0.237 0.025 −0.297 −0.796 2.968 −0.039 0.093 −0.031 0.140 −0.227* 0.747 AVLT-IR −0.173 1.613 −0.010 −0.145 −2.518 7.657 −0.168 0.901 −0.181 1.140 −0.325 1.223 AVLT-SR −0.084 0.790 −0.006 −0.047 −1.559* 4.756* −0.080 0.414 −0.080 0.499 −0.165 0.581 AVLT-LR −0.061 0.500 0.005 −0.196 −0.524 1.999 −0.044 0.150 −0.068 0.326 −0.047 −0.051 SDMT −0.281 2.907 −0.105 0.899 −8.171* 23.727* −0.802* 4.809* −0.323 2.387 −1.086* 4.840 DSF −0.039 0.425 0.017 −0.130 −0.698 2.184* −0.065 0.454 −0.026 0.213 −0.069 0.338 LMT −0.006 −0.113 0.075 −0.658 −1.188 4.261 −0.108 0.327 0.014 −0.394 −0.460 1.599 Abbreviations: MoCA: Montreal Cognitive Assessment; AVLT-IR: auditory verbal learning test-immediate recall; AVLT-SR: auditory verbal learning test-short recall; AVLT-LR: auditory verbal learning test-long recall; SDMT: symbol digit modalities test; DSF: digit span forwards; LMT: logical memory test; TC: total cholesterol; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; TG: triglycerides; Non-HDL-C: non- high-density lipoprotein cholesterol β: unstandardized regression coefficients were obtained from mixed-effects regression models adjusted for sex, age, BMI, smoking and drinking status, diabetes hypertension and coronary artery disease history and per se use of lipid-lowering medication, number of APOE ε4 alleles and intakes of energy, protein, carbohydrates, fat, SFA, PUFA, and MUFA Elementary school: ≤6 years of education; Junior middle school: 7–9 years of education; Senior middle school: 10–12 years of education; College and above: at least college or university; ≥13 years of education) ∗P < 0.05 Abbreviations: MoCA: Montreal Cognitive Assessment; AVLT-IR: auditory verbal learning test-immediate recall; AVLT-SR: auditory verbal learning test-short recall; AVLT-LR: auditory verbal learning test-long recall; SDMT: symbol digit modalities test; DSF: digit span forwards; LMT: logical memory test; TC: total cholesterol; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; TG: triglycerides; Non-HDL-C: non- high-density lipoprotein cholesterol β: unstandardized regression coefficients were obtained from mixed-effects regression models adjusted for sex, age, BMI, smoking and drinking status, diabetes, hypertension and coronary artery disease history and per se use of lipid-lowering medication, number of APOE ε4 alleles and intakes of energy, protein, carbohydrates, fat, SFA, PUFA, and MUFA Elementary school: ≤6 years of education; Junior middle school: 7–9 years of education; Senior middle school: 10–12 years of education; College and above: at least college or university; ≥13 years of education) ∗P < 0.05 possible, their evaluation is needed as targets for interven- tion to reduce the risk of cognitive impairment. Author contribution d d d RX conceived and designed the study, YA performed the analyses and wrote the manuscript. XZ, YW, YW, WL, TW and ZQ helped collect and analyze the data. All authors read and approved the final manuscript. References 1. Loera-Valencia R, Goikolea J, Parrado-Fernandez C, Merino-Serrais P, Maioli S. Alterations in cholesterol metabolism as a risk factor for developing Alzheimer's disease: potential novel targets for treatment. J Steroid Biochem Mol Biol. 2019;190:104–14. 2. Ylilauri MP, Voutilainen S, Lonnroos E, Mursu J, Virtanen HE, Koskinen TT, Salonen JT, Tuomainen TP, Virtanen JK. Association of dietary cholesterol and egg intakes with the risk of incident dementia or Alzheimer disease: the Kuopio Ischaemic heart disease risk factor study. Am J Clin Nutr. 2017; 105(2):476–84. Acknowledgements The authors are indebted to Deqiang Zheng from Department of Epidemiology and Health Statistics of Capital Medical University for their statistical advice. Author details 1 f Strengths of this study include its longitudinal design, analysis of nonlinear effects of both dietary and serum cholesterol with consideration of multiple fractions, use of extensive cognitive tests and including data on the APOE genotype as well as per se use of a lipid-lowering medication for participants and examination measured in midlife, despite the fact that the median age of this cohort was much younger than the usual age of onset for MCI from nearly any cause including AD. Besides, the limitation of this investigation also includes its rela- tively shorter follow-up. 1School of Public Health, Capital Medical University, No.10 Xitoutiao, You An Men Wai, Beijing 100069, China. 2Jincheng People’s Hospital, Jincheng, China. 1School of Public Health, Capital Medical University, No.10 Xitoutiao, You An Men Wai, Beijing 100069, China. 2Jincheng People’s Hospital, Jincheng, China. Received: 9 May 2019 Accepted: 16 December 2019 Received: 9 May 2019 Accepted: 16 December 2019 Received: 9 May 2019 Accepted: 16 December 2019 Discussion have a greater risk of developing AD while APOE ε2 is considered protective [45]. However, the extent to which APOE genotype modulates associations of diet- ary cholesterol/egg intakes and serum cholesterol levels with cognitive decline remains relatively unknown. Our Genetic variability of APOE is dependent on three alleles: ε2, ε3 and ε4, which combine to form six geno- types. It has been established that carriers of APOE ε4 Page 17 of 19 Page 17 of 19 Page 17 of 19 An et al. Molecular Neurodegeneration (2019) 14:51 An et al. Molecular Neurodegeneration (2019) 14:51 An et al. Molecular Neurodegeneration step-wise investigation of potential role of confounder and modifier examined how the cholesterol-cognition associations may vary by APOE ε4. Similar to studies in 1259 middle-aged and older men from Eastern Finland [2], the identified associations of cholesterol and cogni- tive decline were conserved after further adjusting for APOE ε4 among a middle-aged and elderly Chinese, possibly due to prominent distribution of APOE ε3. Abbreviations AD: Alzheimer’s disease; APOE: Apolipoprotein E; AVLT: Auditory Verbal Learning Test; BMI: Body mass index; CHD: Coronary heart disease; DSF: Digit Span Forward; EMCOA: Effects and Mechanism investigation of Cholesterol and Oxysterol on Alzheimer’s disease; FFQ: Food frequency questionnaire; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; LMT: Logical Memory Test; MoCA: Montreal Cognitive Assessment; non-HDL-C: Non-high-density lipoprotein cholesterol; SDMT: Symbol Digit Modalities Test; TC: Total cholesterol; TG: Triglycerides; WMS-RC: Wechsler Memory Scale—Revised, Chinese version The quantity of dietary cholesterol intake and serum lipid profiles may be affected by socio-economic factors [46, 47]. Our analysis provided such evidence that chol- esterol/egg intakes and some serum cholesterol were not uniformly distributed across educational groups. With respect to consumption patterns, subjects with more years of education had more dietary cholesterol and egg intakes, which was different from older Australians with comparable age (55–65) reported by Thorpe et al [48]. They found those with higher consumption of red and processed meat had a lower level of education. When it comes to serum cholesterol, the population with higher levels of education presented a higher prevalence of al- tered cholesterol, inconsistent with Brazilian adult popula- tion aged 45 years old and older [49]. It may be attributed to the fact that participants aged 50–70 with lower educa- tion levels in our country, generally considered as lower socioeconomic status, were more likely to do physically intensive jobs while those with higher education levels were less likely to do physically active jobs and may be adapting unhealthy lifestyles. The subanalysis thus showed differential associations between cholesterol and cognitive function for different educational groups, which would be helpful to promote specific dietary instruction and lipid management according to educational background. Availability of data and materials h d d d l d The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the State Key Program of the National Natural Science Foundation of China (Grant No. 81330065) and National Natural Science Foundation of China (Grant No. 81673149). Ethics approval and consent to participate The study design was ethically approved by the Ethics Committee of Capital Medical University (2013SY35). All participants were provided written informed consent at the beginning of the study. 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The Determination of 10 Compounds of GuiZhi Decoction in Rat Plasma after Oral Administration by HPLC-MS/MS and its Application to a Pharmacokinetic Study
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The Determination of 10 Compounds of GuiZhi Decoction in Rat Plasma after Oral Administration by HPLC-MS/MS and its Application to a Pharmacokinetic Study Huan Gao  China Academy of Chinese Medical Sciences Qin Guo  China Academy of Chinese Medical Sciences Lishi Zhang  China Academy of Chinese Medical Sciences Jiannan Song  China Academy of Chinese Medical Sciences Dong Bai  (  baidong2000@126.com ) China Academy of Chinese Medical Sciences, Beijing https://orcid.org/0000-0001-6129-9550 Xiaoxia Wu  China Academy of Chinese Medical Sciences Research Keywords: Guizhi Decoction, HPLC-MS/MS, Multiple Compounds, Pharmacokinetics, Rat Plasma Posted Date: March 2nd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-254154/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License Read Full License The Determination of 10 Compounds of GuiZhi Decoction in Rat Plasma after Oral Administration by HPLC-MS/MS and its Application to a Pharmacokinetic Study by HPLC-MS/MS and its Application to a Pharmacokinetic Study Huan Gao  China Academy of Chinese Medical Sciences Qin Guo  China Academy of Chinese Medical Sciences Lishi Zhang  China Academy of Chinese Medical Sciences Jiannan Song  China Academy of Chinese Medical Sciences Dong Bai  (  baidong2000@126.com ) China Academy of Chinese Medical Sciences, Beijing https://orcid.org/0000-0001-6129-9550 Xiaoxia Wu  China Academy of Chinese Medical Sciences Research Keywords: Guizhi Decoction, HPLC-MS/MS, Multiple Compounds, Pharmacokinetics, Rat Plasma Posted Date: March 2nd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-254154/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License Read Full License * Correspondence: baidong2000@126.com(D. B.); xiaoxia2311@sina.com(X. W.); 1 Institute of Basic Theory of Traditional Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, 100700, China Full list of author information is available at the end of the article. Abstract Background: Guizhi Decoction (GZD), a traditional Chinese medical formula, has been commonly used to treat fever, sweating, and cold in China. g Methods: The high performance liquid chromatography-tandem mass spectrometry (HPLC- MS/MS) method was established for the determination of 10 compounds, including cinnamic acid, paeoniflorin, albiflorin, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, glycyrrhizic acid, glycyrrhetinic acid, and 6-gingerol. And the specificity, linearity, lower limit of quantification (LLOQ), lower limit of detection (LLOD), precision and accuracy, recovery, matrix effect, and stability were used to verify the HPLC-MS/MS method. This validated method was successfully applied for pharmacokinetic study of the 10 compounds in rat plasma after oral administration of GZD in three doses (40 g crude drug·kg−1, 20 g crude drug·kg−1, 10 g crude drug·kg−1) and intravenous injection of GZD extraction at a dose of 2.0 g crude drug·kg−1.The measurements of pharmacokinetic parameters including AUC0–∞, T1/2, Tmax, Cmax, Vz_F, Cl_F, and MRT, were performed using a non-compartmental model with Winnonlin 8.1 software. Results: The results showed that 10 compounds were detected in plasma after oral administration of GZD. the compounds (except for glycyrrhetinic acid) reached the maximum blood concentration quickly, whose Tmax was about 0.1-0.2 min. And a total of 9 compounds were detected after intravenous injection of GZD. The plasma concentration-time curve of these compounds declines rapidly at the beginning, and then decreased slowly, indicating that the plasma concentration-time curves were double exponential function curves. p Conclusions: In this study, the developed method was suitable for pharmacokinetic analysis of the main compounds of GZD in rat plasma, and may reveal the pharmacodynamic material basis of GZD and provide a reference for the rational use of GZD in the clinic. p Keywords: Guizhi Decoction; HPLC-MS/MS; Multiple Compounds; Pharmacokinetics; Rat Plasma The determination of 10 compounds of GuiZhi Decoction in rat plasma after oral administration by HPLC-MS/MS and its application to a pharmacokinetic study Huan Gao 1,2,+, Qin Guo 1,+, Lishi Zhang 1, Jiannan Song1, Dong Bai 1 *, and Xiaoxia Wu 3 * Background Guizhi Decoction (GZD) is a classical formula from Treatise on Febrile Diseases, a famous ancient Chinese book of traditional Chinese medicine (TCM). Studies have demonstrated that it is mainly used for the treatment of fever, self-sweating, headache, painful stiff nape, and external wind-cold syndrome [1, 2]. GZD is composed of five herbal slices, including 9.0 g of Cinnamomi Ramulus, 9.0 g of Paeoniae Radix Alba, 9.0 g of Rhizoma Zingiberis Recens, 9.0 g of Fructus Jujubae, and 6.0 g of Radix Glycyrrhizae [3-5]. Modern studies have proven that GZD has bidirectional regulating action on the body temperature, blood pressure, secretion of sweat glands, intestinal peristalsis, and immune function [6, 7]. Before this study, we reviewed a large number of pharmacokinetics (PK) studies of the formulae related to GZD. Some researchers have preliminarily discussed the PK characteristics of GZD [4]. There were some PK studies on other Chinese medicine formulae, such as Gualou-Guizhi Decoction, Guizhi-Gancao Decoction, Guizhi-Fuling Capsule, Huangqi-Guizhi- Wuwu Decoction and so on. For example, 11 compounds in plasma were detected after the rats were given Gualou-Guizhi Decoction by gavage, including liquiritin, isoliquiritin, liquirtin apioside, isoliquiritin apioside, liquiritigenin, soliquiritigenin, glycyrrhizic acid, glycyrrhetinic acid, paeonilorin, albiflorin, and paeonilorin sulfonate. These compounds belonged to Radix Glycyrrhizae and Paeoniae Radix Alba [8]. In the PK study of Guizhi-Gancao Decoction in vivo, 7 compounds were detected, including cinnamaldehyde, cinnamic acid, 2-methoxy cinnamic acid, glycyrrhizic acid, glycyrrhetinic acid, liquiritigenin, and isoliquiritin, from Cinnamomi Ramulus and Radix Glycyrrhizae [9]. 6 compounds in plasma were quantified in the PK study of Guizhi-Fuling Capsul, involving Cinnamomi Ramulus and Paeoniae Radix Alba [10]. Moreover, only two compounds (paeoniorin and astragaloside IV) were analyzed in the PK study of Huangqi-Guizhi-Wuwu decoction [11]. It could be seen that these selected compounds were only from individual herbs in the above studies. These compounds were too few to represent PK activities the whole formula. In addition, after oral administration of TCM formula, some compounds could be transformed into metabolites by enzymes in intestinal flora and liver. These metabolites could be absorbed and have pharmacodynamic effects in vivo. In the previous study, researchers had found that the compounds of GZD absorbed into blood were significantly different from the prototype compounds in decoction by HPLC [12]. We thought only prototype compounds could not represent the real effective ingredients in TCM. There is little doubt that attention should also be paid to metabolites in PK research. Background In this study, we detected 10 compounds of GZD in rat plasma after intragastric and intravenous administration, including 9 prototype compounds and 1 metabolite. GZD was composed by five Chinese herbs. In this study, corresponding compounds and their metabolites in each herb were chosen as much as possible, to reveal the overall PK characteristics of GZD. Cinnamic acid was the main compound of Cinnamomi Ramulus, It could combat viral infections, protect neural functions, prevent or slow cognitive decline, and support the immune and digestive systems [13, 14]. So, cinnamic acid was selected as the index compounds in Cinnamomi Ramulus. Paeoniflorin and albiflorin were the main glycosides in Paeoniae Radix Alba, and they were isomers of each other. It had been noted that they could transfer to each other by microorganism [15]. Pharmacological studies have exhibited that they had remarkable effects in the treatment of pain, inflammation, muscle spasms, and neurodegenerative disorder properties [16-19]. So, paeoniflorin and albiflorin were selected. The content of gingerol was high in Rhizoma Zingiberis Recens. It has been found to gingerol had a variety of pharmacological effects, such as analgesic, antipyretic, antiemetic, and anti-inflammatory. So, 6-gingerol was selected. Both of liquiritin and isoliquiritin, liquiritigenin and isoliquiritigenin are isomers. These flavonoids compounds had strong therapeutic effects, including anti-inflammation, vasodilation, inhibition of platelet aggregation and regulation of blood lipids. Liquiritin and isoliquiritin were converted into liquiritigenin and isoliquiritigenin in the intestinal tract, which were subsequently metabolized to the glucuronic acid compounds in vivo [20-22]. Glycyrrhetinic acid was the glycoside and metabolite of glycyrrhizic acid. Both of them exhibited high activities, such as antibacterial and anti-inflammatory effects [23-25].So 6 compounds, including 5 prototypes and 1 metabolite, were chosen to be quantitated in Radix Glycyrrhizae. In this study, a selective HPLC-MS/MS method for the simultaneous determination of multiple compounds of GZD in rat plasma was developed. With this method, the PK process after intragastric and intravenous administration were studied. In this study, we expected that the absorption, metabolism and excretion of GZD in vivo could be systematically studied to reveal the pharmacodynamic process of GZD. Cinnamomi Ramulus, Paeoniae Radix Alba, Rhizoma Zingiberis Recens, Fructus Jujubae and Radix Glycyrrhizae were purchased from Beijing Tongrentang pharmacy (Beijing, China), and were authenticated by Chunsheng Liu, the Professor of Beijing University of Chinese Medicine (Beijing, China), in line with the Pharmacopoeia of the People’s Republic of China (Edition 2015) standards. Cinnamic acid (110,786–201,604), paeoniflorin (110,736–201,741), liquiritin (111,610– 201,106), glycyrrhetinic acid (110,723–201,514), icariin (110,737–200,415), and aesculetin Materials and reagents Cinnamomi Ramulus, Paeoniae Radix Alba, Rhizoma Zingiberis Recens, Fructus Jujubae and Radix Glycyrrhizae were purchased from Beijing Tongrentang pharmacy (Beijing, China), and were authenticated by Chunsheng Liu, the Professor of Beijing University of Chinese Medicine (Beijing, China), in line with the Pharmacopoeia of the People’s Republic of China (Edition 2015) standards. Cinnamic acid (110,786–201,604), paeoniflorin (110,736–201,741), liquiritin (111,610– 201,106), glycyrrhetinic acid (110,723–201,514), icariin (110,737–200,415), and aesculetin Cinnamomi Ramulus, Paeoniae Radix Alba, Rhizoma Zingiberis Recens, Fructus Jujubae and Radix Glycyrrhizae were purchased from Beijing Tongrentang pharmacy (Beijing, China), and were authenticated by Chunsheng Liu, the Professor of Beijing University of Chinese Medicine (Beijing, China), in line with the Pharmacopoeia of the People’s Republic of China (Edition 2015) standards. Cinnamic acid (110,786–201,604), paeoniflorin (110,736–201,741), liquiritin (111,610– Cinnamic acid (110,786–201,604), paeoniflorin (110,736–201,741), liquiritin (111,610– 201,106), glycyrrhetinic acid (110,723–201,514), icariin (110,737–200,415), and aesculetin (110,741–200,506) were purchased from National Institutes for Food and Drug Control (Beijing, China). Isoliquiritin (TT102I), liquiritigenin (TT011L), and glycyrrhizic acid (AB291G) were purchased from Epuresino technology (Tianjin, China) Co., Ltd (Tianjin, China). Albiflorin (Y15D8H50784) was purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). 6-Gingerol (ASB-00007164-005) and isoliquiritigenin (ASB-00009265-005) were purchased from ChromaDex, Inc. (Irvine, CA, USA). The purity of all standards was greater than 98%. Acetonitrile was purchased from Fisher Scientific (Hampton, NH, USA). The chemical structures of the analytes and internal standards (ISs) were shown in Fig. 1. Methanol and formic acid were both purchased from Thermo Fisher Scientific-CN (Beijing, China); acetic acid and ammonium acetate were (110,741–200,506) were purchased from National Institutes for Food and Drug Control (Beijing, China). Isoliquiritin (TT102I), liquiritigenin (TT011L), and glycyrrhizic acid (AB291G) were purchased from Epuresino technology (Tianjin, China) Co., Ltd (Tianjin, China). Albiflorin (Y15D8H50784) was purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). 6-Gingerol (ASB-00007164-005) and isoliquiritigenin (ASB-00009265-005) were purchased from ChromaDex, Inc. (Irvine, CA, USA). The purity of all standards was greater than 98%. Acetonitrile was purchased from Fisher Scientific (Hampton, NH, USA). The chemical structures of the analytes and internal standards (ISs) were shown in Fig. 1. Methanol and formic acid were both purchased from Thermo Fisher Scientific-CN (Beijing, China); acetic acid and ammonium acetate were purchased from Dikma Technologies Inc. (Beijing, China). All reagents were chromatographically pure. pure. Materials and reagents hydrolysis Liquiritin Liquiritigenin hydrolysis Isoliquiritin Isoliquiritigenin Glycyrrhizic acid hydrolysis Glycyrrhetinic acid Icariin (IS1) Aesculetin (IS2) Cinnamic acid Paeoniforin Albiflorin 6-Gingerol Fig.1 Chemical structures of analytes and internal standard (IS) Albiflorin Cinnamic acid Paeoniforin hydrolysis Liquiritin Liquiritigenin Cinnamic acid Paeoniforin Albiflorin Cinnamic acid Albiflorin Albiflorin Paeoniforin hydr Liquiritin olysis Liquiritigenin Liquiritigenin drolysis Isoliquiritigenin Liquiritigenin Liquiritin Isoliquiritin Isoliquiritin Isoliquiritigenin Glycyrrhizic acid hydrolysis Glycyrrhetinic acid Icariin (IS1) Aesculetin (IS2) 6-Gingerol Fig.1 Chemical structures of analytes and internal standard (IS) Isoliquiritigenin Glycyrrhizic acid Glycyrrhetinic acid 6-Gingerol 6-Gingerol Icariin (IS1) Aesculetin (IS2) Fig.1 Chemical structures of analytes and internal standard (IS) HPLC–MS/MS conditions Agilent 1260 with the Rapid Resolution Liquid Chromatography (RRLC) system (Agilent Technologies, Santa Clara, CA, USA) was used. The separation of compounds was carried out on the Agilent SB C18 column (2.1 × 50 mm, 1.8 μm) at 35℃. Agilent 6410 mass spectrometry was equipped with Electron Spray Ionization (ESI), using Multiple Reaction Monitoring (MRM) with negative ion mode detection. The ion source temperature was 350℃; the drying gas was nitrogen (N2, purity of 99.9%); the flow rate was 10 L·min−1; the nebulizing gas pressure was 40 psi; and the capillary voltage was 4.0 kv. As the analytes and IS had different ionization activities, two optimized mobile phases were used separately to ensure the sensitivity. The MS conditions, including the ion pair, fragmentor voltage, and collision energy were all optimized with standard solutions. Condition Ι: the mobile phase was composed of 0.1% aqueous acetic acid (A) and acetonitrile (B) with a gradient program (0.0–1.0 min, 5% B; 1.1–5.0 min, 14% B; 5.1–8.0 min, 20% B; 8.1–11.0 min, 45% B, 11.1–17.0 min, 85% B). Condition ΙΙ: the mobile phase was composed of 0.2 % acetic acid aqueous solution containing 2 mmoL·L−1 ammonium acetate (A) and acetonitrile (B) with a gradient program (0.0–1.0 min, 5%B ; 1.1–8.0 min, 45% B). The flow rate was 0.3 mL·min−1 and the injection volume was 2 μL. The stop time was 8.0 min with the post time of 6.0 min. Preparation of GZD for oral adminstration The volatile oil of Ramulus Cinnamomiand and Rhizoma Zingiberis Recens was extracted by steam distillation with four folds water for 2 h. The residues of the two herbal slices and other three herbal slices were boiled twice with eight folds water for 30 min each time. All those water decoctions were mixed together and concentrated to 1.0 g crude drugs per milliliter. After the volatile oil were added and mixed together, the extraction was stored at 4℃. Animals Animals 30 clean male Sprague–Dawley rats (300 ± 20 g) were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China quality certification number: SCXK (Jing) 2016–0002). The temperature and humidity of the controlled environmental conditions were 23 ± 3℃ and 50% ± 10℃, respectively. The principles of laboratory animal care and all protocols were in accordance with the relevant national legislation and local guidelines and were approved by Animal Care and Use Committee of the Institute of Basic Theory for Chinese Medicine, China Academy of Chinese Medical Sciences (Date: 25 March 2019; No.: SYXK (Jing) 2019-008). Use Committee of the Institute of Basic Theory for Chinese Medicine, China Academy of Chinese Medical Sciences (Date: 25 March 2019; No.: SYXK (Jing) 2019-008). Preparation of GZD extraction for injection GZD solution (1.0 g·mL−1) was centrifuged (15,000 rpm, 15 min, 4℃) and the supernatant was filtered with 0.2 μm filter membranes. Then, 100 mL filtrate was vacuum freeze-dried into powder, which was dissolved with 500 mL sodium chloride injection (0.9%). The solution was filtered with 0.2 μm filter membrane in the super clean bench to remove the bacteria and then packed separately and stored at 4℃. This extraction contained 0.2 g crude drugs per milliliter. Sample preparation In this study, the methods between liquid–liquid extraction (LLE) and protein precipitation (PPT) were compared to extract and purify the compounds to be tested in rat plasma. For LLE, ethyl acetate with or without hydrochloric acid were used as the extract liquor. For PPT, methanol and acetonitrile with or without hydrochloric acid were used to precipitate protein. The mixed reference solution was added into the blank plasma samples and prepared with above methods separately and injected into HPLC-MS/MS. The ratio of the peak area of each compound in the plasma sample to that of in the reference solution with same concentration was taken as the extraction rate. The method with a high extraction rate was chosen to prepare the samples. Precision and accuracy The accuracy and precision of the method were evaluated by intra- and inter-day variations. Standard solutions were added to the blank plasma and prepared at three different concentrations (low, medium, and high). Intra-day precision was evaluated with six replicates at one day, and inter- day precision was evaluated at three days. The precision of each compound was evaluated by relative standard deviation (RSD) value, which should not exceed 15.0%, and the accuracy was estimated with the relative error (RE), which should be within ±15.0%. Stability Stability of the analytes from the plasma samples were investigated by determining three different concentrations (low, medium and high samples) in five replicates under different storage conditions. The stability in plasma was assessed by analyzing (i) samples kept at room temperature (25°C) for 24 h, (ii) samples after three freeze-thaw cycles, (iii) samples after stored at −80℃ for 15 days. Specificity The specificity of the method was assessed by comparing chromatograms of blank plasma samples from six individual rats, blank plasma spiked with the 10 analytes and 2 ISs. The plasma samples were obtained at 30 min or 4 h from the rats after oral administration of the GZD or GZD extraction. Linearity, LLOQ, and LLOD The standard stock solutions of 10 analytes and 2 ISs were prepared in methanol. The initial concentrations of each compounds were 12,750 ng·mL−1 for cinnamic acid, 3248 ng·mL−1 for paeoniflorin, 3488 ng·mL−1 for albiflorin, 3568 ng·mL−1 for 6-gingerol, 3248 ng·mL−1 for liquiritin, 3248 ng·mL−1 for isoliquiritin, 3728 ng·mL−1 for liquiritigenin, 3328 ng·mL−1 for isoliquiritigenin, 3488 ng·mL−1 for glycyrrhizic acid, and 8360 ng·mL−1 for glycyrrhetinic acid. The concentration of 2 ISs were 508 ng·mL−1 for icariin and 638 ng·mL−1 for aesculetin.To prepare the working solutions for calibration samples, 10 series concentrations solutions were prepared by methanol dilution. 20 μL working solution was added to the blank plasma. Plasma calibration curves were constructed using the peak area ratios of the 10 analytes to the ISs, and applying separate weighted (1/χ2) least squares linear regression. The lower limit of quantification (LLOQ) and the lower limit of detection (LLOD) were defined by the signal-to-noise ratio method. LLOQ should be ten times the noise level (S/N ≥ 10), and LLOD should be three times the noise level (S/N ≥ 3). Extraction recovery and matrix Effect The recoveries of the analytes from plasma samples were determined by comparing the peak areas of the analytes in plasma samples after extraction to those of the same concentration of the analytes spiked into the solution extracted from plasma samples. The matrix effects were measured by comparing the peak areas obtained from samples with the analytes spiked after extraction, at three concentration levels (low, middle, and high), to those obtained from standard solutions at the same concentrations. Pharmacokinetic study 30 clean male Sprague–Dawley rats (300 ± 20 g) were divided into oral administration (ig) groups (high, medium, and low dose), intravenous injection (iv) group and blank group. There were 6 rats in each ig group and 5 rats in iv group. All animals were fasted for 12 h but with access to water before experiment. Rats in ig group were given GZD by gavage according to their body weight (4.0 mL·100 g−1). The high dose group was given GZD in a concentration of 1.0 g crude drug·mL−1, which was about 10 times as much as the clinical dosage (40.0 g crude drug·kg−1). The medium dose group was in a concentration of 0.5 g crude drug·mL−1(20.0 g crude drug·kg−1), and the low dose group was in a concentration of 0.25 g crude drug·mL−1 (10.0 g crude drug·kg−1). 300 μL blood samples were collected in Eppendorf tubes from the postorbital venous plexus veins of each rat by capillary tubes before dose (0 h) and after doses at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h. Rats in iv group were injected with GZD extraction via the caudal vein according to their body weight (1.0 mL·100 g−1), which was equivalent to 0.5 times as much as the clinical dosage (2.0g crude drug·kg−1). Blood samples were collected at 0 min, 2 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after injecting GZD extraction. j g Rats in the blank group were used to collect blank blood samples from the abdominal aorta after anesthetized by intraperitoneal injection of 3% sodium pentobarbital in the dosage of 0.5mL·100 1 The blood samples were centrifuged at 12,000 rpm for 10 min at 4℃, and stored at −80℃ unti analysis. Data analysis The validated HPLC-MS/MS methods were applied to analyze the concentrations of 10 compounds in rat plasma after oral or parenteral administration GZD at different times. The pharmacokinetic parameters were obtained by the non-compartmental analysis of plasma concentration versus time data using a non-compartmental model of the Phoenix Winnonlin 8.1 software (Certara, USA). Optimization of chromatographic and Mass conditions The final sample treatment method was as follows: 20 µL IS solution (500 ng·mL−1 of icariin; 600 ng·mL−1 of aesculetin) and 10 µL hydrochloric acid (20 mmol·L−1) were added into each 100 µL plasma sample. The sample was mixed for 1 min and 200 µL methanol was added. Then the mixture was vortex for 3 min. The sample was centrifuged at 12,000 rpm for 15 min at 4℃. 300 µL of the supernatant was collected and evaporated to dryness by vacuum freeze-drying. Finally, the residue was dissolved by 100 µL mobile phase (ACN: H2O = 1:1) and vortexed for 3 min. After centrifuged at 12,000 rpm for 15 min at 4℃, 2 µL aliquot was injected into HPLC- MS/MS system. Table 1 MS/MS parameters of the analytes and internal standards Optimization of chromatographic and Mass conditions In order to obtain high detection sensitivity and good peak symmetry of the analytes, different ratios of formic acid, acetic acid, or ammonium acetate were chosen as the mobile phase annexing additive. Finally, we found that the separation and detection of the analytes were determined with these two phase conditions. The mobile phase used acetonitrile and 0.1% formic acid was suitable for paeoniflorin, albiflorin, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, glycyrrhizic acid, glycyrrhetinic acid, and icariin (IS1), while the mobile phase condition of acetonitrile and 0.2% acetic acid containing 2 mmoL·L−1 ammonium acetate was suitable for cinnamic acid, 6-gingerol, and aesculetin (IS2). The ionization conditions of those compounds, such as capillary voltage, nebulizer pressure, fragmentor voltage, and collision energy, were optimized by using the standard solutions. The optimized ionization conditions for the 10 compounds and 2 ISs were summarized in Table 1. Table 1 MS/MS parameters of the analytes and internal standards Analytes Quantification Transition (m/z) Dwell Time (ms) Fragmentor Voltage (V) Collision Energy (V) Cinnamic acid 147.0 → 103.0 60 62 6 Paeoniflorin 525.2 → 121.0 70 157 37 Albiflorin 525.2 → 121.0 70 157 37 Liquiritin 417.2 → 255.2 70 152 15 Isoliquiritin 417.2 → 255.2 70 152 15 Liquiritigenin 255.1 → 119.0 70 105 24 Isoliquiritigenin 255.1→ 119.0 70 105 24 Glycyrrhizic acid 821.2 → 350.9 70 181 40 Glycyrrhetinic acid 469.4 → 425.5 70 190 41 6-Gingerol 293.1 → 99.1 60 82 6 Icariin (IS1) 721.3 → 367.2 60 120 35 Aesculetin (IS2) 177.1 → 133.0 60 110 15 Table 1 MS/MS parameters of the analytes and internal standards Analytes Quantification Transition (m/z) Dwell Time (ms) Fragmentor Voltage (V) Collision Energy (V) Cinnamic acid 147.0 → 103.0 60 62 6 Paeoniflorin 525.2 → 121.0 70 157 37 Albiflorin 525.2 → 121.0 70 157 37 Liquiritin 417.2 → 255.2 70 152 15 Isoliquiritin 417.2 → 255.2 70 152 15 Liquiritigenin 255.1 → 119.0 70 105 24 Isoliquiritigenin 255.1→ 119.0 70 105 24 Glycyrrhizic acid 821.2 → 350.9 70 181 40 Glycyrrhetinic acid 469.4 → 425.5 70 190 41 6-Gingerol 293.1 → 99.1 60 82 6 Icariin (IS1) 721.3 → 367.2 60 120 35 Aesculetin (IS2) 177.1 → 133.0 60 110 15 Sample pretreatment The absolute recoveries of 10 analytes and 2 ISs after different sample pretreatments could be found in Table 2. Sample pretreatment The absolute recoveries of 10 analytes and 2 ISs after different sample pretreatments could be found in Table 2. The final sample treatment method was as follows: 20 µL IS solution (500 ng·mL−1 of icariin; 600 ng·mL−1 of aesculetin) and 10 µL hydrochloric acid (20 mmol·L−1) were added into each 100 µL plasma sample. The sample was mixed for 1 min and 200 µL methanol was added. Then the mixture was vortex for 3 min. The sample was centrifuged at 12,000 rpm for 15 min at 4℃. 300 µL of the supernatant was collected and evaporated to dryness by vacuum freeze-drying. Finally, the residue was dissolved by 100 µL mobile phase (ACN: H2O = 1:1) and vortexed for 3 min. After centrifuged at 12,000 rpm for 15 min at 4℃, 2 µL aliquot was injected into HPLC- MS/MS system. Table 2 Absolute recoveries of the analytes and internal standards after different sample pretreatments methods (n = 3, mean ± SD). Sample pretreatment Analytes Ethylacetate (%) Ethylacetate + 50 mmol·L−1 HCl(%) MeOH (%) MeOH + 50 mmol·L−1 HCl(%) MeOH + 20 mmol·L−1 HCl(%) MeOH + 10 mmol·L−1 HCl(%) MeOH + 5 mmol·L−1 HCl(%) ACN (%) ACN + 50 mmol·L−1 HCl(%) ACN + 20 mmol·L−1 HCl(%) ACN + 10 mmol·L−1 HCl(%) ACN + 5 mmol·L−1 HCl(%) Cinnamic acid 8.0 ± 0.5 107.4 ± 8.5 102.2 ± 8.6 108.0 ± 11.7 106.8 ± 2.4 101.0 ± 2.7 103.6 ± 8.7 108.3 ± 11.9 100.3 ± 12.9 108.7 ± 17.1 106.9 ± 15.1 105.3 ± 12.4 Paeoniflorin 30.0 ± 3.3 25.7 ± 7.2 64.8 ± 7.4 72.4 ± 4.2 81.8 ± 7.6 85.8 ± 3.1 70.0 ± 9.7 64.7 ± 9.5 71.6 ± 8.9 84.9 ± 3.8 85.6 ± 6.3 94.9 ± 7.9 Albiflorin 46.5 ± 0.7 42.5 ± 8.9 62.9 ± 13.2 106.6 ± 12.1 111.5 ± 2.3 117.9 ± 1.1 100.6 ± 7.4 73.0 ± 3.5 96.4 ± 16.7 120.1 ± 10.6 109.5 ± 10.8 118.8 ± 2.1 Liquiritin 40.9 ± 8.3 91.2 ± 12.6 97.4 ± 13.7 92.9 ± 8.7 108.2 ± 2.5 111.3 ± 2.2 105.5 ± 14.4 116.4 ± 17.6 93.2 ± 13.9 108.1 ± 16.5 113.7 ± 15.4 115.2 ± 15.0 Isoliquiritin 77.3 ± 6.7 102.8 ± 8.6 61.3 ± 10.7 94.5 ± 10.4 100.2 ± 1.9 102.1 ± 1.8 95.8 ± 6.6 62.7 ± 16.8 96.6 ± 10.0 106.8 ± 19.6 82.7 ± 12.8 103.5 ± 10.4 Liquiritigenin 103.9 ± 7.7 98.2 ± 8.6 76.2 ± 12.3 97.3 ± 0.1 107.3 ± 1.3 110.9 ± 1.0 102.5 ± 11.8 75.6 ± 15.6 98.1 ± 9.4 112.0 ± 8.3 108.7 ± 11.7 114.5 ± 16.5 Isoliquiritigenin 105.4 ± 6.3 75.9 ± 6.3 49.3 ± 10.5 106.3 ± 0.7 109.9 ± 2.2 115.4 ± 1.9 99.9 ± 7.7 58.6 ± 11.2 115.8 ± 10.5 126.6 ± 17.6 109.5 ± 7.6 113.8 ± 2.5 Glycyrrhizic acid 0.0 ± 0.0 0.0 ± 0.0 101.8 ± 17.2 12.3 ± 0.6 52.6 ± 5.0 59.9 ± 0.9 63.5 ± 1.3 77.9 ± 1.9 17.2 ± 23.6 44.1 ± 9.6 64.4 ± 14.6 63.2 ± 4.1 Glycyrrhetinic acid 36.0 ± 7.1 48.7 ± 4.4 36.2 ± 6.9 55.2 ± 7.7 59.4 ± 1.2 58.6 ± 1.5 59.0 ± 9.6 39.3 ± 9.4 63.2 ± 14.3 64.6 ± 3.6 61.7 ± 4.3 65.6 ± 15.1 6-Gingerol 130.0 ± 17.6 6.0 ± 4.2 84.2 ± 12.4 102.3 ± 8.7 100.4 ± 14.8 111.7 ± 1.9 101.4 ± 7.8 89.2 ± 7.7 100.3 ± 11.7 108.5 ± 12.9 100.0 ± 13.5 108.7 ± 12.4 IS1 94.2 ± 9.6 89.2 ± 10.8 72.1 ± 12.1 94.1 ± 6.0 114.4 ± 12.8 120.2 ± 2.6 105.9 ± 15.0 82.2 ± 13.1 104.0 ± 8.5 120.2 ± 17.5 114.1 ± 10.0 124.4 ± 7.6 IS2 80.1 ± 10.2 2.5 ± 0.0 51.3 ± 4.0 98.3 ± 5.7 82.0 ± 1.2 62.6 ± 0.3 50.0 ± 2.7 47.0 ± 6.8 75.9 ± 10.2 61.9 ± 0.2 42.3 ± 5.4 40.6 ± 2.6 Note: MeOH: protein precipitation with methanol; ACN: protein sediment by acetonitrile; Ethyl acetate: extracting with ethyl acetate; HCl: adding hydrochloric acid; MeOH + 50 mmol/L HCl: protein precipitation with methanol under acidification. Precision and accuracy The results of precision and accuracy at the three different concentration levels were presented in Table 4. The intra- and inter-day RSD values were below 6.9% and 9.6% respectively, while the corresponding RE values ranged from -10.8% to 5.3%. All the assay values were within the acceptable criteria. Linearity, LLOQ, and LLOD The results showed that the 10 compounds had good linearity in the corresponding concentration range. The regression equation, linear range and correlation coefficient (r), LLOQ, and lower limit of detection (LLOD) of each compound were listed in Table 3. specificity Under the developed chromatographic and mass conditions, the sample chromatograms of blank plasma, the standard sample solution, the drug-containing plasma samples after oral administration of GZD, and the solution of GZD were presented in Fig. 2. The method had high specificity and could be used for the qualitative determination of these compounds in plasma. Matrix effect and extraction recovery Average recoveries of investigated targets ranged from 85.4% to 113.9%. The RSD values of all analytes were lower than 15.3%, which indicated that there was no significant loss of the compounds among the process of the protein precipitation in the plasma samples. The matrix effect of each compound ranged from 85.4% to 116.8%, indicating that there was no obvious matrix interference. The results showed that the method was accurate and acceptable. The data of extraction recovery and matrix effect were listed in Table 5. Method validation Method validation Method validation Sample pretreatment lytes and internal standards after different sample pretreatments methods (n = 3, mean ± SD). ute recoveries of the analytes and internal standards after different sample pretreatments methods (n = 3, mean ± SD). Table 2 Absolute recoveries of the analytes and internal standards after different sample pretreatments me Note: MeOH: protein precipitation with methanol; ACN: protein sediment by acetonitrile; Ethyl acetate: extracting with ethyl acetate; HCl: adding hydrochloric acid; MeOH + 50 mmol/L methanol under acidification. Stability After storage at -80℃ for 15 days and three freeze-thaw cycles, the stability RSD values of 10 compounds in plasma were all less than 9.9% (Table 6). The results showed that the detected analytes were all satisfied with the criteria under all conditions, so the samples were stable during the test process. 1 x10 0 2 4 6 - MRM (147.00 -> 103.00) B1.d C (%) A i i i Ti ( i 4 5 6 7 1 x10 0 1 2 3 - MRM (525.20 -> 121.00) Blank.d C (%) A i i i Ti ( i 3 4 5 6 1 x10 0 1 2 3 - MRM (417.20 -> 255.20) Blank.d C (%) A i i i Ti ( i 10 11 12 13 14 15 1 x10 0 2 4 - MRM (417.20 -> 255.20) Blank.d C (%) A i i i Ti ( i 3 4 5 6 2 x10 0 0.5 1 - MRM (255.10 -> 119.00) Blank.d 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (821.20 -> 350.90) Blank.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (469.40 -> 425.50) Blank.d C (%) A i i i Ti ( i 15 16 17 2 x10 0 0.25 0.5 0.75 1 - MRM (293.10 -> 99.10) B1.d C (%) A i i i Ti ( i 4 5 6 7 2 x10 0 0.5 1 - MRM (721.30 -> 367.20) Blank.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (177.10 -> 133.00) 180513010.d Counts (%) vs. Stability Acquisition Time (min 4 5 6 7 A 2 x10 0 0.5 1 - MRM (147.00 -> 103.00) 5m.d C (%) A i i i Ti ( i 4 5 6 7 2 x10 0 0.5 1 - MRM (525.20 -> 121.00) 5min.d C (%) A i i i Ti ( i 3 4 5 6 2 x10 0 0.5 1 - MRM (417.20 -> 255.20) 5min.d C (%) A i i i Ti ( i 3 4 5 6 2 x10 0 0.5 1 - MRM (417.20 -> 255.20) 5min.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (255.10 -> 119.00) 1h.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (821.20 -> 350.90) 1h.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (469.40 -> 425.50) 8h.d C (%) A i i i Ti ( i 15 16 17 2 x10 0 0.5 1 - MRM (293.10 -> 99.10) 5m.d C (%) A i i i Ti ( i 4 5 6 7 2 x10 0 0.5 1 - MRM (721.30 -> 367.20) 1h.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (177.10 -> 133.00) 1h.d Counts (%) vs. Stability Acquisition Time (min 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11 12 C 2 x10 0 0.5 1 - MRM (147.00 -> 103.00) 180521004.d C (%) A i i i Ti ( i 4 5 6 7 2 x10 0 0.5 1 - MRM (525.20 -> 121.00) 180521004.d C (%) A i i i Ti ( i 3 4 5 6 2 x10 0 0.5 1 - MRM (417.20 -> 255.20) 180521004.d C (%) A i i i Ti ( i 3 4 5 6 2 x10 0 0.5 1 - MRM (417.20 -> 255.20) 180521004.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (255.10 -> 119.00) 180521004.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.25 0.5 0.75 - MRM (821.20 -> 350.90) 180521004.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (469.40 -> 425.50) 1806119003 C (%) A i i i Ti ( i 15 16 17 2 x10 0 0.5 1 - MRM (293.10 -> 99.10) 180521004.d C (%) A i i i Ti ( i 4 5 6 7 2 x10 0 0.5 1 - MRM (721.30 -> 367.20) 180521004.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (177.10 -> 133.00) 180521004.d Counts (%) vs. Acquisition Time (min 4 5 6 7 D 1 2 3 4 5 6 7 8 10 2 x10 0 0.5 1 - MRM (417.20 -> 255.20) QC.d 3 4 5 6 2 x10 0 0.5 1 - MRM (821.20 -> 350.90) QC.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (469.40 -> 425.50) QC.d C (%) A i i i Ti ( i 15 16 17 2 x10 0 0.5 1 - MRM (177.10 -> 133.00) 180520041.d Counts (%) vs. Stability Acquisition Time (min 4 5 6 7 2 x10 0 0.5 1 - MRM (147.00 -> 103.00) q1.d 4 5 6 7 2 x10 0 0.5 1 - MRM (417.20 -> 255.20) QC.d 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (525.20 -> 121.00) QC.d 3 4 5 6 2 x10 0 0.5 1 - MRM (255.10 -> 119.00) QC.d 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (293.10 -> 99.10) q1.d 4 5 6 7 2 x10 0 0.5 1 - MRM (721.30 -> 367.20) QC.d C (%) A i i i Ti ( i 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 B 1 x10 0 2 4 6 - MRM (147.00 -> 103.00) B1.d C (%) A i i i Ti ( i 4 5 6 7 1 x10 0 1 2 3 - MRM (525.20 -> 121.00) Blank.d C (%) A i i i Ti ( i 3 4 5 6 1 x10 0 1 2 3 - MRM (417.20 -> 255.20) Blank.d C (%) A i i i Ti ( i 10 11 12 13 14 15 1 x10 0 2 4 - MRM (417.20 -> 255.20) Blank.d C (%) A i i i Ti ( i 3 4 5 6 2 x10 0 0.5 1 - MRM (255.10 -> 119.00) Blank.d 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (821.20 -> 350.90) Blank.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (469.40 -> 425.50) Blank.d C (%) A i i i Ti ( i 15 16 17 2 x10 0 0.25 0.5 0.75 1 - MRM (293.10 -> 99.10) B1.d C (%) A i i i Ti ( i 4 5 6 7 2 x10 0 0.5 1 - MRM (721.30 -> 367.20) Blank.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (177.10 -> 133.00) 180513010.d Counts (%) vs. Acquisition Time (min 4 5 6 7 A 1 1 2 5 C (%) A i i i Ti 10 11 12 13 14 - MRM (469.40 -> 425.50) QC.d 10 10 10 11 12 D C B A Fig. Stability Acquisition Time (min 4 5 6 7 2 x10 0 0.5 1 - MRM (147.00 -> 103.00) q1.d 4 5 6 7 2 x10 0 0.5 1 - MRM (417.20 -> 255.20) QC.d 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (525.20 -> 121.00) QC.d 3 4 5 6 2 x10 0 0.5 1 - MRM (255.10 -> 119.00) QC.d 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (293.10 -> 99.10) q1.d 4 5 6 7 2 x10 0 0.5 1 - MRM (721.30 -> 367.20) QC.d C (%) A i i i Ti ( i 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 B Fig. 2 Multiple reaction monitoring mode (MRM) chromatograms of cinnamic acid (1), paeoniflorin (2), albiflorin (3), liquiritin (4), isoliquiritin (5), liquiritigenin (6), isoliquiritigenin (7), glycyrrhizic acid (8), glycyrrhetinic acid (9), 6-gingerol (10), icariin (11; IS1), and aesculetin (12; IS2) . blank rat serum (A), blank rat serum spiked with the analytes and ISs (B), rat serum samples at 5 min, 1 h, or 8 h after oral administration of GZD (C), and GZD after 1000 times dilution with methanol(D). Stability Acquisition Time (min 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11 12 C n 2 x10 0 0.5 1 - MRM (147.00 -> 103.00) 180521004.d C (%) A i i i Ti ( i 4 5 6 7 2 x10 0 0.5 1 - MRM (525.20 -> 121.00) 180521004.d C (%) A i i i Ti ( i 3 4 5 6 2 x10 0 0.5 1 - MRM (417.20 -> 255.20) 180521004.d C (%) A i i i Ti ( i 3 4 5 6 2 x10 0 0.5 1 - MRM (417.20 -> 255.20) 180521004.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (255.10 -> 119.00) 180521004.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.25 0.5 0.75 - MRM (821.20 -> 350.90) 180521004.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (469.40 -> 425.50) 1806119003 C (%) A i i i Ti ( i 15 16 17 2 x10 0 0.5 1 - MRM (293.10 -> 99.10) 180521004.d C (%) A i i i Ti ( i 4 5 6 7 2 x10 0 0.5 1 - MRM (721.30 -> 367.20) 180521004.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (177.10 -> 133.00) 180521004.d Counts (%) vs. Acquisition Time (min 4 5 6 7 D 1 2 3 4 5 6 7 8 10 2 x10 0 0.5 1 - MRM (417.20 -> 255.20) QC.d 3 4 5 6 2 x10 0 0.5 1 - MRM (821.20 -> 350.90) QC.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (469.40 -> 425.50) QC.d C (%) A i i i Ti ( i 15 16 17 2 x10 0 0.5 1 - MRM (177.10 -> 133.00) 180520041.d Counts (%) vs. Stability 2 x10 0 0.5 1 - MRM (147.00 -> 103.00) 5m.d C (%) A i i i Ti ( i 4 5 6 7 2 x10 0 0.5 1 - MRM (525.20 -> 121.00) 5min.d C (%) A i i i Ti ( i 3 4 5 6 2 x10 0 0.5 1 - MRM (417.20 -> 255.20) 5min.d C (%) A i i i Ti ( i 3 4 5 6 2 x10 0 0.5 1 - MRM (417.20 -> 255.20) 5min.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (255.10 -> 119.00) 1h.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (821.20 -> 350.90) 1h.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (469.40 -> 425.50) 8h.d C (%) A i i i Ti ( i 15 16 17 2 x10 0 0.5 1 - MRM (293.10 -> 99.10) 5m.d C (%) A i i i Ti ( i 4 5 6 7 2 x10 0 0.5 1 - MRM (721.30 -> 367.20) 1h.d C (%) A i i i Ti ( i 10 11 12 13 14 15 2 x10 0 0.5 1 - MRM (177.10 -> 133.00) 1h.d Counts (%) vs. Stability (ng·mL−1) RSD % RE % Cinnamic acid 51 51.3 ± 0.9 2.0 0.6 47.8 ± 4.1 8.8 −6.2 204 209.4 ± 9.6 5.1 2.7 203.0 ± 17.2 8.8 −0.5 812 818.6 ± 31.3 4.3 0.3 815.6 ± 22.7 2.9 −0.1 Paeoniflorin 51 50.8 ± 1.9 4.2 0.1 53.1 ± 3.5 6.8 4.6 203 202.9 ± 10.0 5.5 −0.1 200.1 ± 13.1 6.8 −1.4 812 807.6 ± 38.5 5.3 −0.6 812.6 ± 21.4 2.7 0.1 Albiflorin 55 54.5 ± 1.1 2.2 −0.1 51.9 ± 4.5 8.9 −4.8 218 218.0 ± 7.6 3.9 0.0 220.6 ± 12.0 5.6 1.2 872 886.5 ± 39.0 4.9 1.7 870.6 ± 20.4 2.4 −0.2 Liquiritin 51 53.2 ± 2.4 5.0 4.8 48.5 ± 4.5 9.6 −4.5 203 198.2 ± 6.4 3.6 −2.4 205.4 ± 10.6 5.3 1.2 812 812.9 ± 22.1 3.0 0.1 811.5 ± 21.1 2.7 −0.1 Isoliquiritin 51 49.4 ± 1.0 2.3 −2.6 46.7 ± 3.9 8.7 −8.1 203 208.1 ± 12.8 6.9 2.5 208.1 ± 17.1 8.5 2.5 812 810.6 ± 44.0 6.1 −0.2 802.7 ± 40.5 5.2 −1.2 Liquiritigenin 58 55.6 ± 2.0 4.0 −4.5 54.3 ± 4.3 8.2 −6.7 233 232.5 ± 5.6 2.7 −0.2 242.8 ± 20.5 8.7 4.2 932 850.1 ± 52.5 6.9 −8.8 888.0 ± 41.2 4.8 −4.7 Isoliquiritigenin 51 47.3 ± 1.7 4.1 −6.9 45.3 ± 3.8 8.8 −10.8 203 207.4 ± 9.5 5.1 2.2 207.5 ± 15.7 7.8 2.2 812 793.3 ± 44.8 6.3 −2.3 794.9 ± 32.1 4.2 −2.1 Glycyrrhizic acid 55 57.4 ± 1.3 2.6 5.3 52.0 ± 3.8 7.5 −4.7 218 213.3 ± 7.2 3.8 −2.2 223.2 ± 13.9 6.4 2.4 872 808.5 ± 22.5 3.1 −7.3 876.2 ± 33.0 3.9 0.5 Glycyrrhetinic acid 52 49.5 ± 1.0 2.2 −5.3 47.5 ± 2.5 5.4 −9.1 209 216.3 ± 11.1 5.8 3.5 216.2 ± 15.6 7.5 3.4 836 834.4 ± 35.3 4.7 −0.2 830.2 ± 29.0 3.6 −0.7 6-Gingerol 56 51.6 ± 2.1 4.6 −7.5 53.7 ± 3.8 7.3 −3.6 223 201.4 ± 1.6 0.9 −9.7 220.7 ± 18.1 8.5 −1.0 892 833.8 ± 35.6 4.8 −6.5 890.3 ± 20.1 2.3 −0.2 Table 3 Calibration curve, linear range, lower limit of detection (LLOD), and lower limit of quantification (LLOQ) of 10 analytes (n = 3) Analytes Regression equation Liner range (ng·mL-1) r LLOQ (ng·mL-1) LLOD (ng·mL-1) Cinnamic acid y = 0.9385x + 0.0344 6-6120 0.9995 6 2 Paeoniflorin y = 0.3622x + 0.0136 6-1624 0.9994 6 2 Albiflorin y = 0.3895x - 0.0395 13-1635 0.9996 13 3 Liquiritin y = 2.5403x + 0.0443 1-1624 0.9996 1 1 Isoliquiritin y = 2.2788x + 0.0393 3-1624 0.9997 3 1 Liquiritigenin y = 1.8926x - 0.0016 4-1864 0.9993 4 1 Isoliquiritigenin y = 3.0726x + 0.0336 3-812 0.9991 3 1 Glycyrrhizic acid y = 0.4481x + 0.0487 7-1744 0.9993 7 3 Glycyrrhetinic acid y = 0.4370x + 0.0427 4-2090 0.9993 4 2 6-Gingerol y = 1.0994x + 0.0392 7-1784 0.9983 7 3 Table 4 Intra-day and inter-day precision and accuracy data of the 10 compounds spiked Table 3 Calibration curve, linear range, lower limit of detection (LLOD), and lower limit of quantification (LLOQ) of 10 analytes (n = 3) Table 4 Intra-day and inter-day precision and accuracy data of the 10 compounds spiked in rat plasma (mean ± SD; 3 replicates per day for 3 days) Table 4 Intra-day and inter-day precision and accuracy data of the 10 compounds spiked in rat plasma (mean ± SD; 3 replicates per day for 3 days) Analytes Spiked conc. Note: RSD: relative standard deviation; RE: refractive error. Note: RSD: relative standard deviation; RE: refractive error. Stability 2 Multiple reaction monitoring mode (MRM) chromatograms of cinnamic acid (1), paeoniflorin (2), albiflorin (3), liquiritin (4), isoliquiritin (5), liquiritigenin (6), isoliquiritigenin (7), glycyrrhizic acid (8), glycyrrhetinic acid (9), 6-gingerol (10), icariin (11; IS1), and aesculetin (12; IS2) . blank rat serum (A), blank rat serum spiked with the analytes and ISs (B), rat serum samples at 5 min, 1 h, or 8 h after oral administration of GZD (C), and GZD after 1000 times dilution with methanol(D). Table 3 Calibration curve, linear range, lower limit of detection (LLOD), and lower limit of quantification (LLOQ) of 10 analytes (n = 3) Analytes Regression equation Liner range (ng·mL-1) r LLOQ (ng·mL-1) LLOD (ng·mL-1) Cinnamic acid y = 0.9385x + 0.0344 6-6120 0.9995 6 2 Paeoniflorin y = 0.3622x + 0.0136 6-1624 0.9994 6 2 Albiflorin y = 0.3895x - 0.0395 13-1635 0.9996 13 3 Liquiritin y = 2.5403x + 0.0443 1-1624 0.9996 1 1 Isoliquiritin y = 2.2788x + 0.0393 3-1624 0.9997 3 1 Liquiritigenin y = 1.8926x - 0.0016 4-1864 0.9993 4 1 Isoliquiritigenin y = 3.0726x + 0.0336 3-812 0.9991 3 1 Glycyrrhizic acid y = 0.4481x + 0.0487 7-1744 0.9993 7 3 Glycyrrhetinic acid y = 0.4370x + 0.0427 4-2090 0.9993 4 2 6-Gingerol y = 1.0994x + 0.0392 7-1784 0.9983 7 3 Table 4 Intra-day and inter-day precision and accuracy data of the 10 compounds spiked in rat plasma (mean ± SD; 3 replicates per day for 3 days) Analytes Spiked conc. (ng·mL−1) Intra-Day (n = 5) Inter-Day (n = 5) Conc. (ng·mL−1) RSD % RE % Conc. Note: RSD: relative standard deviation; RE: refractive error. Stability (ng·mL−1) Intra-Day (n = 5) Inter-Day (n = 5) Conc. (ng·mL−1) RSD % RE % Conc. (ng·mL−1) RSD % RE % Cinnamic acid 51 51.3 ± 0.9 2.0 0.6 47.8 ± 4.1 8.8 −6.2 204 209.4 ± 9.6 5.1 2.7 203.0 ± 17.2 8.8 −0.5 812 818.6 ± 31.3 4.3 0.3 815.6 ± 22.7 2.9 −0.1 Paeoniflorin 51 50.8 ± 1.9 4.2 0.1 53.1 ± 3.5 6.8 4.6 203 202.9 ± 10.0 5.5 −0.1 200.1 ± 13.1 6.8 −1.4 812 807.6 ± 38.5 5.3 −0.6 812.6 ± 21.4 2.7 0.1 Albiflorin 55 54.5 ± 1.1 2.2 −0.1 51.9 ± 4.5 8.9 −4.8 218 218.0 ± 7.6 3.9 0.0 220.6 ± 12.0 5.6 1.2 872 886.5 ± 39.0 4.9 1.7 870.6 ± 20.4 2.4 −0.2 Liquiritin 51 53.2 ± 2.4 5.0 4.8 48.5 ± 4.5 9.6 −4.5 203 198.2 ± 6.4 3.6 −2.4 205.4 ± 10.6 5.3 1.2 812 812.9 ± 22.1 3.0 0.1 811.5 ± 21.1 2.7 −0.1 Isoliquiritin 51 49.4 ± 1.0 2.3 −2.6 46.7 ± 3.9 8.7 −8.1 203 208.1 ± 12.8 6.9 2.5 208.1 ± 17.1 8.5 2.5 812 810.6 ± 44.0 6.1 −0.2 802.7 ± 40.5 5.2 −1.2 Liquiritigenin 58 55.6 ± 2.0 4.0 −4.5 54.3 ± 4.3 8.2 −6.7 233 232.5 ± 5.6 2.7 −0.2 242.8 ± 20.5 8.7 4.2 932 850.1 ± 52.5 6.9 −8.8 888.0 ± 41.2 4.8 −4.7 Isoliquiritigenin 51 47.3 ± 1.7 4.1 −6.9 45.3 ± 3.8 8.8 −10.8 203 207.4 ± 9.5 5.1 2.2 207.5 ± 15.7 7.8 2.2 812 793.3 ± 44.8 6.3 −2.3 794.9 ± 32.1 4.2 −2.1 Glycyrrhizic acid 55 57.4 ± 1.3 2.6 5.3 52.0 ± 3.8 7.5 −4.7 218 213.3 ± 7.2 3.8 −2.2 223.2 ± 13.9 6.4 2.4 872 808.5 ± 22.5 3.1 −7.3 876.2 ± 33.0 3.9 0.5 Glycyrrhetinic acid 52 49.5 ± 1.0 2.2 −5.3 47.5 ± 2.5 5.4 −9.1 209 216.3 ± 11.1 5.8 3.5 216.2 ± 15.6 7.5 3.4 836 834.4 ± 35.3 4.7 −0.2 830.2 ± 29.0 3.6 −0.7 6-Gingerol 56 51.6 ± 2.1 4.6 −7.5 53.7 ± 3.8 7.3 −3.6 223 201.4 ± 1.6 0.9 −9.7 220.7 ± 18.1 8.5 −1.0 892 833.8 ± 35.6 4.8 −6.5 890.3 ± 20.1 2.3 −0.2 N t RSD l ti t d d d i ti RE f ti Table 5 Extraction recovery and matrix effect data of the 10 compounds in rat plasma (n = 5) Analytes Spiked conc. Stability (ng·mL−1) Extraction Recovery (%) Matrix Effect (%) Mean RSD Mean RSD Cinnamic acid 51 97.9 15.3 96.1 8.9 204 96.3 8.7 103.3 8.5 812 110.9 4.8 90.3 6.0 Paeoniflorin 51 94.1 5.8 111.0 8.3 203 99.4 2.4 96.8 2.2 812 108.2 7.1 92.6 4.8 Albiflorin 55 95.9 9.8 102.4 10.5 218 104.8 5.8 85.4 9.4 872 113.9 9.4 88.7 7.5 Liquiritin 51 106.3 6.2 100.1 6.2 203 91.0 4.0 104.5 4.0 812 91.8 5.3 116.8 9.5 Isoliquiritin 51 87.6 11.3 106.4 3.9 203 110.3 6.6 93.0 4.0 812 102.0 5.4 98.1 2.5 Liquiritigenin 58 97.1 3.9 97.8 2.6 233 101.8 2.3 97.8 2.0 932 105.5 7.3 103.3 6.5 Isoliquiritigenin 51 92.6 5.9 108.6 4.6 203 105.2 4.9 105.8 3.2 812 97.2 2.7 113.7 7.4 Glycyrrhizic acid 55 100.7 5.4 114.8 9.0 218 89.1 5.9 105.7 4.3 872 87.5 8.8 94.7 12.7 Glycyrrhetinic acid 52 88.2 8.3 94.0 7.2 209 88.1 8.2 105.4 4.7 836 94.5 3.9 106.2 4.4 6-Gingerol 56 85.4 7.9 111.3 6.7 223 88.0 6.9 88.6 9.1 892 109.5 4.3 86.5 8.0 Table 5 Extraction recovery and matrix effect data of the 10 compounds in rat plasma (n = 5) Analytes Spiked conc. (ng·mL−1) Extraction Recovery (%) Matrix Effect (%) Mean RSD Mean RSD Cinnamic acid 51 97.9 15.3 96.1 8.9 204 96.3 8.7 103.3 8.5 812 110.9 4.8 90.3 6.0 Paeoniflorin 51 94.1 5.8 111.0 8.3 203 99.4 2.4 96.8 2.2 812 108.2 7.1 92.6 4.8 Albiflorin 55 95.9 9.8 102.4 10.5 218 104.8 5.8 85.4 9.4 872 113.9 9.4 88.7 7.5 Liquiritin 51 106.3 6.2 100.1 6.2 203 91.0 4.0 104.5 4.0 812 91.8 5.3 116.8 9.5 Isoliquiritin 51 87.6 11.3 106.4 3.9 203 110.3 6.6 93.0 4.0 812 102.0 5.4 98.1 2.5 Liquiritigenin 58 97.1 3.9 97.8 2.6 233 101.8 2.3 97.8 2.0 932 105.5 7.3 103.3 6.5 Isoliquiritigenin 51 92.6 5.9 108.6 4.6 203 105.2 4.9 105.8 3.2 812 97.2 2.7 113.7 7.4 Glycyrrhizic acid 55 100.7 5.4 114.8 9.0 218 89.1 5.9 105.7 4.3 872 87.5 8.8 94.7 12.7 Glycyrrhetinic acid 52 88.2 8.3 94.0 7.2 209 88.1 8.2 105.4 4.7 836 94.5 3.9 106.2 4.4 6-Gingerol 56 85.4 7.9 111.3 6.7 223 88.0 6.9 88.6 9.1 892 109.5 4.3 86.5 8.0 Table 6 Stability of 10 compounds under various conditions (n = 5). Analytes Spiked conc. Stability Note: Mean: measured concentration; RSD: relative standard deviation. Stability (ng·mL−1) 12 h at 4℃ (%) Three Freeze– Thaw Cycles (%) 15 Days at −80℃ (%) Mean RSD Mean RSD Mean RSD Cinnamic acid 52 97.5 8.4 92.1 4.0 101.1 8.4 209 111.2 5.1 102.5 6.3 109.4 8.0 836 111.4 6.1 99.9 1.9 99.5 4.6 Paeoniflorin 51 88.8 6.2 89.6 4.0 104.6 7.7 203 85.6 6.3 103.3 4.4 97.5 3.1 812 99.5 8.5 99.9 2.7 100.1 4.7 Albiflorin 55 100.7 9.9 96.2 6.5 90.6 1.9 218 90.1 8.5 100.5 4.4 104.2 8.3 872 102.6 6.8 100.0 1.3 101.2 4.7 Liquiritin 51 92.2 5.8 89.7 2.7 93.3 0.6 203 103.6 5.6 103.2 5.2 106.7 5.1 812 98.2 5.8 99.8 3.8 99.7 2.0 Isoliquiritin 51 109.1 4.6 87.2 1.6 104.8 5.2 Table 6 Stability of 10 compounds under various conditions (n = 5). Table 6 Stability of 10 compounds under various conditions (n = 5). Analytes Spiked conc. (ng·mL−1) 12 h at 4℃ (%) Three Freeze– Thaw Cycles (%) 15 Days at −80℃ (%) Mean RSD Mean RSD Mean RSD Cinnamic acid 52 97.5 8.4 92.1 4.0 101.1 8.4 209 111.2 5.1 102.5 6.3 109.4 8.0 836 111.4 6.1 99.9 1.9 99.5 4.6 Paeoniflorin 51 88.8 6.2 89.6 4.0 104.6 7.7 203 85.6 6.3 103.3 4.4 97.5 3.1 812 99.5 8.5 99.9 2.7 100.1 4.7 Albiflorin 55 100.7 9.9 96.2 6.5 90.6 1.9 218 90.1 8.5 100.5 4.4 104.2 8.3 872 102.6 6.8 100.0 1.3 101.2 4.7 Liquiritin 51 92.2 5.8 89.7 2.7 93.3 0.6 203 103.6 5.6 103.2 5.2 106.7 5.1 812 98.2 5.8 99.8 3.8 99.7 2.0 Isoliquiritin 51 109.1 4.6 87.2 1.6 104.8 5.2 203 90.9 5.4 104.0 4.5 95.8 7.9 812 96.1 7.6 99.8 4.0 100.9 1.3 Liquiritigenin 58 95.4 9.5 89.1 2.6 95.6 7.9 233 102.9 3.4 103.4 6.1 109.4 6.3 932 108.3 7.3 99.8 4.5 99.3 2.1 Isoliquiritigenin 51 109.5 8.8 90.7 2.5 97.5 8.8 203 90.2 5.2 102.9 5.6 101.5 2.3 812 97.5 6.7 99.9 5.1 99.9 4.0 Glycyrrhizic acid 54.555 99.3 5.8 89.8 4.4 98.0 1.8 218 101.0 1.8 103.2 2.1 104.7 4.3 872 102.9 5.7 99.9 5.8 99.8 1.6 Glycyrrhetinic acid 52 100.4 8.5 90.5 5.1 103.6 7.5 209 99.5 6.8 105.2 6.2 108.3 3.7 836 105.6 6.4 99.7 2.9 99.6 4.2 6-Gingerol 56 98.8 5.1 87.5 4.6 97.5 7.0 223 99.3 4.6 103.9 4.3 100.1 4.1 892 103.5 7.0 99.8 3.5 100.0 0.3 Note: Mean: measured concentration; RSD: relative standard deviation. Pharmacokinetic results The validated HPLC-MS/MS methods were applied to the pharmacokinetic study of the 10 compounds in rat plasma after oral administration and intravenous injection of GZD. The pharmacokinetic parameters were calculated by Phoenix Winnonlin 8.1 software (Certara, USA). Mean blood concentration–time curves (C-T) of the 10 compounds taken orally were displayed in Fig. 3, and the main pharmacokinetic parameters were listed in Table 7. Simultaneously, the pharmacokinetic results of the compounds administered intravenously were mainly shown in Fig. 4 and Table 8. After oral administration of GZD, 10 compounds were detected in plasma. Except for glycyrrhetinic acid (metabolite), the other compounds reached the maximum blood concentration quickly, whose Tmax was about 0.1-0.2 min. This pharmacokinetics phenomenon coincided with the characteristic of GZD as the Jiebiao formula (TCM term), which took effect quickly. All the AUC and Cmax values of those 10 compounds at different doses were positively correlated with the dose, and T1/2 was independent with the dose, indicating that the compounds were in line with the first- order kinetic process in vivo. A total of 9 compounds were detected after intravenous injection of GZD. The plasma concentration-time curve of these compounds declines rapidly at the beginning, and then decreased slowly, indicating that the plasma concentration-time curves were double exponential function curves. Initially, these compounds did not reach the dynamic balance (the effects of distribution and elimination combined together). After a period of time, only the elimination process existed in vivo. The metabolites, glycyrrhetinic acid, was detected in plasma. However, it could not be fitted with winnonlin software due to its concentration was too low.The V_F values of paenoiflorin, liquiritigenin, and isoliquiritigenin were higher than those of other compounds, which indicated they had broad distribution in tissues.Glycyrrhizic acid had the longest T1/2 and MRT. The compounds eliminated slowly in vivo. Fig. 3 Mean blood concentration–time curves of the 10 analytes in rat plasma after oral administration of GZD (H: high dose, 40.0 g crude drug·kg−1, M: medium dose, 20.0 g crude drug·kg−1, L: low dose, 10.0 g crude drug·kg−1; each point represents mean + SD, n = 6). Fig. 3 Mean blood concentration–time curves of the 10 analytes in rat plasma after oral administration of GZD (H: high dose, 40.0 g crude drug·kg−1, M: medium dose, 20.0 g crude drug·kg−1, L: low dose, 10.0 g crude drug·kg−1; each point represents mean + SD, n = 6). Fig. Pharmacokinetic results 4 Mean blood concentration–time curves of the 9 analytes in rat plasma after intravenous injection of GZD at a dose of 2.0 g crude drug·kg−1 (each point represents mean ± SD, n = 5). Fig. 4 Mean blood concentration–time curves of the 9 analytes in rat plasma after intravenous injection of GZD at a dose of 2.0 g crude drug·kg−1 (each point represents mean ± SD, n = 5). Discussion The validated HPLC-MS/MS method was successfully applied to determination of 10 compounds, including 9 prototype compounds and 1 metabolite in rat plasma after oral administration and intravenous injection. Besides, we also studied the bioavailability of all measured substances in plasma based on the pharmacokinetics results to explore the absorption and utilization of GZD in vivo. Ramulus Cinnamomi was the sovereign drug in GZD. The AUC of cinnamic acid(i.g.) were bigger than other prototypic compounds, while the content of cinnamic acid in decoction was much lower. It meant that cinnamic acid was more easily absorbed into blood. Paeoniae Radix Alba was the assistant drug in GZD. Several papers had reported that there was a mutual transformation between paeoniflorin and albiflorin[26]. The ratios of AUC and Cmax of paeoniflorin and albiflorin were basically the same as the ratios of content in decoction (2:1) after oral or intravenous administration. This indicated that the two compounds might not be transferred into each other in vivo. Radix Glycyrrhizae was the envoy drug in GZD. In this study, 6 compounds were detected in rat plasma after oral administration. The content of liquiritin was higher in GZD than that of other compounds, but its Cmax and AUC values were low, indicating that it was not easily absorbed into the body. The Cmax and AUC values of liquiritigenin were about twice that of liquiritin in vivo, but the content of liquiritigenin was much lower in GZD (1:100). The result suggested that liquiritin could be converted into liquiritigenin in vivo. Moreover, the C-T diagram of liquiritigenin showed a double peak, which might due to the transformation of liquiritin in the intestinal tract. The AUC ratio of liquiritin to liquiritigenin in the plasma during intravenous administration was similar to the ratio of their content in GZD, indicating that liquiritin could not be transformed into liquiritigenin by liver. Combining the results of oral administration, it could be speculated that the transformation process mainly occurred in intestines.Similar to liquiritin, glycyrrhizic acid had high content in GZD, but the values of Cmax and AUC were low after oral administration, indicating that it was not easily absorbed. The Cmax and AUC of glycyrrhetinic acid were about 60-100 times than that of glycyrrhizic acid, which shown that glycyrrhetinic acid were more easily absorbed into the body than glycyrrhizic acid. The Tmax of glycyrrhetinic acid was about 8h, indicating that the speed of conversion was slow. Discussion After intravenous injection, the AUC ratio of glycyrrhetinic acid to glycyrrhizic acid was significantly lower than the ratio of the two compounds after oral administration, suggesting that the liver could not convert glycyrrhizic acid into glycyrrhetinic acid effectively. We speculated that after GZD injected into blood, only a small part of glycyrrhizic acid was excreted into intestine by the bile of the liver, and metabolized into glycyrrhetinic acid by intestinal flora, then re-absorbed into the blood. Rhizoma Zingiberis Recens were the supplementary drug in GZD. The Vz_F of 6-gingerol was relatively high , indicating that the compound was widely distributed in the body or accumulated in some tissues. Different from cinnamic acid, the T1/2 of 6-gingerol (about 3h) was greater than the T1/2 of cinnamic acid (about 0.5h), and the Cl_F and MRT values were higher than those of cinnamic acid, indicating that the elimination of 6-gingerol in the body was slow. g g g y Based on the determination of the main compounds of GZD, we studied the pharmacokinetics of the main effective compounds of GZD through blood. These compounds are likely to be the basis of the pharmacodynamics of Guizhi Decoction. These studies will provide information to help clarify the biologically active ingredients and the mechanism of action of Chinese medicine prescriptions. On the basis of compound chemical components research of GZD, we studied the major effective compounds observed in blood through the pharmacokinetics research, which were the mostly possible material basis of GZD[27]. Moreover, the research help to clarify the biologically active ingredients and mechanism of action in TCM prescriptions. Pharmacokinetic results Table 7 Pharmacokinetic parameters of 10 compounds in rat plasma after oral administration of GZD in three doses (H: high dose, 40.0 g crude drug·kg−1, M: medium dose, 20.0 g crude drug·kg−1, L: low dose, 10.0 g crude drug·kg−1; n = 6) Analytes Dose AUC0–∞ T1/2 Tmax Cmax Vz_F Cl_F MRT0–t (h·μg·L−1) (h) (h) (μg·L−1) (L·kg−1) (L·h−1·kg−1) (h) Cinnamic acid H 1059.3±221.1 0.5±0.1 0.1±0.0 4027.6±1046.7 5.2±3.5 3.3±0.6 0.5±0.1 M 396.5±124.3 0.3±0.1 0.1±0.1 1004.0±253.0 1.9±0.8 4.8±2.0 0.4±0.1 L 186.4±63.8 0.2±0.0 0.1±0.0 831.3±250.1 1.3±0.4 5.1±2.0 0.2±0.1 Paeoniflorin H 353.1±123.8 2.1±0.4 0.1±0.1 221.3±64.1 952.8±338.6 319.8±93.3 2.6±0.5 M 175.2±46.7 1.8±0.4 0.1±0.0 100.7±31.9 618.2±170.1 316.5±94.3 2.0±0.2 L 46.8±8.7 2.8±3.4 0.5±0.7 31.0±25.1 858.5±599.4 519.8±60.2 4.2±4.8 Albiflorin H 138.9±55.9 1.8±0.3 0.1±0.1 107.1±42.1 992.9±335.6 394.8±159.7 2.3±0.6 M 67.1±33.1 1.5±0.4 0.1±0.0 52.6±19.3 886.5±381.0 426.6±179.3 1.9±0.5 L 46.8±8.7 1.6±1.2 0.5±0.7 31.0±25.1 1037.8±1304.7 262.4±51.4 2.2±1.1 Liquiritin H 47.1±11.3 0.9±0.3 0.1±0.0 37.6±7.9 2834.8±574.8 2417.7±450.2 1.3±0.3 M 27.6±12.8 0.9±0.5 0.1±0.1 19.2±2.3 2554.0±656.6 2277.9±794.6 1.3±0.6 L 12.3±1.7 0.5±0.1 0.5±0.4 13.7±4.1 1626.9±280.2 2261.8±335.8 0.8±0.1 Isoliquiritin H 8.0±3.7 0.9±0.5 0.2±0.1 7.8±0.9 2984.1±1349.0 2491.4±898.8 1.3±0.6 M 5.0±1.8 1.2±1.6 0.1±0.1 4.7±1.3 1976.4±1291.6 1779.6±940.7 1.8±2.3 L 1.5±0.1 1.1±0.8 0.2±0.2 2.2±0.4 1084.1±70.7 2584.6±212.1 1.2±1.0 Liquiritigenin H 102.0±39.2 3.3±1.9 0.6±0.5 42.3±17.4 192.8±57.9 36.7±8.5 4.2±2.3 M 72.3±0.3 4.1±0.4 0.8±0.4 19.9±7.6 170.9±16.8 29.1±0.1 5.8±0.8 L 16.1±9.7 3.9±0.1 0.8±0.3 13.4±6.8 198.2±46.1 43.0±3.9 4.3±1.7 Isoliquiritigenin H 4.5±0.9 0.9±0.2 0.1±0.0 6.8±3.4 299.7±98.4 227.7±51.8 1.2±0.3 M 2.9±1.1 1.1±0.8 0.1±0.1 2.7±1.6 356.2±101.2 211.1±95.3 2.0±1.6 L 1.1±0.1 0.6±0.3 0.2±0.4 1.8±0.7 196.4±120.1 229.0±31.6 0.8±0.4 Glycyrrhizic acid H 174.8±36.9 3.0±0.3 0.2±0.2 73.6±20.9 530.9±116.0 120.9±24.2 4.2±0.4 M 109.9±43.0 3.2±1.0 0.5±0.8 44.7±25.6 334.2±173.9 107.2±49.8 3.2±1.5 L 54.8±18.5 3.8±2.1 0.5±0.8 15.3±10.0 540.7±194.0 105.1±47.9 5.8±3.1 Glycyrrhetinic acid H 18958.4±6548.3 6.6±2.3 8.0±0.0 2000.2±854 10.5±3.2 1.2±0.4 12±2.6 M 9781.7±2315.9 6.4±1.9 8.0±0.0 1021.9±425.1 10.3±4.7 1.1±0.3 11.6±0.8 L 3275.4±993.6 6.2±2.9 7.3±3.0 283.8±115.9 14.1±6.4 1.7±0.4 12.3±4.7 Table 7 Pharmacokinetic parameters of 10 compounds in rat plasma after oral administration of GZD in three doses (H: high dose, 40.0 g crude drug·kg−1, M: medium dose, 20.0 g crude drug·kg−1, L: low dose, 10.0 g crude drug·kg−1; n = 6) Table 8 Pharmacokinetic parameters of 10 compounds in rat plasma after intravenous injection of GZD at a dose of 2.0g crude drug·kg−1 (n = 6) Analytes C0 AUC0–∞ T1/2 Vss Cl_F MRT0–t (μg·L−1) (h·μg·L−1) (h) (L·kg−1) (L·h−1·kg−1) (h) Cinnamic acid 106.4±40.3 821.6±310.7 0.2±0.0 0.2±0.0 1.8±0.9 0.3±0.0 Paeoniflorin 2671.4±354 11038.6±2175.8 0.5±0.2 0.6±0.2 2.0±0.3 0.6±0.0 Albiflorin 987.3±226.3 4386.1±2358.1 0.2±0.0 0.7±0.4 2.6±0.7 0.2±0.1 Liquiritin 2072.0±588.0 285.2±59.0 0.2±0.0 2.2±0.5 21.2±4.4 0.3±0.0 Isoliquiritin 68.1±18.8 570.8±200.6 0.1±0.1 1.1±0.5 12.8±4.2 0.1±0.0 Liquiritigenin 11.8±1.1 57.3±7.0 0.1±0.0 0.7±0.1 4.1±0.4 0.2±0.0 Glycyrrhizic acid 5802.2±458.7 10327.2±791.6 2.3±0.6 0.3±0.0 0.2±0.0 2.5±0.3 6-Gingerol 12.9±2.6 84.4±25.4 0.1±0.0 0.6±0.2 6.7±1.3 0.1±0.0 Table 8 Pharmacokinetic parameters of 10 compounds in rat plasma after intrave drug·kg−1 (n = 6) ounds in rat plasma after intravenous injection of GZD at a dose of 2.0g crude etic parameters of 10 compounds in rat plasma after intravenous injection of GZD Authors’contributions H.G., Q. G., D.B. and X.W. designed the research and performed the HPLC experiment; L.Z., H.G. and Q. G. performed the animal experiment; H.G., Q. G., and M.B. analyzed the data and wrote the paper. All authors read and approved the final manuscript. Author details 1 Institute of Basic Theory of Traditional Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, 100700, China 1 Institute of Basic Theory of Traditional Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, 100700, China 1 Institute of Basic Theory of Traditional Chinese Medicine, China Academy of Chinese Medica Sciences, Beijing, 100700, China 2 College of pharmacy, Shaanxi University of Chinese Medicine, Xianyang, 712000, China 3 Medical Experiment Center, China Academy of Traditional Chinese Medicine Sciences, Beijing, 100700, China College of pharmacy, Shaanxi University of Chinese Medicine, Xianyang, 712000, China 3 Medical Experiment Center, China Academy of Traditional Chinese Medicine Sciences, Beijing, 100700, China + Huan Gao and Qin Guo contributed equally to this work. Competing interests Competing interests The authors declare no conflict of interest. The authors declare no conflict of interest. Acknowledgements This study was financially supported by the Special Funding for Basic Scientific Research Business Expenses of Central Public Welfare Scientific Research Institutes (No. YZ202027). y y pp y p g Expenses of Central Public Welfare Scientific Research Institutes (No. YZ202027). Ethics approval and consent to participate pp p p The animal study was approved by Animal Care and Use Committee of the Institute of Basic Theory for Chinese Medicine, China Academy of Chinese Medical Sciences (Date: 25 March 2019; No.: SYXK (Jing) 2019-008). Conclusions In this study, the HPLC-MS/MS analysis method was established for the quantification and determination of 10 compounds in GZD and 10 compounds (including 9 prototype compounds and 1 metabolites) in rat plasma. The results showed that the compounds in GZD were absorbed into the body with a first-order kinetics. At the dose of the study, the exposure of these compounds was proportional to the dose. The absorption was rapid, as the tmax were about 0.1~0.2h.The results suggested that the intestinal digestion played an important role in the pharmacological effects of GZD. The active compounds of GZD might the metabolisms, rather than the prototypes. This process might affect the pharmacodynamics effects of GZD. We expected this results would help us to reveal the pharmacodynamic material basis of GZD and provide a reference for the rational use of GZD in the clinic. Consent to publish All authors consent to publication of this study in Chinese Medicine. Availability of data and materials The datasets used in this article are available from the corresponding author upon request. List of abbreviations GZD: Guizhi Decoction; TCM: traditional Chinese medicine; HPLC-MS/MS: high-performance liquid chromatography-tandem mass spectrometry; LLOQ: lower limit of quantification; LLOD: lower limit of detection; PK: pharmacokinetics; MRM: multiple reaction monitoring; IS: internal standard; RSD: relative standard deviation; RE: refractive error. Funding Funding This work was supported by the Youth Project of National Natural Science Foundation (No. 81403282) and the Natural Science Foundation of Beijing (No. 7133251). INTEGR MED 2013;19:826-35. [3] Wu XX, Zhang LS, Gao H, Song JN, Bai D. Antipyretic Mechanism of Guizhitang and Its Effective Components Based on Network Pharmacology. Chin J Exp Tradit Med Formulae 2018;24:190-7. [4] Chen YR, Gao CL, Ma YM, Qiu FR. Pharmacokinetic study of multiple active constituents after oral gavage of Guizhi decoction in rats using a LC-MS/MS method. Eur J Drug Metab Pharmacokinet 2013;38:283-93. [5] Huo H, Tan Y, Zhou A. 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[27] Wang K, Liu L, Yang Y, Liu X, Zhang L, Xu W, et al. An effective UFLC-MS/MS method used to study pharmacokinetics of major constituents of Fukeqianjin formula in rat plasma. Chin Med 2020;15:74. Figures Figures Figure 1 Chemical structures of analytes and internal standard (IS) Fi 2 Figure 2 Multiple reaction monitoring mode (MRM) chromatograms of cinnamic acid (1), paeoni§orin (2), albi§orin (3), liquiritin (4), isoliquiritin (5), liquiritigenin (6), isoliquiritigenin (7), glycyrrhizic acid (8), glycyrrhetinic acid (9), 6-gingerol (10), icariin (11; IS1), and aesculetin (12; IS2) . blank rat serum (A), blank rat serum spiked with the analytes and ISs (B), rat serum samples at 5 min, 1 h, or 8 h after oral administration of GZD (C), and GZD after 1000 times dilution with methanol(D). Figure 2 Multiple reaction monitoring mode (MRM) chromatograms of cinnamic acid (1), paeoni§orin (2), albi§orin (3), liquiritin (4), isoliquiritin (5), liquiritigenin (6), isoliquiritigenin (7), glycyrrhizic acid (8), glycyrrhetinic acid (9), 6-gingerol (10), icariin (11; IS1), and aesculetin (12; IS2) . blank rat serum (A), blank rat serum spiked with the analytes and ISs (B), rat serum samples at 5 min, 1 h, or 8 h after oral administration of GZD (C), and GZD after 1000 times dilution with methanol(D). Figure 3 Mean blood concentration–time curves of the 10 analytes in rat plasma after oral administration of GZ (H: high dose, 40.0 g crude drug·kg−1, M: medium dose, 20.0 g crude drug·kg−1, L: low dose, 10.0 g cru drug·kg−1; each point represents mean + SD, n = 6). Figure 3 Figure 3 Mean blood concentration–time curves of the 10 analytes in rat plasma after oral administration of GZD (H: high dose, 40.0 g crude drug·kg−1, M: medium dose, 20.0 g crude drug·kg−1, L: low dose, 10.0 g crude drug·kg−1; each point represents mean + SD, n = 6). Mean blood concentration–time curves of the 10 analytes in rat plasma after oral administration of GZD (H: high dose, 40.0 g crude drug·kg−1, M: medium dose, 20.0 g crude drug·kg−1, L: low dose, 10.0 g crude drug·kg−1; each point represents mean + SD, n = 6). Figure 4 Mean blood concentration–time curves of the 9 analytes in rat plasma after intravenous injection of GZD at a dose of 2.0 g crude drug·kg−1 (each point represents mean ± SD, n = 5). Figure 4 Mean blood concentration–time curves of the 9 analytes in rat plasma after intravenous injection of GZD at a dose of 2.0 g crude drug·kg−1 (each point represents mean ± SD, n = 5).
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Hallazgos radiológicos de la tuberculosis pulmonar y su correlación con la presencia de cultivo positivo para Mycobacterium tuberculosis
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Hallazgos radiológicos de la tuberculosis pulmonar y su correlación con la presencia de cultivo positivo para Mycobacterium tuberculosis TESIS DOCTORAL Presentada por: ANTONIO NAVARRO BALLESTER Dirigida por: DR. SANTIAGO F. MARCO DOMÉNECH DR. LUIS LIZÁN TUDELA Castellón, 2017 AGRADECIMIENTOS - A mi hija, Sofía Jinqiu, quien ha traído la alegría a nuestra casa y nos ha bendecido con su sonrisa perenne. - A mi esposa Laura, compañera y confidente en los buenos y en los malos momentos. - A mis padres Antonio y Antonia, así como a mi hermano Paco, quienes han sido mi apoyo incondicional desde el principio de mis días. - A mis directores de tesis, los doctores Santiago F. Marco Doménech y Luis Lizán Tudela, maestros de los que he aprendido mucho durante el desarrollo de esta tesis. - A la Dra. María Victoria Ibáñez Gual, sin la cual hubiera sido imposible manejar la inmensidad de datos recogidos en este estudio, llevando a buen puerto su análisis estadístico. - A todos los pacientes que han participado en el estudio, puesto que sin ellos, nada de esto hubiera sido posible. ÍNDICE 1. INTRODUCCIÓN…………………………………………………………………...1 1.1. Historia de la tuberculosis……………………………………………………….3 1.1.1. Historia del diagnóstico…………………………………..……………5 1.1.2. Sanatorios tuberculosos……………………………………………….6 1.1.3. Vacuna contra la tuberculosis……………………………………..….6 1.1.4. Cirugía para la tuberculosis………………………………………..….6 1.1.5. Fármacos antituberculosos…………………………………………...6 1.2. Microbiología de la tuberculosis.………………..…….………………………..6 1.3. Patogenia………………………………………………………………….....……8 1.3.1. Transmisión………………………………………………………….….8 1.3.2. Instauración de la infección………………………………….……..…9 1.4 Factores de riesgo……………………………………...……………………….10 1.4.1. VIH y tuberculosis…………………………………………………....10 1.4.2. Alcoholismo y tuberculosis………………………………………..…11 1.4.3. Diabetes y tuberculosis…………………………………………..…..12 1.4.4. Tabaquismo y tuberculosis…………………………………………..12 1.4.5. Enfermedad renal crónica y tuberculosis………………………….13 1.4.6. Gastrectomía y tuberculosis……………………………………..…..13 1.4.7. Trasplante y tuberculosis………………………………………….....14 1.4.8. Enfermedades hematológicas y tuberculosis…………………….14 1.4.9. Anemia y tuberculosis………………………………………………..15 1.5. Diagnóstico………………………………………………………………………15 1.5.1. Clínica……………………………………………………………….....15 1.5.2. Radiografía de tórax………………………………………................16 1.5.3. Microscopía…………………………………………………………....17 1.5.4. Cultivo bacteriológico………………………………………………...17 1.5.5. Interferon Gamma Release Assays (IGRA).….………………..….18 1.5.6. Reacción en cadena de la polimerasa (PCR)…………………….19 1.6. Complicaciones de la tuberculosis pulmonar……………………………….19 1.6.1. Complicaciones parenquimatosas……………………....................19 1.6.2. Complicaciones relacionadas con la vía aérea….………………..20 1.6.3. Complicaciones vasculares……………………………...................22 1.6.4. Complicaciones mediastínicas………………………………………22 1.6.5. Complicaciones pleurales……………………………………………23 1.6.6. Complicaciones en pared torácica……………………...................24 2. HIPÓTESIS Y OBJETIVOS……………………………………….....................25 2.1. Hipótesis de trabajo…………………………………………………………….27 2.2. Objetivos…………………………………………………………………………27 3. MATERIAL Y MÉTODOS.……………………………......................................29 3.1. Material…………………………………………………………………………..31 3.1.1. Pacientes………………………………………………………………31 3.2. Métodos………………………………………………………………………….33 3.2.1. Variables epidemiológicas y antecedentes de enfermedad…………………………………………………………….....................33 3.2.2. Variables clínicas……………………………………………………..38 3.2.3. Variables en radiografía de tórax……………………………………40 3.2.4. Variables en tomografía computarizada……………………………44 3.2.5. Evaluación microbiológica……………………………………………53 3.2.6. Análisis estadístico……………………………………………………54 4. RESULTADOS……….……………………………………………………………57 4.1. Análisis descriptivo de las variables epidemiológicas de todos los pacientes y de sus características epidemiológicas, clínicas y radiológicas……………………………………………..…………………………….59 4.1.1. Variables epidemiológicas………………………............................59 4.1.2. Variables clínicas……………………………………………………..77 4.1.3. Variables en radiografía de tórax…………………….....................84 4.1.3.1. Hallazgos pulmonares……………………………………...84 4.1.3.2. Hallazgos mediastínicos……………………………………92 4.1.3.3. Hallazgos pleurales…………………………………………94 4.1.4. Variables en tomografía computarizada……………......................95 4.1.3.1. Hallazgos pulmonares……………………………………...95 4.1.3.2. Hallazgos mediastínicos………………………………….145 4.1.3.3. Hallazgos pleuropericárdicos……...……………………..148 4.2. Análisis multivariante………………………………………………………….153 4.2.1. Variables epidemiológicas………………………………………….153 4.2.2. Variables clínicas……………………………………………………154 4.2.3. Variables en radiografía de tórax………………………………….154 4.2.4. Variables en tomografía computarizada…………………………..155 4.2.5. Modelo global………………………………………………………..156 5. DISCUSIÓN………………………………………………………………………161 5.1. Variables epidemiológicas……………………………………………163 5.2. Variables clínicas………………………………………………………168 5.3. Variables en radiografía de tórax…………………………………….169 5.3.1. Hallazgos pulmonares…………………………..................169 5.3.2. Hallazgos mediastínicos…………………………………….170 5.3.3. Hallazgos pleurales……………………………...................170 5.4. Variables en tomografía computarizada…..………………………...171 5.4.1. Hallazgos pulmonares…………………………..................171 5.4.2. Hallazgos mediastínicos…………………………………….175 5.4.3. Hallazgos pleuropericárdicos………………………………176 5.5. Análisis multivariante……………………………………...................176 5.6. Limitaciones del estudio………………………………………………177 6. CONCLUSIONES………………………………………………………………..179 7. BIBLIOGRAFÍA…………………………………………………………………..183 ANEXO 1…………………………………………………………………………….199 ANEXO 2…………………………………………………………………………….201 ANEXO 3…………………………………………………………………………….203 1. INTRODUCCIÓN 1 2 La tuberculosis (TB), también llamada tisis en el pasado, es una común y en algunos casos fatal enfermedad infecto-contagiosa, causada por agentes del grupo Mycobacterium complex, por lo general por el Mycobacterium tuberculosis o bacilo de Koch. Dicho microorganismo es un bacilo ácidoalcohol resistente aerobio de crecimiento intracelular facultativo (macrófagos) (1). Es la infección crónica más importante del mundo en cuanto a morbilidad y mortalidad (2), siendo su localización más frecuente, con diferencia, el aparato respiratorio, aunque puede afectar a cualquier órgano. En 2013, 9 millones de personas enfermaron de tuberculosis y 1,5 millones murieron por esta enfermedad en el mundo, siendo una de las cinco causas principales de muerte en las mujeres entre los 15 y los 44 años (3). La tuberculosis afecta principalmente a los adultos jóvenes pero todos los grupos de edad están en riesgo. Con todo, más del 95% de las muertes por tuberculosis ocurrieron en países de ingresos bajos y medianos (3). En 2013, aproximadamente medio millón de niños (de 0 a 14 años) enfermaron de tuberculosis, y una media de 80.000 niños seronegativos murieron por esta causa (3). Las personas infectadas por el VIH tienen entre 26 y 31 veces más probabilidades de enfermar de tuberculosis (4). El riesgo de desarrollar tuberculosis activa también es mayor en las personas aquejadas de otros trastornos que deterioran el sistema inmunitario. El tabaquismo aumenta por 2,5 el riesgo de sufrir tuberculosis, incrementando también el riesgo de morir por esa causa. Más del 20% de la incidencia mundial de tuberculosis puede atribuirse al tabaco, siendo un factor de riesgo de tuberculosis, independiente de otros factores de riesgo socioeconómicos, Por tanto, controlar la epidemia de tabaquismo ayudará a controlar la epidemia de tuberculosis. Veinte años después de que la OMS declarara la TB una emergencia mundial de salud pública, se han hecho importantes progresos hacia la consecución de las metas mundiales fijadas para 2015 en el contexto de los “Objetivos de Desarrollo del Milenio” (5). A dos años de esa fecha, en el “Informe mundial sobre la tuberculosis 2013” y en su suplemento “Cuenta regresiva para 2015” se evaluaron los progresos realizados en la consecución de las metas para 2015 y las medidas de máxima prioridad necesarias para alcanzarlas y seguir progresando más allá de ellas (3). 1.1 Historia de la tuberculosis La tuberculosis es una enfermedad que ha acompañado a la humanidad ya desde sus orígenes, siendo considerada, por tanto, una de las patologías más antiguas de la historia. Se cree que antes de afectar al ser humano, fue una enfermedad endémica en animales en el Paleolítico. En base a la existencia de ciertos hallazgos paleopatológicos óseos atribuibles a la tuberculosis como son los acuñamientos vertebrales (Enfermedad de Pott), la reacción perióstica y la osteomielitis, se ha podido determinar su presencia en yacimientos humanos ubicados cronológicamente en la prehistoria (6). 3 En el 2001 se consiguió demostrar la existencia de DNA de Mycobacterium tuberculosis en muestras de lesiones óseas sugestivas de la enfermedad, en cadáveres momificados de la necrópolis de Thebes-West y de Abydos, en el Egipto predinástico (3500-2650 a.C.) (7). Gracias a un estudio paleomicrobiológico del 2004, se logró detectar DNA de Mycobacterium tuberculosis en dos esqueletos encontrados en Rössberga (Suecia), pertenecientes al Neolítico, coincidiendo con el inicio de la domesticación de algunos animales (3200-2300 a.C.) (8). Durante algunos años, el yacimiento más antiguo donde se había conseguido demostrar la enfermedad se ubicaba en Liguria (Italia), siendo datados los restos humanos en una fase más temprana del Neolítico, aproximadamente en el 4.000 a.C. (9-10). No obstante, un estudio reciente ha permitido confirmar la existencia de DNA de la micobacteria en los restos de una mujer y de un niño, pertenecientes al yacimiento neolítico de Atlit-Yam (9250-8160 a.C.), ahora sumergido en el mar mediterráneo próximo a la costa levantina (11). Aunque otros trabajos del pasado siglo hayan encontrado indicios de esta enfermedad en yacimientos más antiguos que los citados (12), lo cierto es que sólo el análisis mediante técnicas de biología molecular permite sostener afirmaciones al respecto. Un hecho intrigante y que habla de los orígenes remotos de esta patología es el hallazgo de lesiones óseas compatibles pero no específicas de tuberculosis en un espécimen de Homo erectus encontrado en Turquía y que vivió a mediados del Pleistoceno (490.000 a 510.000 a.C.) (13). Es probable que la primera cita bibliográfica que se pueda hallar en relación a la tuberculosis se encuentre en los libros del Antiguo Testamento, en los que se hace referencia al impacto de la enfermedad en la población judía en Egipto, región con gran prevalencia de la infección. En los primeros textos médicos fue descrita con los nombres de consunción o tisis (phtisis), atribuyéndole el término “tisis” a Hipócrates, médico que contribuyó en gran medida al conocimiento de la enfermedad. El mismo Hipócrates observó en el siglo V a. d. C. que los contactos estrechos con los pacientes con tisis llevaba a contraer la enfermedad, identificando a la tuberculosis como una causa frecuente de enfermedad en su tiempo, que afectaba a la población de 18 a 35 años y que casi siempre era fatal (14). Aristóteles (siglo IV a.d.C.), otro científico de la antigua Grecia habló “del peligro del contagio de la consunción a través de la respiración”. Galeno, por su parte, en el siglo III definió la tisis como una “ulceración de los pulmones, tórax o garganta, acompañada de tos, fiebre y consunción del cuerpo por el pus” (15). Hasta el siglo XVII, apenas se avanzó en el conocimiento de la tuberculosis. Parece difícil concretar la incidencia que hubo de la enfermedad durante la Edad Media europea, aunque indirectamente puede deducirse que fue elevada debido a la difusión de la ceremonia del “toque real”, basada en la creencia del poder sanador del rey. Dicho “poder” iba dirigido a sanar todo tipo de enfermedades, aunque pronto se restringió a la escrófula, término que englobaba varias formas de tuberculosis no pulmonar, especialmente la ganglionar cervical (16). 4 Los avances más significativos de la época pueden atribuirse a la práctica de autopsias. Sylvius asoció los tubérculos o nódulos descubiertos en varios tejidos durante las autopsias a los síntomas de la tisis que los pacientes habían padecido en vida y creyó erróneamente que eran ganglios linfáticos pulmonares que habían sufrido cambios degenerativos análogos a los observados en los ganglios cervicales en la escrófula. Más tarde fue Morgagni quien mantuvo que dichos nódulos eran estructuras nuevas (15). El propio Morgagni, al igual que Desault, propuso en sus respectivos trabajos que el esputo era la principal fuente de infección. No obstante, esta teoría fue olvidada hasta mucho después. Durante el Romanticismo se asoció a la tuberculosis a las clases altas, a las mujeres y a los jóvenes, e incluso se le atribuyó de forma errónea un componente hereditario (15). En el siglo XIX, tener la infección por tuberculosis estigmatizaba a las familias, ocultándose los casos de la enfermedad y convirtiéndose a los afectados en motivo de vergüenza. Por tanto, en esta época es promovida la marginalidad y la exclusión social en estos enfermos. 1.1.1. Historia del diagnóstico En lo referente al diagnóstico, el primer método objetivo fue la percusión torácica, desarrollada por Leopold Auenbrugger en 1761, aunque sus descubrimientos no tuvieron mucha repercusión en la época y sería en años posteriores cuando serían tenidos en cuenta (17). Uno de los avances más importantes en cuanto al diagnóstico de la tuberculosis vino de la mano del francés Laënnec. En 1818 descubrió el estetoscopio, publicando un tratado en el que analizaba todos los sonidos que captaba, acuñando términos como estertores, crepitantes o soplos. Además, estudió la patocronia de esta enfermedad, desde la aparición de un tubérculo hasta la aparición de una caverna, afirmando la unicidad del proceso a través de lesiones de apariencia diferente (18). Años después, la auscultación fue considerada junto con la percusión el método diagnóstico más fiable para las enfermedades torácicas, entre ellas la tuberculosis, hasta el descubrimiento de los rayos X a finales de siglo. En 1882, Robert Koch consiguió identificar el bacilo responsable en la enfermedad en muestras de pacientes infectados. Junto con esto, el médico alemán consiguió otros hitos entre los que se encuentran conseguir cultivar el bacilo in vitro, así como conseguir la producción de la enfermedad tras la inoculación del cultivo puro en animales de experimentación (de cuyas lesiones tuberculosas pudo aislar de nuevo el bacilo). En 1891, Koch dio a conocer un remedio para el tratamiento de la tuberculosis, denominado tuberculina. Se trataba de un extracto obtenido a partir de un cultivo puro de bacilos tuberculosos. Los primeros ensayos demostraron que la tuberculina no era útil como remedio para la enfermedad, no obstante, sí que lo fue como método diagnóstico (19). Koch fue galardonado por su descubrimiento con el Premio Nobel en 1905. 5 1.1.2 Sanatorios tuberculosos En el siglo XIX el concepto de mantener a los pacientes con tuberculosis aislados en un sanatorio comenzó a extenderse. El primero fue establecido en Silesia en 1859 por Hermann Brehmer, obteniendo un gran éxito. En 1884, Edward Livingston Trudeau creo el primer sanatorio en los Estados Unidos. Las personas infectadas fueron aisladas de la sociedad y tratadas con reposo y una nutrición cuidada (20). 1.1.3. Vacuna contra la tuberculosis En la década de 1880 Louis Pasteur empezó a desarrollar vacunas contra el ántrax, cólera de las gallinas, y, más tarde, la rabia. En 1908, los científicos franceses Albert Calmette y Camille Guerin consiguieron hacer crecer el bacilo de Koch en varios medios para disminuir su virulencia y aumentar la capacidad de producir inmunidad. Esto llevó a la ahora famosa vacuna llamada BCG, que lleva el nombre de los dos fundadores. Dicha vacuna se introdujo en 1921 (21). 1.1.4. Cirugía para la tuberculosis Antes de que se encontraran antibióticos eficaces contra la tuberculosis, el tratamiento quirúrgico de la tuberculosis era común y a menudo salvaba vidas. El Dr. James Carson, un médico escocés (1821), inició el tratamiento mediante el drenaje de derrame pleural y consiguió prolongar la vida de algunos pacientes. Estas técnicas precursoras evolucionaron, pero debido a la falta de eficacia se desvanecieron después de la llegada de los fármacos antituberculosos (22). 1.1.5. Fármacos antituberculosos Se utilizaron antibióticos contra la tuberculosis por primera vez en 1944 después del descubrimiento de la estreptomicina. El uso de este agente por sí mismo lo único que consiguió fue generar resistencia a los antibióticos en los pacientes tratados, problema que sigue siendo importante en nuestros días. Mejores resultados produjo el desarrollo del PAS (ácido para-aminosalicílico). En esos tiempos el PAS era un fármaco oral, a diferencia de estreptomicina. Más tarde aparecieron fármacos más eficaces como la isoniazida (1950) y posteriormente con rifampicina (23). En la actualidad existen menos de 20 agentes con actividad contra las micobacterias. 1.2. Microbiología de la tuberculosis El orden de los Actinomycetales incluye la familia Mycobacteriaceae, Actinomycetaceae, Streptomycetaceae y Nocardiaceae. La familia Mycobacteriaceae contiene un solo género, el género Mycobacterium, del que en sus orígenes sólo se conocían dos especies: el bacilo de la lepra o Mycobacterium leprae y el bacilo tuberculoso o Mycobacterium tuberculosis. En la actualidad, dentro del género Mycobacterium ya se han descrito más de 120 especies de micobacterias diferentes (24). 6 Las micobacterias son bacterias aerobias y no móviles (con excepción de la especie Mycobacterium marinum, que ha mostrado ser móvil dentro de los macrófagos). Tienen ácido-alcohol resistencia, no producen endosporas ni cápsulas y son biológicamente similares a las bacterias Gram positivas (aunque frente a la tinción de Gram, las micobacterias son débilmente positivas o no se tiñen). En algunos casos, estos bacilos pueden formar filamentos ramificados. Aunque las micobacterias no parecen encajar en la categoría Gram-positiva desde un punto de vista empírico, se clasifican como bacterias ácido-resistentes Gram-positivas. Todas las especies de Mycobacterium comparten una característica pared celular, más gruesa que la de muchas otras bacterias, hidrofóbica, cerosa, y rica en ácidos micólicos. La pared celular es rica en lípidos, lo que hace que su superficie sea hidrófoba y confiere a las micobacterias resistencia frente a muchos desinfectantes y las tinciones de laboratorio. Las micobacterias son capaces de sobrevivir durante semanas o meses sobre objetos inanimados, siempre que estén protegidas de la luz solar, y son más resistentes a los ácidos, álcalis y desinfectantes que el resto de las bacterias no formadoras de esporas. Resisten la desecación y la congelación, pero la luz ultravioleta y el calor (>65º C durante 30 minutos) las inactivan (25). Debido a que la pared celular de las micobacterias es compleja y a que este grupo de microorganismos es exigente desde el punto de vista nutricional, la mayoría crecen lentamente, se dividen cada 12 a 24 horas y se necesitan hasta 8 semanas antes de poder detectar el crecimiento en los cultivos de laboratorio. Además, algunas especies tienen también ciclos de reproducción muy largos. M. leprae puede tardar más de 20 días para completar un ciclo de división (por comparación, algunas cepas de E. coli toman sólo 20 minutos), aunque jamás se ha podido aislar de manera artificial a esta especie, haciendo que el cultivo en laboratorio sea un proceso lento. Algunas de las especies pueden ser extremadamente difíciles de cultivar y puede llevar más de dos años desarrollar su cultivo. Las micobacterias se pueden clasificar en 3 grupos desde el punto de vista diagnóstico y tratamiento:    Complejo Mycobacterium tuberculosis, que puede causar la tuberculosis: M. tuberculosis, M. bovis, M. africanum, M. microti y M. Canetti. M. leprae y M. lepromatosis, que causa lepra o enfermedad de Hansen. Las micobacterias no tuberculosas (MNT) son el resto de micobacterias que pueden producir afectación pulmonar similar a la de la tuberculosis, además de linfadenitis, patología cutánea o enfermedad diseminada. A su vez, Runyon propuso una clasificación de las MNT basado en la tasa de crecimiento, la producción de pigmento amarillo y en si este pigmento se producía en la oscuridad o sólo después de la exposición a la luz.    Fotocromógenos: producen pigmentos pero sólo después de la exposición a la luz. Los ejemplos incluyen M. kansasii, M. marinum y M. Simiae. Escotocromógenos: producen pigmento sólo en condiciones de oscuridad. Ejemplos: M. scrofulaceum y M. szulgai. No cromógenos: incluye un grupo de patógenos oportunistas prevalentes llamado M. avium complex (MAC). Otros ejemplos son M. 7  ulcerans, M. xenopi, M. malmoense, M. terrae y M. haemophilum genavense. Micobacterias de crecimiento rápido: no producen pigmento: M. chelonae, M. abscessus, M. fortuitum y M. peregrinum. Otros ejemplos de micobacterias que también pueden producir enfermedad en humanos, aunque de forma más rara, son M. smegmatis y M. flavescens. Mycobacterium tuberculosis complex engloba a un grupo de micobacterias que presentan más del 95% de homología en su DNA y que está compuesto, además de M. tuberculosis, por Mycobacterium bovis, M. bovis BCG (una variante de laboratorio utilizada en vacunación y en instilaciones vesicales en pacientes con neoplasia de vejiga), Mycobacterium africanum (principal causante de la TB en África tropical), Mycobacterium microti (causante de la TB en roedores y otros mamíferos) y Mycobacterium canetii (24). Habitualmente se utiliza el término de M. tuberculosis o bacilo tuberculoso como sinónimo de todas ellas, constituyendo el aislamiento de cualquiera una evidencia diagnóstica de infección por tuberculosis en el paciente analizado. El Mycobacterium tuberculosis es un bacilo aeróbico, no móvil, no productor de esporas, por lo general ligeramente curvado, con una longitud de 2-4 micras y 0,3-0,5 micras de diámetro (26). Se caracteriza por ser una bacteria ácidoalcohol resistente (BAAR) y esto es debido al alto contenido en lípidos que tienen en su pared celular. Este hecho impide que penetren los colorantes habituales de anilina, por lo que no suelan poder verse en la tinción de Gram, y hace que para poder visualizarla sean necesarios colorantes especiales (arilmetanos), pero que una vez teñidas no se decoloran con una mezcla de alcohol y ácido. 1.3. Patogenia 1.3.1. Transmisión En más del 98% de los casos, la infección es causada por la inhalación de las secreciones respiratorias emitidas por un individuo con enfermedad tuberculosa pulmonar con esputo positivo para M. tuberculosis (27). No todos los casos desarrollarán la enfermedad. Existen dos factores que determinan esta evolución: la facilidad de exposición a un adulto enfermo y las condiciones inmunológicas propias del huésped. Los niños menores de un año son los que tienen más probabilidades de desarrollar formas graves de la enfermedad, como la tuberculosis miliar y la meningitis tuberculosa (28). La transmisión de la infección tuberculosa es por vía aérea en la mayoría de las ocasiones. El acceso del bacilo al organismo por otras vías de entrada (digestiva, cutánea o mucosa) requiere de situaciones anómalas en el huésped como son la pérdida de la continuidad de las superficies expuestas. Los factores que influyen en la transmisión aérea de la infección son los siguientes (25): 8 A) La fuente o reservorio emisor del agente infeccioso: en general, se trata de una persona enferma en los países desarrollados, siendo menos frecuente que sea un animal infectado. En pacientes bacilíferos, la infección es exportada mediante los aerosoles de la respiración, al hablar, al toser o estornudar. Los principales bacilíferos son los enfermos activos de tuberculosis laríngea y pulmonar, los enfermos portadores de tuberculosis con cepas más virulentas y los inmunodeprimidos. B) El vehículo transmisor: Las denominadas “gotas de Flügge” son unas gotículas de saliva mayores de 10 μ que transportan los bacilos tuberculosos y permiten la infección persona-persona (29). Por su parte, los “núcleos goticulares de Wells” gracias a su pequeño tamaño (1-10 μ), son capaces de mantenerse suspendidos en el aire (también las gotas de Flügge, aunque más raramente por su mayor tamaño). Con un golpe de tos se pueden llegar a emitir hasta 3.000 núcleos goticulares infecciosos. La presencia de bacilos ácidoalcohol resistentes en el esputo es el principal indicador del potencial de transmisión (30). C) El medio transmisor: El medio transmisor está constituido por el aire interpuesto entre el emisor de gotículas y el receptor de la infección. Dependerá de factores como son los metros cúbicos del espacio habitable compartido, la distancia entre la fuente emisora y el contacto, y el tiempo compartido. 1.3.2. Instauración de la infección Por tanto, la infección se produce cuando una persona susceptible inhala núcleos de gotitas que contienen bacilos tuberculosos. Como la distribución de núcleos de gotitas inhalados está determinada por el patrón ventilatorio y los volúmenes de los diversos lóbulos pulmonares, el sitio de implantación se produce preferentemente en el campo medio pulmonar y en zonas pulmonares inferiores, aunque cualquier lóbulo puede verse afectado (31). Una vez alojado en el alvéolo, el M. tuberculosis es ingerido por los macrófagos alveolares. La resistencia al establecimiento de la infección tuberculosa tiene un componente genético (32), dependiendo el curso de la enfermedad de la interacción entre el poder microbicida inherente del macrófago alveolar y la virulencia del bacilo ingerido (33). Si el macrófago alveolar no puede destruir o inhibir M. tuberculosis, los bacilos se multiplican dentro de su entorno intracelular, haciendo que el macrófago y su progenie terminen estallando. El ciclo continúa, siendo los bacilos liberados ingeridos por otros macrófagos alveolares y los nuevos monocitos serán reclutados desde la sangre. Durante este período de crecimiento rápido, el bacilo de la tuberculosis se propaga a través de los canales linfáticos a los ganglios linfáticos hiliares y mediastínicos regionales y a través del torrente sanguíneo a localizaciones más distantes en el cuerpo. La fase logarítmica de crecimiento bacilar se detiene con el desarrollo de la inmunidad mediada por células y con la hipersensibilidad de tipo retardado en la semana 2-10 después de la infección inicial (33, 34). Por lo general, la inmunidad específica consigue limitar aún más la multiplicación de los bacilos, permitiendo que el anfitrión permanezca asintomático y que las lesiones se curen (34). Algunos de los bacilos permanecen latentes y viables durante muchos años, conociéndose esta 9 condición como “Infección de TB latente”. En aproximadamente el 5% de los individuos infectados, la inmunidad es inadecuada, desarrollándose la forma clínicamente activa dentro del primer año tras la infección (34). 1.4. Factores de riesgo Los pacientes inmunocomprometidos tienen una mayor prevalencia de la tuberculosis que la población general (35) y también tienen más probabilidades de estar infectados con tuberculosis multirresistente (36). Además, el patrón de la enfermedad es diferente en pacientes inmunocomprometidos, que tienen una mayor prevalencia de afectación extrapulmonar. En un estudio, 38% de los pacientes inmunocomprometidos con tuberculosis tenían afectación pulmonar solamente, el 30% sólo tenía afectación extrapulmonar, y el 32% tienen tanto afectación pulmonar como extrapulmonar (37). Incluso cuando hay afectación pulmonar, una respuesta inmune limitada puede dar lugar a hallazgos radiográficos de tórax normales (38). Son muchos los factores de riesgo conocidos para el desarrollo de tuberculosis activa, incluyendo condiciones que se asocian con defectos de los linfocitos T y/o de la función de los macrófagos. Entre dichos factores encontramos algunos como la desnutrición, el abuso de drogas y alcohol, y otras condiciones médicas como la insuficiencia renal crónica, la diabetes mellitus, la silicosis, derivación yeyunoileal y gastrectomía subtotal, así como el tratamiento con corticosteroides u otras terapias inmunosupresoras (39). 1.4.1. VIH y tuberculosis El virus de la inmunodeficiencia humana (VIH) responsable del Síndrome de la Inmunodeficiencia Adquirida (SIDA) produce una alteración de la inmunidad celular lo suficientemente grave como para posibilitar el desarrollo de infecciones oportunistas que son generalmente endógenas y/o tumores, que definen la situación del SIDA (40). Aunque en las últimas décadas se ha producido un descenso de la TB en muchos países occidentales, la extensión de la epidemia VIH en todo el mundo ha permitido que su interacción con la TB haya modificado su curva de descenso en los países que habían conseguido dicha disminución (41), o que en los países en vías de desarrollo donde la TB era endémica (42), dicho problema se haya agravado. La co-infección con el VIH y M. tuberculosis es el factor de riesgo más fuerte conocido, tanto para la progresión inmediata como tardía de la infección de tuberculosis activa (43, 44). El riesgo de progresión de la enfermedad en las personas co-infectadas es del 5 -10% por año, lo cual contrasta con el riesgo del 5 - 10% por vida para las personas VIH-negativas (45, 46). Es posible que, además de aumentar la susceptibilidad individual a desarrollar TB activa tras la infección por Mycobacterium tuberculosis, la asociación de TB y VIH también aumenta las tasas de transmisión de TB en cualquier ámbito de la comunidad, poniendo en peligro la salud y la supervivencia de los individuos VIH-negativos. En varios países, el VIH se ha asociado con brotes epidémicos 10 de TB, y muchos de los brotes notificados involucraron cepas multirresistente, con las dificultades desde el punto de vista epidemiológico que esto conlleva (47). Desde el punto de vista inmunológico, la disminución de CD4, citocinas T1 e interferón alfa en pacientes con VIH favorece el desarrollo de TB. Por su parte, la TB ejerce estimulación de macrófagos e incrementa la producción de interleucina 1 y 6 además del TNF alfa, lo que unido a la estimulación inmunológica con aumento de CD4 y de los correceptores para VIH (CxCR4 y CCr5), favorece el desarrollo del virus de VIH (48). 1.4.2. Alcoholismo y tuberculosis Parece existir una fuerte asociación entre el consumo de alcohol y el riesgo de desarrollar TB. La prevalencia de los trastornos por consumo de alcohol entre los pacientes diagnosticados de TB ha oscilado entre el 10% y el 50% según demuestran los resultados de estudios realizados en diferentes países (49-52). En el caso inverso, se ha demostrado también un fuerte vínculo entre alcoholismo y tuberculosis al analizar la prevalencia de TB en grupos de población con trastornos relacionados con el consumo de alcohol. Como reportó Jones y cols. (53) la prevalencia de la tuberculosis pulmonar activa entre los usuarios de servicios sociales (entre los cuales los trastornos por uso de alcohol fue el principal problema) en los EE.UU. en la década de 1950 fue de 55 veces la prevalencia de la población general (2.220 / 100.000 vs. 40 / 100.000). Friedman y cols. (54) por su parte reportaron una prevalencia 46 veces mayor entre las personas con trastornos por uso de alcohol en Nueva York en la década de 1980 (1.500 / 100.000 vs. 32 / 100.000). En un trabajo algo más actual se seleccionó un cohorte de personas con trastornos por uso de alcohol que fueron seguidos de forma prospectiva durante 8 años, la incidencia de TB fue 464 / 100.000 personas por año, lo que constituyó 9 veces la incidencia en la población de referencia en la misma franja de edad en Nueva York (55). No obstante, los resultados de estos estudios no deben ser tomados al pie de la letra puesto que existieron factores de confusión que pudieron distorsionar la medida de asociación entre estas dos variables. Existen dos posibles causas, no excluyentes, para explicar la asociación entre la infección por tuberculosis y las conductas adictivas relacionadas con el alcohol: - Relación entre el abuso de alcohol y la pertenencia a clases sociales más bajas, así como los patrones de interacción social que conlleva (56). - Sistema inmunológico debilitado por el consumo crónico de alcohol como factor que conduce a un mayor riesgo a contraer la infección (57). Este último puede ser a través de efectos tóxicos directos del alcohol en el sistema inmunológico, o indirectamente a través del déficit en micro y macronutrientes que propicia (58). También puede predisponer a otras condiciones médicas relacionadas con el alcohol, tales como tumores malignos y la depresión. 11 1.4.3. Diabetes y tuberculosis Se prevé que el número de personas en el mundo con diabetes (DM) aumente a 366 millones en 2030, con un incremento más acentuado en países de bajos a medianos ingresos (59). Al mismo tiempo, estos países son los que poseen una mayor prevalencia de tuberculosis en el mundo (60). En la primera mitad del siglo XX, cuando el tratamiento antidiabético era limitado, la DM parecía ser un factor de riesgo bien reconocido clínicamente para la tuberculosis (61). La fuerza de cualquier forma de asociación entre la DM, la disglucemia, y el riesgo de la tuberculosis sigue siendo objeto de debate. Algunos estudios han demostrado que el sistema inmune innato se ve afectada por altos niveles de glucosa en sangre (62), y que las personas con DM tienen un mayor riesgo de neumonía (63). Un metaanalisis del 2008 presentó una evidencia robusta para soportar esta asociación, tras llevar a cabo una revisión de 13 estudios observacionales previos (64). Dicho estudio dilucidó que la DM se asocia a un mayor riesgo de TB, con estimaciones de riesgo relativo que varían de 1.2 a 7,8. En cuanto a la disglicemia, Leung et al. (65) encontraron que entre las personas de edad avanzada con diabetes, aquellos con HbA1c ≥7% tenían un riesgo relativo de TB de 3,1 (IC del 95% 1.6 a 5.9) en comparación con aquellos con HbA1c <7%. La patogénesis de la asociación entre la DM y la tuberculosis es bastante compleja. No está claro si la DM aumenta la susceptibilidad a la infección inicial de TB o si hace más probable la reactivación de la enfermedad latente. Se cree que la DM produce una alteración en la acción de los monocitos, así como en los mecanismos de reconocimiento celular del huésped, lo que conlleva una alteración de la respuesta inmune innata (66). 1.4.4. Tabaquismo y tuberculosis El tabaco es la principal causa evitable de muerte en el mundo (67), y el fumar influye negativamente en los esfuerzos para prevenir y tratar no sólo la TB sino también la infección por VIH. El tabaquismo se relaciona con un aumento de riesgo de ser infectado por el Mycobacterium tuberculosis y de desarrollar enfermedad pulmonar activa, además de que propicia resultados pobres en el tratamiento tuberculostático (68). Se estima que hasta un 16% de todos los casos de TB se podrían evitar si la exposición al tabaco (activa y pasiva) fuera eliminada (69). La prevalencia de tabaquismo entre los pacientes con TB es mayor que la prevalencia en la población general (70). Adicionalmente, la mortalidad en pacientes tuberculosos que fuman es mayor. Algunos de los mecanismos biológicos que pueden explicar el efecto del tabaquismo sobre el riesgo de infección de la TB incluyen, entre otros, disminución de la función ciliar, alteraciones en el número de macrófagos y la respuesta, así como la disminución de las células CD4 y CD8 que producen interferón gamma y TNF alfa (71). Hay algunas pruebas que indican efectos dañinos del humo en fumadores pasivos sobre el sistema inmunitario innato de los pulmones, así como la respuesta inmunológica adaptativa, lo que lleva a 12 una mayor susceptibilidad a la infección por M. tuberculosis y a la progresión a TB activa (72). Sin embargo, el vínculo entre la exposición al humo ajeno y la tuberculosis aun hoy no es comprendida del todo. 1.4.5. Enfermedad renal crónica y tuberculosis La alteración de la inmunidad en las personas con enfermedad renal crónica (ERC) es el principal factor de riesgo para el desarrollo de la TB. En los últimos años, la tasa de incidencia de la tuberculosis ha ido en aumento en esta población debido a la inmunosupresión, sobre todo en personas que han sido sometidos a trasplante renal (73). La inmunodeficiencia observada en la ERC se produce como resultado de las situaciones de inflamación crónica, del uso de terapias de reemplazo renal y de la insuficiencia renal crónica, así como la existencia de comorbilidades (74). Por otra parte, el estado nutricional, la deficiencia de vitamina D y el hiperparatiroidismo son factores que contribuyen también al deterioro de la inmunidad en este tipo de pacientes (75). El diagnóstico de la TB en pacientes en diálisis puede ser difícil debido a la mayor frecuencia de afectación extrapulmonar, lo que puede dar lugar a manifestaciones clínicas atípicas y otros síntomas que no son específicos. Tanto es así que algunos estudios han descrito un mayor porcentaje de casos con TB extrapulmonar en comparación con la forma pulmonar, siendo la linfadenitis tuberculosa la forma más frecuente (76). La prueba cutánea de la tuberculina es ampliamente utilizada para detectar la infección latente por Mycobacterium tuberculosis. Sin embargo, han sido informadas de algunas limitaciones, incluyendo la anergia como consecuencia de la inmunidad deteriorada en los pacientes con enfermedad renal crónica, lo cual conduce a posibles resultados falsos negativos, y a una baja sensibilidad de la prueba (74). La TB es la segunda causa de muerte de etiología infecciosa en ERC (73). Los pacientes en diálisis tienen una mayor prevalencia de infección latente por TB que aquellos con enfermedad renal crónica grave. La edad avanzada, las lesiones residuales de TB previa en la radiografía, el aumento de nivel de albúmina sérica y la diálisis a largo plazo son predictores de la infección latente por TB (77). 1.4.6. Gastrectomía y tuberculosis En varios estudios clásicos (78, 79, 80) fue observado un aumento en la incidencia de tuberculosis en pacientes intervenidos de gastrectomía. Parece existir también una correlación entre la tuberculosis y el síndrome de Dumping (vaciado rápido del contenido estomacal al duodeno o yeyuno tras cirugía gástrica). El antecedente de gastrectomía también puede influir en la reactivación de la tuberculosis en pacientes con una infección latente, ya que puede causar un mal estado nutricional y una alteración asociada en la inmunidad. Además, los pacientes que han sido sometidos a una gastrectomía pueden sufrir de desnutrición secundaria a una pobre ingesta de alimentos y a la mala absorción 13 intestinal. La desnutrición, per se, ya es un reconocido factor de riesgo para el desarrollo de la infección (81). Se ha observado que los pacientes gastrectomizados presentan un menor nivel de leucocitos en sangre en respuesta a infecciones, en comparación con los controles. Esta diferencia no fue estadísticamente significativa en situación de reposo pero si en condiciones de estrés como en el ejercicio o tras la inyección de adrenalina, observándose en los pacientes gastrectomizados un menor incremento en las cifras de leucocitos (especialmente polimorfonucleares) (79). 1.4.7. Trasplante y tuberculosis La incidencia de la tuberculosis entre los receptores de trasplante de órgano sólido (TOS) depende de su incidencia en la población general. España tiene una alta incidencia de tuberculosis, una alta prevalencia de la infección tuberculosa latente (> 25%) (82), y una gran actividad de TOS (83). La frecuencia de la tuberculosis activa en los receptores de TOS en regiones de baja prevalencia (<20 casos de tuberculosis por cada 100 000 habitantes) varía de 0,26% a 6,5% (84). Según un estudio de cohorte prospectivo llevado a cabo por RESITRA (Spanish Network of Infection in Transplantation) con una población de 4.388 pacientes (85), la incidencia de tuberculosis obtenida en pacientes con TOS (512 casos por cada 100.000 pacientes por año) no sólo fue mayor que la de la población general (18,9 casos por cada 100.000 pacientes por año), sino que también fue superior a la observada en los pacientes sometidos a trasplante alogénico de células madre hematopoyéticas (135,6 casos por 100.000 receptores de trasplantes por año) y a los candidatos a TOS (80 casos por cada 100.000 candidatos por año). Por lo tanto, los pacientes sometidos a TOS deben ser considerados de alto riesgo para el desarrollo de la tuberculosis, aunque existen diferencias en la incidencia entre los diferentes tipos de trasplantes de órganos, siendo mayor el riesgo de infección tuberculosa en casos de trasplante pulmonar y en receptores ancianos. 1.4.8. Enfermedades hematológicas y tuberculosis A diferencia de los paciente con neoplasias sólidas en los que la infección tuberculosa suele ser diagnosticada como complicación del propio tumor, en los pacientes con neoplasias hematológicas es más frecuente el diagnóstico de tuberculosis durante el tratamiento quimioterápico, probablemente por la utilización de corticoides y quimioterapias que producen una gran inmunosupresión (86). Algún estudio ha demostrado que el tratamiento de algunas enfermedades hematológicas, en concreto el trasplante de precursores hematopoyéticos, aumenta el riesgo de TB hasta de nueve a trece veces. Esto es debido al debilitamiento de la inmunidad mediada por células producido por el tratamiento farmacológico en la fase de acondicionamiento pretrasplante, por los agentes inmunosupresores y por la enfermedad de injerto contra huésped (87). 14 Lo expuesto sugiere que la detección de infección tuberculosa latente y el tratamiento de la misma deben ser considerados en pacientes con enfermedades hematológicas previo al inicio del tratamiento con esteroides y quimioterapia. 1.4.9. Anemia y tuberculosis La TB puede causar diversas alteraciones de laboratorio como la anemia (88), aumento de la velocidad de sedimentación globular, disminución de la albúmina sérica, hiponatremia, alteración de la función hepática, leucocitosis e hipocalcemia (89,90). Existen estudios que han documentado la asociación entre anemia y TB, sin embargo, estos estudios incluyeron sólo un pequeño número de pacientes y los resultados no fueron uniformes (89). Se estima que la anemia ocurre en 1-8% de la población mundial (91). La prevalencia de la anemia en pacientes con TB oscila entre el 30-94% (92). Se ha demostrado que la anemia es más probable que ocurra en pacientes con TB en comparación con los controles sanos (93). Más importante aún, la anemia se asocia con formas más graves de tuberculosis y con un peor pronóstico y mortalidad (94). Existe escasa literatura e inconsistente acerca de la relación entre la anemia y la conversión de esputo, aunque parece ser que la anemia al inicio del tratamiento de la tuberculosis se asocia significativamente con un retraso en la conversión de esputo en pacientes con esputo positivo para TB. El mecanismo causal aun no es conocido del todo (92). La deficiencia de hierro es el cuadro deficitario de micronutrientes más común en el mundo, y numerosos estudios han evaluado la asociación entre los niveles séricos de hierro y la anemia por deficiencia de hierro (95, 96). No obstante, es un tema aun controvertido: mientras que algunos estudios han demostrado que la deficiencia de hierro aumenta la susceptibilidad a procesos infecciosos, otros han obtenido evidencias de que el exceso de hierro es más dañino para el cuerpo humano que la deficiencia del mismo, y que la deficiencia de hierro puede proteger contra la infección (97). 1.5. Diagnóstico 1.5.1. Clínica a) Manifestaciones generales: Las manifestaciones clínicas de la TB son inespecíficas y muy variables. Se puede distinguir las manifestaciones generales de aquellas que se relacionan con un órgano o sistema concreto. Entre las manifestaciones generales encontraremos un cuadro subagudo o crónico caracterizado por:      Adelgazamiento. Astenia. Anorexia. Febrícula de predominio vespertino. Sudoración nocturna. 15 Pese a la escasa especificidad de los síntomas referidos, éstos deben inducirnos a incluir a la TB en el diagnóstico diferencial, activando la realización de pruebas que nos permitan conseguir un diagnóstico precoz, con los beneficios para el paciente que ello conlleva. b) Manifestaciones en el sistema respiratorio: Cualquier parte del cuerpo humano puede verse afecto por la TB. No obstante, en el 85-90% se manifiesta como TB pulmonar (98). En algunos pacientes, como los infectados por VIH, existe una mayor presencia de manifestaciones extrapulmonares de la TB (69%) (98). Dado que la vía de entrada de la infección es la vía aérea, las manifestaciones respiratorias son las más frecuentes en la tuberculosis pulmonar. De ellas, la tos es el síntoma más común. Esta tos suele ser seca durante las primeras semanas o meses pero tiende a convertirse en productiva y mucoide o mucopurulenta. Se distinguen dos formas de tuberculosis, la primaria y posprimaria, pudiendo establecerse diferencias en cuanto a la clínica observada en cada una de ellas. La forma primaria de tuberculosis pasa inadvertida en la mayoría de los casos, dependiendo ello de varios factores, siendo el estado inmunitario el más importante. En muchas ocasiones es diagnosticada al realizar la prueba de la tuberculina en pacientes asintomáticos o con clínica leve. Los síntomas que puede producir son astenia, pérdida de peso y dolor torácico, pudiendo acompañarse de fiebre. Otras manifestaciones como la tos con expectoración, sudoración nocturna o hemoptisis son inhabituales en esta forma de TB, siendo casi exclusivas de la forma posprimaria. La semiología de consolidación en la auscultación también es un hallazgo raro en la forma primaria de tuberculosis (99). Un meta-análisis llevado a cabo por van’t Hoog et al demostró que la tos de larga duración (> 2 semanas) tiene una especificidad del 96% para tuberculosis activa pero una sensibilidad del 24%. Esta sensibilidad aumenta hasta el 56% si se utiliza para el diagnóstico la tos de cualquier duración (100). Según un estudio llevado a cabo en la población de Los Ángeles, entre los pacientes con tuberculosis pulmonar confirmada, el 75% presentó una tos de duración variable (el 52% tenía una tos que duró más de 2 semanas), el 58% presentó fatiga, el 50% experimentó una sensación distérmica (fiebre no termometrada), el 46% informó de sudores, el 43% refirió pérdida de peso, el 41% tuvo dolor torácico y, por último, el 23% presentó hemoptisis (101). 1.5.2. Radiografía de tórax La radiografía de tórax ha sido una parte fundamental del diagnóstico de la tuberculosis durante más de un siglo. En individuos inmunocompetentes, es raro encontrar una radiografía de tórax normal cuando existe una tuberculosis pulmonar activa. En un estudio realizado sobre una población de 518 pacientes, sólo un 4.8% presentó un radiografía de tórax normal (102). Por desgracia, esta alta sensibilidad se acompaña de una baja especificidad. No obstante, existen algunos patrones radiológicos que en la radiografía de tórax se consideran sugestivos de infección por TB. Entre éstos encontraríamos las 16 consolidaciones en el lóbulo superior o las lesiones cavitarias (103). Se ha calculado que la especificidad de la radiografía de tórax para el diagnóstico de TB usando estos hallazgos es del 63%, aunque descartar a aquellos pacientes que no presentan consolidación de localización en lóbulos superiores o lesiones cavitadas disminuye la sensibilidad hasta el 73% (103). Los pacientes con TB coinfectados por el VIH son menos propensos a tener hallazgos típicos de TB en la radiografía de tórax, especialmente cuando existe una disminución en las cifras de linfocitos CD4 (104), pudiendo presentar un patrón radiológico equiparable al observado en pacientes con tuberculosis primaria. Estas diferencias serán mejor abordadas en la sección dedicada a tomografía computarizada (TC). La naturaleza a veces subjetiva de la interpretación de la radiografía de tórax también supone un desafío. Incluso entre los radiólogos experimentados se han detectado altos niveles de variabilidad inter-observador (105). 1.5.3. Microscopía El examen microscópico directo para la detección de bacilos ácidoresistentes (baciloscopía de BAAR) aun hoy constituye una de las piedras angulares del diagnóstico de la TB. Algunas de las ventajas de la baciloscopia son su rapidez, bajo coste y la necesidad de escasos requerimientos técnicos. Sin embargo, hay varios factores que pueden afectar el rendimiento de un frotis tales como el tiempo de la recogida, el procesamiento de esputo y la técnica utilizada. Algunas de las herramientas comúnmente utilizadas para el diagnóstico de la tuberculosis en entornos con recursos limitados, son la observación directa tras la tinción con técnica de Ziehl-Neelsen y la microscopía de fluorescencia directa. Se ha comparado ambas técnicas, demostrándose que dejaron sin diagnosticar el 64 y el 20% de los casos, respectivamente (106). Estos datos soportan que el uso aislado de la tinción de Ziehl-Neelsen conlleva un infradiagnóstico de la infección por TB, siendo fundamental detectar nuevos métodos diagnósticos que permitan un diagnóstico de sospecha, fiable y accesible, y que produzca el menor número posible de falsos negativos. En general, se estima que es necesaria una carga de TB de 1 × 104 bacilos por ml para que sea detectable con un frotis (105). Una revisión sistemática encontró que las técnicas con fluorescencia son un 10% más sensibles que las técnicas de tinción estándar (Ziehl-Neelsen), con una especificidad similar. El uso de la tinción fluorescente también ha demostrado reducir el tiempo de lectura a 1 minuto para un frotis frente a los 4 minutos de la técnica convencional (107). La OMS recomendó en 2009 que la microscopía convencional debería ser sustituida por la microscopía fluorescente del LED, lo que ha llevado a un uso más generalizado en los últimos años (108). 1.5.4. Cultivo bacteriológico El cultivo de micobacterias tradicionalmente se ha realizado en medios sólidos, por lo general Lowenstein-Jensen. La sensibilidad del cultivo es mayor que la del frotis, pudiendo detectar muestras que poseen 1 x 102 bacilos por ml. Otra de las ventajas de esta técnica es su capacidad para distinguir entre micobacterias tuberculosas y no tuberculosas, indistinguibles por microscopía 17 directa. Desgraciadamente, el tiempo de espera de 4-6 semanas necesario para el crecimiento de las colonias de micobacterias constituye uno de los mayores inconvenientes del cultivo (105). Los sistemas de cultivos líquidos constituyen el método ordinario de diagnóstico de la tuberculosis y tratamiento de pacientes con esa infección en los países industrializados. Con una mayor sensibilidad y menores demoras, los sistemas de cultivos líquidos pueden mejorar considerablemente el tratamiento de los pacientes (109). El cultivo bacteriológico tanto en medio sólido como líquido supone también una buena oportunidad para valorar la sensibilidad de la cepa a los diferentes fármacos antituberculosos. 1.5.5. Interferon Gamma Release Assays (IGRA) El IGRA es una prueba que se desarrolló con el objetivo de conseguir una prueba más específica que mejorara el test cutáneo de la tuberculina gracias a la no utilización del bacilo de Calmette-Guerin, evitando así falsos positivos en pacientes vacunados contra la TB. El interferón gamma es una molécula importante para el control de la infección tuberculosa, y su participación es imprescindible en la respuesta inmune protectora frente al Mycobacterium tuberculosis. Esta citoquina, producida por los linfocitos T CD4+, CD8+ y NK, activa los macrófagos infectados, con la consiguiente liberación de IL-1 y TNF-α, que limitan el crecimiento y la multiplicación de las micobacterias. Para la realización de esta IGRA, las células del paciente se incuban con antígenos específicos de tuberculosis y se mide la liberación de interferóngamma en respuesta a dichos antígenos. Existen dos pruebas comerciales que son usadas en la actualidad, el TSPOT.TB, que utiliza células mononucleares aisladas de sangre y el QuantiFERON-TB, que utiliza la sangre sin filtrar. La primera generación de QuantiFERON-TB, aprobada en el año 2001, detectaba la liberación de interferón gamma en respuesta a PPD. En el año 2004, fue aprobada la segunda generación de esta prueba diagnóstica, denominada QuantiFERONTB Gold, la cual ya no utilizaba como antígenos micobacterianos el PPD, sino péptidos sintéticos que simulan antígenos más específicos y que están ausentes en M. bovis BCG y en la mayoría de las micobacterias no tuberculosas (excepto M. kansasii, M. marinum, M. szulgai). En la actualidad ya se comercializa la tercera generación de esta prueba, denominada QuantiFERON-TB Gold In Tube (QFT-GIT), que incorpora un tercer antígeno micobacteriano: el TB 7.7, y tubos específicamente diseñados para recoger la muestra de sangre (110). Existe un gran interés en la utilización de IGRAs no sólo para evaluar la presencia de tuberculosis latente sino también de la forma activa. Sin embargo, en una revisión sistemática de los estudios de pacientes con sospecha de tuberculosis activa, la QFT-GIT tuvo una sensibilidad del 69% y una especificidad del 52%, mientras que el T-SPOT.TB tuvo una sensibilidad 18 combinada del 83% y una especificidad del 61%. La comorbilidad con VIH y SIDA disminuyó la sensibilidad a 60% y 76%, respectivamente (111) Sobre la base de estos datos, la OMS aconseja que "ni IGRAs ni la prueba cutánea de la tuberculina deben ser utilizados para el diagnóstico de la tuberculosis activa." (112). 1.5.6. Reacción en cadena de la polimerasa (PCR) Existen varias técnicas en relación con la amplificación del ADN y del ARN que han demostrado ser más sensibles y específicas que los métodos convencionales para la detección rápida de Mycobacterium tuberculosis en el esputo y en otras muestras de fluidos corporales. Para este propósito, la PCR es un método alternativo rápido y eficaz que permite confirmar la presencia de este microorganismo en muestras biológicas a los 1-3 días, lo cual contrasta con las 2-6 semanas de las técnicas tradicionales de cultivo. Además, esta técnica presenta una alta especificidad y una potencial alta sensibilidad, aunque los resultados de los estudios realizados al respecto hasta la fecha son algo heterogéneos (109, 113). Otra de las ventajas de esta modalidad diagnóstica es que permite detectar el M. tuberculosis en muestras de tejido a pesar de haber sido expuestas al formol u otras sustancias que impiden la posibilidad del cultivo. Algunas de las desventajas de la PCR, en comparación con otros métodos diagnósticos de tuberculosis, son su elevado coste y la poca disponibilidad en áreas sanitarias menos desarrolladas. Aunque la PCR presenta especificidad y valor predictivo negativo más bajo de lo deseable en muestras de individuos VIH positivos, la técnica puede todavía ser ventajosa en comparación con los métodos convencionales para el diagnóstico rápido de tuberculosis pulmonar paucibacilar. Hasta la fecha, no ha habido ningún otro método más eficaz cuando la combinación de hallazgos clínicos, radiológicos y microbiológicos convencionales no establece el diagnóstico (103). 1.6. Complicaciones de la tuberculosis pulmonar 1.6.1. Complicaciones parenquimatosas La cavitación es un sello distintivo de la tuberculosis pulmonar. Las cavitaciones residuales de paredes delgadas pueden ser vistas tanto en la enfermedad activa como en la inactiva. Tras el tratamiento quimioterápico antituberculoso, dichas cavidades pueden desaparecer, aunque se dan casos en que su pared se vuelve más fina y permanece como una formación quística con contenido aéreo, incluso después de la eliminación de la infección del organismo. Las cavitaciones tuberculosas se curan mediante dos procesos diferentes, abiertos y cerrados, dependiendo del estado del bronquio de drenaje. En la forma abierta, existe un bronquio a través del que drena el contenido de la cavidad, quedando una pared libre de Mycobacterium tuberculosis tras el tratamiento antituberculoso y que sufrirá fibrosis con epitelización posterior. La pared puede ser fina o gruesa variando de 1 cm a menos de 1 mm, pudiendo 19 ser difícil distinguir a veces estas cavidades de bullas enfisematosas, quistes o neumatoceles. Estas cavidades pueden estar asociadas con complicaciones como hemoptisis, invasión fúngica o infección secundaria por microorganismos piógenos que resulta en un absceso pulmonar; rara vez se produce la reinfección por Mycobacterium tuberculosis. Además, la rotura de la cavidad puede dar lugar a un neumotórax espontáneo. La luz del bronquio de drenaje quedará patente de forma residual en controles radiológicos posteriores. En la forma cerrada de curación, el bronquio de drenaje se ocluye y la cavitación da lugar a la formación de una atelectasia o cicatriz parenquimatosa. 1.6.2. Complicaciones relacionadas con la vía aérea La enfermedad pulmonar obstructiva crónica (EPOC) es una patología caracterizada por una limitación del flujo aéreo que no es completamente reversible. La limitación del flujo aéreo es generalmente progresiva y está asociada con una respuesta inflamatoria anormal de los pulmones a partículas o gases nocivos, y cuya causa fundamental es el tabaquismo. Aunque la EPOC afecta a los pulmones, también produce consecuencias sistémicas significativas. Las partículas nocivas responsables del desarrollo de la EPOC lesionan el epitelio de las vías respiratorias y conduce a la inflamación específica de la vía aérea y cambios estructurales. Una vez que se eliminan estos agentes, los mecanismos reparativos del sistema respiratorio deben, idealmente, devolver a las vías respiratorias a su estructura y función normal. En general, se cree que un proceso de reparación inadecuado puede jugar un papel clave en el desarrollo de la obstrucción crónica del flujo aéreo en algunos, pero no todos, los fumadores. En muchas ocasiones, la mayor parte de los cambios inflamatorios continúan a pesar de que cede la agresión externa. Este fracaso en el control de la reacción inflamatoria bronquial podría contribuir tanto a los cambios sistémicos como bronquiales en el curso de la enfermedad, conllevando la posterior degradación de la matriz pulmonar. Además de la inflamación bronquial persistente, otros fenómenos importantes que intervienen en la iniciación y progresión de la enfermedad incluyen el estrés oxidativo y el desequilibrio proteasa-antiproteasa. Varios estudios han establecido que la obstrucción de vías respiratorias en la EPOC se debe a los cambios que afectan a las pequeñas vías aéreas y el parénquima pulmonar, mientras que la contribución de la remodelación epitelial en la vía aérea proximal es menos clara. La disminución del volumen espiratorio forzado en un segundo (FEV1) en la EPOC se relaciona principalmente con el engrosamiento de las paredes de las pequeñas vías aéreas de conducción y con la obstrucción de estas mismas vías aéreas por exudado mucoso (114, 115). Existe una fuerte asociación positiva entre la tuberculosis pulmonar y la presencia de enfermedades respiratorias crónicas (incluyendo tanto la EPOC como las bronquiectasias). Las asociaciones más fuertes entre la TB y la EPOC se han registrado en países con mayores tasas de incidencia de tuberculosis, como África del Sur y Filipinas. La asociación entre la tuberculosis y EPOC también suele ser más evidente entre los adultos más jóvenes (<40 años) (116). Esto puede explicarse por la historia natural de la EPOC: a diferencia de la EPOC relacionada con el tabaquismo, la EPOC relacionada 20 con la tuberculosis se produce en adultos jóvenes puesto que son los más afectados por esta infección y el daño pulmonar asociado se produce durante el proceso de la enfermedad aguda, lo que explica por qué su contribución relativa a la EPOC es mayor en el grupo de edad más joven, especialmente en las zonas endémicas de TB. Existen estudios que han documentado una asociación más fuerte entre la TB y la EPOC entre las personas que nunca han fumado (117). Aunque la EPOC pueda poseer etiologías diferentes, no se puede decir que exista un fenotipo EPOC exclusivo relacionado con cada una de sus causas, siendo el aspecto histológico y el patrón espirométrico en muchos casos superponibles. La anormalidad en la función pulmonar a menudo se correlaciona con el grado de deterioro radiológico, con el número de episodios de tuberculosis y con la presencia de otras comorbilidades respiratorias. En ocasiones, es observable una combinación de un patrón obstructivo con un restrictivo (118). La bronquiectasia es una dilatación anormal y permanente de los bronquios. La tuberculosis es una de sus causas más importantes conocidas, pudiendo estar presente las bronquiectasias en varias fases de la infección. Tanto es así que aparecen hasta en el 85 % de los pacientes que han sido tratados previamente de una tuberculosis (119). Los factores que pueden estar relacionados con la aparición de bronquiectasias en la tuberculosis pulmonar son principalmente las atelectasias y la fibrosis pulmonar. La fibrosis que aparece en la curación de la tuberculosis suele conllevar una retracción del parénquima pulmonar que conlleva una dilatación de los bronquios y, por tanto, contribuye a la formación de bronquiectasias. También se puede dar que la estenosis bronquial producida por la inflamación tuberculosa con su posterior fibrosis mural de los bronquios puede conducir a una retención de secreciones que cuando se sobreinfectan por bacterias pueden producir la destrucción y dilatación de las vías respiratorias. Es importante recordar que en los adultos la tuberculosis suele afectar predominantemente a los lóbulos superiores en pacientes inmunocompetentes, existiendo la particularidad de que el drenaje de las secreciones se realiza cuando las bronquiectasias presentan localización superior, siendo la sobreinfección por microorganismos piógenos poco habitual (“bronquiectasia seca”). La compresión de los bronquios por ganglios linfáticos agrandados produce consecuencias similares a la obstrucción intraluminal. La tuberculosis puede favorecer el desarrollo del carcinoma broncogénico por mecanismos locales (inflamación crónica y degeneración maligna de la cicatriz), aunque en muchas ocasiones se da la coexistencia de ambas entidades. Además, en ocasiones el carcinoma puede conducir a la reactivación de la tuberculosis, tanto por la erosión de un foco encapsulado de infección, como por la inmunodepresión que produce en el paciente. Por lo tanto, el aumento de tamaño de un nódulo en los controles de imagen sucesivos en un paciente con tuberculosis pulmonar debe hacer sospechar la coexistencia de un carcinoma pulmonar (120). La broncolitiasis es una complicación poco frecuente de la tuberculosis pulmonar, que consiste en la presencia de masas ganglionares calcificadas 21 dentro de los bronquios. Dicha complicación se produce por la erosión de la pared del bronquio y la posterior extrusión de un ganglio linfático adyacente calcificado al interior de la luz bronquial. Algunos hallazgos que suelen acompañar a la broncolitiasis son la atelectasia del segmento o lóbulo pulmonar correspondiente, la neumonitis obstructiva o la hiperinsuflación local. Los síntomas que asocia pueden incluir tos, hemoptisis o síntomas relacionados con la obstrucción bronquial. La tuberculosis endobronquial se puede producir o bien por inoculación directa de los bacilos de las lesiones parenquimatosas de pulmón, o bien por la infiltración de la vía aérea por bacilos de los ganglios linfáticos mediastínicos adyacentes. A pesar del tratamiento adecuado con tuberculostáticos, la tuberculosis puede causar una gran obstrucción de las vías respiratorias con estenosis, hecho que sucede con más frecuencia en el sexo femenino. La afectación estenótica de los bronquios principales es rara, siendo la localización más común el bronquio principal izquierdo debido, probablemente, a que este bronquio se comprime fácilmente por el arco de la aorta y los ganglios linfáticos adyacentes. 1.6.3. Complicaciones vasculares La hemoptisis es un síntoma comúnmente asociado a la tuberculosis y que se suele relacionar con cavitaciones o bronquiectasias. La inflamación crónica característica de las lesiones tuberculosas conduce a neovascularización y a un mayor aporte vascular de las arterias sistémicas cercanas. Estos vasos colaterales recién formados tienen una pared arterial débil y son propensos a la ruptura. El aneurisma de las arterias bronquiales (aneurisma de Rasmussen) ha sido reportado en alrededor del 7% de los pacientes con tuberculosis pulmonar que presentan hemoptisis masiva, siendo la colateralidad e hipertrofia de las arterias bronquiales un hallazgo común en los estudios angiográficos (121). 1.6.4. Complicaciones mediastínicas La adenitis mediastínica es una manifestación frecuente de la tuberculosis pulmonar primaria en pacientes en edad pediátrica, disminuyendo su incidencia progresivamente con la edad. A nivel histológico se produce la formación de granulomas caseificantes en los ganglios linfáticos, que más comúnmente involucra el lado derecho. En la etapa activa, los ganglios tienen baja atenuación central en TC, lo cual se corresponde con una caseificación o necrosis licuefactiva; periféricamente se observa una zona hiperdensa e hipercaptante, en relación con tejido de granulación con hipervascularidad inflamatoria en el análisis histopatológico. Con el tratamiento, los ganglios cambian en apariencia, adoptando una densidad homogénea en TC para, finalmente, hacerse imperceptible o convirtiéndose en una lesión residual compuesta por tejido fibrótico, calcificaciones y sin áreas de baja atenuación (120). Tal y como documentó Gawne-Cain et al, existen diferencias estadísticamente significativas entre las adenopatías calcificadas producidas por la tuberculosis y la sarcoidosis, en lo que respecta a su tamaño, distribución y patrón de calcificación (122). 22 La afectación esofágica en la infección por tuberculosis es rara y suele ser secundaria a la transmisión del bacilo desde una adenopatía adyacente, siendo la zona más frecuentemente afecta la subcarinal debido a su proximidad a los ganglios patológicos. Cuando las adenopatías erosionan la pared esofágica o bronquial adyacente, esto da lugar a una fístula que se suele manifestar como una colección gaseosa localizada dentro del mediastino. Los síntomas de presentación pueden incluir fiebre, tos, pérdida de peso, disfagia, dolor torácico o de espalda (120). La afectación tuberculosa del pericardio es rara, pudiendo producirse por propagación desde una neumonía adyacente o la extensión directa desde una infección localizada en la pleura visceral o en la pared torácica. También es posible la afectación pericárdica por siembra hematógena en el contexto de una tuberculosis miliar. No obstante, la causa más frecuente de pericarditis tuberculosa es por la infiltración de bacilos desde las adenopatías adyacentes, fundamentalmente desde las localizadas en cadena subcarinal. En cuanto a la clínica, no es raro que el paciente esté asintomático cuando el derrame es pequeño. Suele tener un inicio insidioso con pérdida de peso y fatiga, pero también es posible un desarrollo agudo y dramático con taponamiento cardiaco (123). 1.6.5. Complicaciones pleurales Aunque la tuberculosis es considerada una enfermedad en esencia crónica, la pleuritis tuberculosa puede presentarse como una enfermedad aguda, con una duración que puede ser menor de 1 semana. Por norma general, el derrame se produce cuando un foco parenquimatoso subpleural o un ganglio linfático afecto se abren al espacio pleural, lo cual se suele dar 3-6 meses después de la infección primaria. Además de la transmisión del bacilo por contigüidad, existe la posibilidad de que el derrame pleural se produzca por diseminación hematógena. La pleuritis tuberculosa progresa para convertirse en empiema tuberculoso crónico, que puede definirse como persistente, de paredes groseras y líquido purulento que contiene bacilos de la tuberculosis pero que en ocasiones es difícil de cultivar (120). La presencia de un fibrotórax, que se traduce por la existencia de un engrosamiento pleural difuso sin derrame objetivable por TC es un buen indicador de inactividad (120). El quilotórax es la acumulación de linfa en el espacio pleural, a consecuencia de la disrupción del flujo linfático torácico. La linfa es grasa emulsificada que es transportada en forma de triglicéridos, los cuales aparecen en altas concentraciones en el espacio pleural. El seudoquilotórax es un derrame pleural crónico con elevada concentración de colesterol (por encima de los 200 mg/dl). Aunque comparte la apariencia lechosa del quilotórax, no está relacionado con el daño linfático y no tiene triglicéridos en su composición. El colesterol del seudoquilotórax puede originarse de la degradación de la pared celular de leucocitos o glóbulos rojos secuestrados en el espacio pleural (124). 23 Los síntomas de presentación más comunes son la tos no productiva, fiebre, dolor torácico pleurítico y disnea, los cuales se pueden dar en la mayoría de los pacientes. Existen otros síntomas como la pérdida de peso, malestar general y disnea que son más habituales en las formas crónicas de derrame. Los derrames pleurales tuberculosos suelen ser unilaterales y en un tercio de los casos co-existentes con enfermedad del parénquima objetivable radiológicamente. La fístula broncopleural consiste en una comunicación anormal permanente que se establece entre el árbol bronquial y el espacio pleural, y que se presenta en la evolución de una enfermedad respiratoria grave como síndrome de distrés respiratorio agudo, crisis bronquial obstructiva severa, absceso pulmonar o pleuroneumonía complicada. La fístula broncopleural debida a la tuberculosis sigue generalmente a un trauma o a un procedimiento quirúrgico, pero también puede ocurrir de forma espontánea. El neumotórax secundario a tuberculosis a menudo se relaciona con una afectación pulmonar severa y extensa, anticipando la aparición de fístula broncopleural y empiema. Se produce en aproximadamente el 5% de los pacientes con tuberculosis posprimaria, por lo general, en relación con la enfermedad cavitaria avanzada, pero rara vez en la tuberculosis miliar (120). La patogénesis implica infiltrados pleurales caseosos que se someten a la licuefacción, lo que resulta en la necrosis pleural y ruptura. 1.6.6. Complicaciones en pared torácica La tuberculosis en ocasiones implica el esternón, a la articulación esternoclavicular o una costilla, lo que lleva a la destrucción ósea y formación de abscesos localizados. Esta participación puede producirse por extensión directa de una lesión tuberculosa pleuropulmonar o por diseminación hematógena desde un foco distante. Cuando la descarga espontánea de empiema través de la pleura parietal en la pared torácica se forma un absceso subcutáneo, produciéndose lo que se denomina necessitatis (125). 24 2. HIPÓTESIS Y OBJETIVOS 25 26 2.1. HIPÓTESIS DE TRABAJO Es conocido por la literatura médica y por nuestra propia experiencia que a pesar que el frotis de esputo con tinción acido-alcohol resistente es el instrumento más utilizado para el diagnóstico provisional de tuberculosis, sólo tiene una sensibilidad ligeramente superior al 70%, lo cual lleva al infradiagnóstico de esta enfermedad. La hipótesis de trabajo que plantea este estudio es desarrollar un modelo predictivo sobre la presencia de tuberculosis pulmonar activa utilizando datos epidemiológicos, de radiografía simple y TC torácica. 2.2. OBJETIVOS Los objetivos primarios de esta tesis son: 1. Estudiar las variables de TC de tórax que pueden estar asociadas a la presencia de un cultivo positivo para Mycobacterium tuberculosis. 2. A través de una regresión logística multivariante, determinar las variables que, en conjunto, pueden predecir un cultivo positivo para tuberculosis, elaborando un sistema de puntuación predictiva que permita establecer el diagnóstico con relativa seguridad. Los objetivos secundarios son: 1. Estudiar las variables epidemiológicas que pueden presentar una mayor prevalencia en los pacientes con tuberculosis pulmonar. 2. Establecer los signos o síntomas cuya presencia ha sido más habitual en los pacientes con tuberculosis pulmonar activa. 3. Determinar qué hallazgos en radiografía simple de tórax pueden asociarse con más fiabilidad a la presencia de cultivo positivo para tuberculosis. 4. Estudiar qué hallazgos radiológicos son sugestivos de la presencia de infección por micobacterias atípicas, en detrimento de la infección por M. tuberculosis. 27 28 3. MATERIAL Y MÉTODOS 29 30 En esta tesis se ha efectuado un estudio observacional, retrospectivo, descriptivo y analítico de variables clínicas y epidemiológicas, así como los hallazgos radiológicos vistos en radiografía simple y TC de tórax realizadas por sospecha clínica de tuberculosis pulmonar en el Servicio de Radiología Diagnóstica y Terapéutica del Hospital Universitario General de Castellón entre el 1 de enero de 2005 y el 31 de Diciembre de 2014. Durante estos 10 años se llevaron a cabo 1.549 estudios de TC de tórax en pacientes con sospecha de tuberculosis pulmonar. 3.1. MATERIAL 3.1.1. Pacientes Inicialmente, se revisaron los registros del Servicio de Microbiología y Parasitología del Hospital Universitario General de Castellón, seleccionando a todos los pacientes a los que se les realizó un cultivo en medio específico para M. tuberculosis (medio Lowenstein-Jensen) en el periodo comprendido entre el 01 de enero de 2005 y el 31 de diciembre de 2014, independientemente de cuál hubiera sido el resultado de dicho cultivo. Sólo fueron seleccionados aquellos cultivos realizados sobre muestras procedentes de vía respiratoria (esputo, lavado broncoalveolar y broncoaspirado); aunque también fue aceptado el cultivo del líquido pleural, descartando a aquellos de diferente procedencia. Nuestro hospital es un centro de tercer nivel, de referencia en la provincia de Castellón, con una población de influencia que en los años que duró el estudio osciló entre los 543.432 y los 604.564 habitantes (126) (Tabla 0). Por tanto, entre los pacientes recogidos en esta primera muestra no sólo estaban los afincados en Castellón de la Plana, sino también aquellos derivados desde otros hospitales de la provincia de Castellón. Tabla 0. Cifras oficiales de población del Instituto Nacional de Estadística en el periodo del 2005 al 2014 en la provincia de Castellón. En un segundo paso, se revisaron las historias clínicas de todos los pacientes con cultivo en medio Lowestein-Jensen, seleccionando a aquellos a los que se les hubiera realizado una TC de tórax ante la sospecha de infección tuberculosa, siempre y que ésta hubiera sido realizada antes de obtenerse la 31 confirmación microbiológica (resultado del cultivo). Se seleccionó a estos pacientes independientemente de cuál hubiera sido el resultado de la TC. Para ello, el servicio de informática realizó un cruce entre la base de datos de cultivos y la base de datos de TCs de tórax del mismo periodo, obteniendo una muestra de menor tamaño con respecto a la inicial. Con la muestra resultante se aplicaron criterios de exclusión, separando a los pacientes que no presentaran una referencia clara al episodio de tuberculosis en su historia clínica y a aquellos cuyo resultado de cultivo hubiera sido indeterminado o técnicamente imposible. También fueron descartados los pacientes cuyo TC no poseyera imágenes o no fuera accesible mediante nuestro sistema de archivo y recuperación de imágenes (PACS). Una vez obtenida la muestra definitiva, se elaboró un listado de los pacientes, en el que sólo se incluyó la fecha de la toma de muestras para el cultivo, el número SIP (Sistema de Identificación Poblacional) y el NHC (Número de Historia Clínica), eliminando cualquier otra característica que pudiera permitir su identificación o afiliación en un grupo de edad o género determinado. Siguiendo dicha lista, se revisó la historia clínica de cada paciente, obtenida en formato electrónica a través de los programas “Alta Hospitalaria” o “Abucasis”, o bien en su formato físico, a través del servicio de consulta facilitado por el Hospital. Se implementó en cada caso un formulario específico (anexo 1), en el que se incluyeron diferentes datos clínicos y epidemiológicos (edad, género, procedencia, antecedentes de enfermedad, analítica, síntomas). Tras esto, se reordenó la lista con la fecha de cultivo, SIP y NHC, utilizada para la recolección de datos clínicos y epidemiológicos, obteniendo una en la que los pacientes presentaban un orden diferente. De este modo se pretendía evitar posibles sesgos de medición. Una vez obtenido el nuevo listado, se revisaron las pruebas de imagen de cada paciente, siempre que se hubiera realizado en un periodo de un mes antes o un mes después de la toma de muestras para el cultivo. Se mostró especial atención a las TC. Se elaboró un formulario (anexo 2) diferente al descrito para los datos clínicos y epidemiológicos, el cual fue implementado con los hallazgos radiológicos observados en cada individuo durante el episodio de tuberculosis pulmonar por un radiólogo con más de 10 años de experiencia en radiología torácica. No sólo se recogieron los datos de TC sino también los derivados de la radiografía simple, siempre y que se hubieran realizado dentro del mismo episodio asistencial. Los hallazgos de la radiografía y de la TC fueron recogidos en bloques diferenciables dentro del formulario utilizado. La ausencia de radiografía de tórax no fue considerado un criterio válido para excluir al paciente del estudio. Tras haber revisado todas las historias clínicas y pruebas de imagen de todos los pacientes del estudio y tras haber completado los dos formularios en cada individuo, se procedió a introducir los datos en una base de datos elaborada en el programa Excel 2011 (Microsoft, Redmond, WA, USA). Con la base de datos completa, se introdujo una casilla adicional en la que se aportó los resultados del cultivo. 32 3.2. MÉTODOS En esta tesis se han evaluado 1.549 estudios de TC torácico en total, realizados durante 10 años a pacientes con sospecha de tuberculosis pulmonar. Este estudio se realizó de conformidad con la Declaración de Helsinki y recibió la aprobación del Comité de Ética del Hospital Universitario General de Castellón. Parámetros técnicos: TC: Todos los estudios se realizaron con un equipo multidetector de tomografía computarizada (Aquilion RXL de 16 detectores o con Aquilion 64 de 64 detectores, ambos de Toshiba Medical System). Los pacientes no necesitaban ninguna preparación especial, simplemente 6 horas de ayuno previo a la inyección del contraste intravenoso. No se les administró contraste oral ni medicación previa al estudio. Se realizó una técnica de adquisición helicoidal, con un campo de estudio (FOV) grande, que se extendía desde la porción subglótica de la tráquea hasta diafragma como mínimo, con cortes de 5 mm de grosor e intervalo de reconstrucción de 5 mm, incluyendo reconstrucciones coronales y sagitales automáticas. Todas las exploraciones fueron realizadas a 120kV con 220 mAs, así como con la aplicación de baja dosis de exposición a la radiación siempre que fue posible. Todos los pacientes recibieron un medio de contraste no iónico intravenoso de baja osmolaridad que contenía una alta concentración de yodo equivalente a entre 320 y 370 mg de Iodo/mL. Se inyectaron 80 ml de contraste cuando se utilizó el aparato de 16 detectores y 60 ml de contraste cuando se usó el aparato de 64 detectores. El medio de contraste intravenoso se administró por medio de una bomba de inyección a una velocidad de 3 ml/s en pacientes estudiados con el aparato de 16 detectores y de 2 ml/s cuando se usó el aparato de 64 detectores, a través de un catéter intravenoso de calibre 20G insertado en una vena del brazo. Con el fin de reducir la dilución del medio de contraste y la acumulación en el espacio muerto venoso periférico, todas las inyecciones fueron seguidas por una descarga de 30 ml de solución salina administrada a la misma velocidad de inyección que el contrate, a los 50 segundos de terminar la administración del contraste intravenoso. Se han recogido variables epidemiológicas, clínicas, de radiografía simple y de TC torácico. 3.2.1. Variables epidemiológicas y antecedentes de enfermedad Fueron recogidas las siguientes variables epidemiológicas, reflejadas en el anexo 1: 33 - Sexo: Hombre o mujer. - Edad: Edad en años en el momento de realizar la TC. - Grupo al que pertenece el paciente: Se asignó a los pacientes a diferentes grupos en función de su edad y del resultado del cultivo, quedando clasificados del siguiente modo: - G1: menores de 18 años con tuberculosis. - G2: menores de 18 años con cultivo negativo. - G3: pacientes de 18-65 años con tuberculosis. - G4: pacientes de 18-65 años con cultivo negativo. - G5: mayores de 65 años con tuberculosis. - G6: mayores de 65 años con cultivo negativo. - G7: Se añadió un séptimo grupo, formado por los pacientes que presentan un cultivo positivo para micobacterias atípicas, independientemente de su edad. - País de procedencia: País en el que nació el paciente, independientemente de su nacionalidad actual. Otros datos como etnia o grupo religioso de pertenencia no son recogidos en este estudio. - Institucionalización en centros penitenciarios: Fueron identificados aquellos pacientes que estuvieran recluidos en alguna institución penitenciaria. Para detectar a estos individuos, se revisó la historia clínica y el centro que solicitó las exploraciones o que derivó al paciente en caso que acabara ingresado. Los dos centros penitenciarios pertenecientes a esta provincia son el Centro Penitenciario Castellón (Castellón de la Plana) y el Centro Penitenciario Castellón II (Albocasser). - Anemia: Se consideró que existía anemia cuando se producía un descenso de la masa eritrocitaria que resulta insuficiente para aportar el oxígeno necesario a las células. En nuestro estudio definimos anemia (127) cuando se observó una cifra de hemoglobina inferior a 130 g/l (8 mmol/l) en el varón o a 120 g/l (7.4 mmol/l) en la mujer. Como caso excepcional, en mujeres embarazadas se consideró cifra de referencia los 110 g/l (6.8 mmol/l). 34 - Antecedente de tuberculosis pulmonar previa: Descrito específicamente en historia clínica, independientemente de los hallazgos en las exploraciones radiológicas que sugirieran cambios cicatriciales postinfección tuberculosa pulmonar. - Antecedente de cirugía: Referidos en historia clínica, especificando los pacientes que fueron sometidos a tratamiento quirúrgico. Se mostró especial interés en los antecedentes de gastrectomía, colectomía, neumonectomía, esplenectomía o hepatectomía, omitiendo otras operaciones de escasa relevancia como apendicectomía, amigdalectomía o colecistectomía, por ejemplo. - Antecedente de trasplante de órgano sólido (especificando el tipo): Se consideró cualquier antecedente de trasplante, independientemente de la fecha en la que se realizó puesto que conllevan terapia inmunosupresora a largo plazo. - Diabetes mellitus: Es en realidad un grupo de enfermedades metabólicas de base genética que se caracterizan fundamentalmente por la hiperglucemia. Los efectos de esta patología son originados por un defecto en la secreción de insulina, de su acción o de la combinación de ambos. Para el diagnóstico de diabetes se aplicaron los criterios de ADA (128), que incluyen: 1. Síntomas típicos: poliuria, polidipsia, pérdida de peso sin motivo aparente y una glucemia casual (en cualquier momento del día sin tener en cuenta el tiempo transcurrido desde la última ingesta) igual o superior a 200 mg/dl (1.1 mmol/l). 2. Glucemia en ayunas (8 horas, mínimo) igual o superior a 126 mg/dl (7 mmol/dl). 3. Glucemia mayor o igual a superior a 200 mg/dl a las 2 horas de la sobrecarga de glucosa durante una prueba de tolerancia oral a la glucosa, consistente en la administración de una dosis oral de glucosa y la práctica de extracciones secuenciales de sangre para determinar la glucemia. Con todo, en nuestro estudio aceptamos como válido el antecedente de diabetes mellitus especificado en la historia clínica. - Alcoholismo: El término alcoholismo recoge, en realidad, distintos tipos de entidades nosológicas. La OMS distingue (129):   Consumo de riesgo: patrón de ingesta alcohólica que incrementa el riesgo de consecuencias negativas para uno mismo o para terceros. Consumo dañino: ingesta que comporta daños físicos, mentales o sociales. 35   Dependencia alcohólica: conjunto de alteraciones conductuales, cognitivas y fisiológicas que pueden aparecer después del consumo repetido. Trastornos relacionados con el alcohol: conjunto de alteraciones físicas, mentales o de conducta social en las que el alcohol es parte del nexo causal de dicha alteración. En el presente estudio aceptamos el diagnóstico de alcoholismo siempre que hubiera sido establecido como tal en la historia clínica. - Terapia con fármacos esteroideos: Los corticosteroides o corticoides son una variedad de hormonas del grupo de los esteroides (producida por la corteza de las glándulas suprarrenales) y sus derivados. Estas sustancias pueden sintetizarse artificialmente y tienen aplicaciones terapéuticas, utilizándose principalmente debido a sus propiedades antiinflamatorias e inmunosupresoras y a sus efectos sobre el metabolismo. Esta categoría hace alusión específicamente a los fármacos corticoides tomados por vía oral, excluyendo los administrados en forma de inhaladores, habituales en el tratamiento del EPOC. Se consideró antecedente positivo para la toma de corticoides orales o sistémicos en caso de que éstos estuvieran pautados antes de la obtención de la muestra para cultivo. - Enfermedades hematológicas: Se revisaron los antecedentes de enfermedades hemato-oncológicas, fuente originaria de trastornos de la inmunidad. También se registraron los casos de enfermedades hematológicas raras no oncológicas tales como insuficiencias medulares (Aplasia medular adquirida y anemia de Fanconi), inmunodeficiencias primarias y neutropenia crónica severa. - Enfermedad renal crónica (ERC): Puede afectar en sus diferentes estadios a cerca del 10 % de la población española (130). Según las actuales guías KDIGO (Kidney Disease Improving Global Outcomes), la ERC se define por la presencia durante al menos tres meses de, al menos, uno de los siguientes ítems:   FGe (filtrado glomerular estimado) inferior a 60 ml/min/1,73 m2. Lesión renal. La lesión renal se puede poner de manifiesto directamente a partir de alteraciones histológicas en la biopsia renal o indirectamente por la presencia de albuminuria, alteraciones en el sedimento urinario o a través de técnicas de imagen (130).  Albuminuria: la determinación de proteinuria incluye no solo la cuantificación de albúmina, sino también la de proteínas de bajo peso molecular, como proteínas de origen tubular o cadenas ligeras de inmunoglobulinas. 36    Sedimento urinario: la presencia en el sedimento urinario de hematuria y/o leucocituria durante más de tres meses, una vez se ha descartado la causa urológica o la infección de orina (incluida la tuberculosis urinaria), puede ser también indicio de ERC. Imágenes radiológicas patológicas persistentes en el tiempo: La ecografía renal permite, en primer lugar, descartar la presencia de patología obstructiva de la vía urinaria, pero también identificar anormalidades estructurales que indican la presencia de daño renal. Los quistes renales simples aislados no son un criterio por ellos mismos de daño renal. Se considera signos sugestivos de ERC la pérdida de tamaño de los riñones, la disminución del espesor parenquimatoso renal, la pérdida de la diferenciación corticomedular o la hiperecogenicidad de la cortical renal objetivable por ecografía, entre otros hallazgos. Alteraciones histológicas sugestivas de ERC. La prevalencia de la ERC aumenta por el envejecimiento de la población, el incremento de la prevalencia de sus factores de riesgo, como la enfermedad cardiovascular, la diabetes mellitus, la hipertensión arterial o la obesidad. Para este estudio, se consideró antecedente positivo para ERC si así venía reflejado en la historia clínica del individuo. - Colagenopatías: Incluyen una serie de enfermedades inflamatorias de curso crónico, etiología desconocida y patogenia autoinmune. Se caracterizan por una alteración inmunopatológica del tejido conectivo de diversos órganos y sistemas. Alguno de los ejemplos más comunes son el lupus eritematoso sistémico, la artritis reumatoide, la esclerodermia o la miastenia gravis. En muchas ocasiones conllevan un compromiso multisistémico y pueden ser potencialmente fatales. Una gran diversidad de agentes infecciosos puede afectar a los pacientes con colagenopatías que son tratados con corticosteroides en dosis inmunosupresoras o con antagonistas del factor de necrosis tumoral (FNTα). Dichos microorganismos incluyen bacterias intracelulares, Mycobacterium tuberculosis, hongos geográficos capaces de establecer infecciones granulomatosas, herpes zoster y el virus de la hepatitis B (pudiendo producirse la reactivación de una hepatitis crónica (131). - Enfermedad pulmonar obstructiva crónica (EPOC): Se caracteriza por la presencia de obstrucción crónica, progresiva y poco reversible al flujo aéreo, causada fundamentalmente por una reacción inflamatoria frente al humo del tabaco. Se prefiere el término de EPOC a otros como el de bronquitis crónica o enfisema porque EPOC engloba a éstos dos y describe mejor la enfermedad obstructiva observable en los fumadores. La bronquitis crónica se caracteriza clínicamente por la presencia de tos y expectoración durante más de 3 meses al año. Por su parte, la definición del enfisema es anatomopatológica y se fundamenta en la observación de un agrandamiento anormal de los espacios aéreos distales a los bronquiolos terminales con destrucción de la pared alveolar, sin que exista una fibrosis 37 manifiesta de los tabiques alveolares (132). Consideramos que el paciente tenía EPOC si estaba descrito así en la historia clínica. - Virus de inmunodeficiencia humana (VIH): Este virus pertenece a la familia Retroviridae. Se trata de un virus de ARN monocatenario positivo con envuelta, cápside icosaédrica y un diámetro de aproximadamente 100-110 nm. El VIH consta de dos cepas reconocidas (VIH-1 y VIH-2), y contiene un enzima denominado transcriptasa inversa o retrotranscriptasa, gracias al cual integra su información genética en el ADN de la célula huésped. El contagio se produce por vía sexual, aunque también es posible por contacto con sangre o fluidos orgánicos de un individuo infectado. Esta infección está caracterizada por una progresiva depresión del sistema inmunitario debido al ataque del virus a las células del organismo hospedador, principalmente a los linfocitos T CD4+. La infección aguda o síndrome retroviral agudo (SRA) es, con frecuencia, asintomática, aunque puede manifestar síntomas similares a los de la gripe o la mononucleosis (normalmente en el caso de VIH-1). Tras esto tiene lugar un periodo de latencia clínica o fase crónica que puede durar hasta varias décadas (en función de la recepción y efectividad del tratamiento) y suele cursar de forma asintomática. Finalmente, el impacto sobre el sistema inmunitario es tal que el individuo empieza a ser susceptible a una serie de infecciones entre las que destaca la tuberculosis, aunque también la candidiasis, herpes zoster, neumonía por pneumocystis jirovecii, encefalopatías y toxoplasmosis. En esta fase, también son más frecuentes algunas neoplasias como el linfoma o el sarcoma de Kaposi. Esta es la etapa más grave y se denomina síndrome de inmunodeficiencia adquirida (SIDA) (133). Se consideró caso positivo para infección por VIH si así venía recogido en la historia clínica. - Tabaquismo: El hábito tóxico de mayor trascendencia en neumología es el hábito tabáquico, que debe ser investigado en cualquier paciente afecto de patología respiratoria. Para cuantificar la cantidad de tabaco fumada por el paciente se utiliza el término “paquetes/año”, aceptada internacionalmente. Para calcular el número de paquetes/año en un individuo, se debe multiplicar el número de paquetes que fuma cada día por el número de años que ha estado fumando dicha cantidad de tabaco. Parece más difícil realizar este cálculo en caso de que el paciente consuma el tabaco a través de otros formatos diferentes al cigarrillo, como sucede en los usuarios de puros o pipa. En nuestro estudio, establecimos tres categorías, especificando si el paciente es fumador activo, lo fue en otra época o no lo ha sido nunca, no cuantificando el grado de consumo de tabaco que tiene o ha tenido. 3.2.2. Variables clínicas - Fiebre: La fiebre es uno de los principales motivos de consulta, no sólo en el ámbito de la atención primaria sino también en los servicios de urgencias hospitalarios. 38 Se entiende por fiebre a la elevación de la temperatura axilar por encima de los 38 ºC. - Febrícula: Se da si la temperatura axilar es mayor de 37 ºC y no excede los 37.9 ºC. Hay que tener en consideración que existe un ritmo circadiano de temperatura corporal, siendo ésta más baja por la mañana y mayor al final de la tarde. - Pérdida de peso: Se consideró significativa la pérdida involuntaria de un 5 % del peso corporal en un periodo de 6 meses, en ausencia de dietas u otra causa que lo justifique. - Tos: Se trata del síntoma más frecuente del paciente de neumología (132). De etiología variada, siempre deben ser estudiados aspectos como el tiempo de instauración (aguda o crónica), frecuencia (predominio diurno, nocturno, tos estacional, etc.), intensidad (permite o no dormir al paciente por la noche) y productividad (tos con o sin expectoración). - Dolor torácico: Se valora la presencia o ausencia de dolor torácico. La enfermedad parenquimatosa pulmonar pura no suele asociar dolor torácico, a no ser que asocie una afectación pleural. Otras estructuras mediastínicas que pueden producir dolor torácico son la tráquea y las estructuras osteomusculares de la pared torácica. - Sudoración nocturna: Muchos autores han intentado dar una definición exacta para este fenómeno, sin embargo, aún hoy no existe consenso al respecto. Para este trabajo se utilizó la definición de Mold y cols. (134), según la cual, sólo se debe considerar que existe el evento cuando no hay una temperatura y/o humedad ambiental que lo justifique y, además, la sudoración nocturna se ha prolongado en el tiempo durante al menos 1 mes. - Disnea: La disnea es la sensación subjetiva de falta de aire junto a la percepción de un trabajo respiratorio excesivo. Una vez detectada, debe establecerse si depende del sistema respiratorio o del sistema cardiovascular. La presencia de tos y expectoración crónica sugieren un origen broncopulmonar. Al contrario, algunos síntomas como la intolerancia al decúbito o el dolor de tipo anginoso suelen orientar a un origen cardiovascular cuando acompañan a la disnea. No se ha valorado el origen de la disnea, sólo su presencia o ausencia. 39 - Hemoptisis: Consiste en la emisión por vía oral de sangre procedente del aparato respiratorio. Antes de asentar su diagnóstico, es necesario descartar otros orígenes como el otorrinolaringológico o el digestivo. Suele conllevar la presencia de una enfermedad grave como neoplasia broncopulmonar, tuberculosis o bronquiectasias. - Mal estado general: Se define por la presencia de una sensación de debilidad y disconfort general. Consiste en una sensación de pérdida del estado basal de salud e impresión subjetiva de enfermedad. Es un síntoma inespecífico que debe ser diferenciado de la sensación de fatiga o la astenia. - Síndrome constitucional: El síndrome constitucional supone la asociación de astenia, anorexia y pérdida significativa de peso (definida como una disminución involuntaria del 5 % del peso corporal total en 6 meses). Su etiología puede ser múltiple y diversa, por lo que se requiere una valoración multidimensional e integral (historia clínica, examen físico y un panel de pruebas complementarias). 3.2.3. Variables en radiografía simple de tórax Para definir cada uno de los conceptos y apariencia radiológica de las diferentes presentaciones de la tuberculosis pulmonar, recurrimos a lo descrito en el “Glosario de términos para imagen torácica” de la Fleischner Society (135). 3.2.3.1. Hallazgos pulmonares: - Nódulo pulmonar: En la radiografía de tórax se representa como una opacidad redondeada, bien o mal definida, que mide hasta 3 cm de diámetro. Se evaluó la presencia o ausencia de nódulos y, así como su aparición única o múltiple (más de un nódulo). - Masa pulmonar: Una masa en radiografía simple de tórax es cualquier lesión pulmonar, pleural o mediastínica vista como una opacidad mayor de 3 cm de diámetro (independientemente de sus características del contorno, borde o densidad). 40 Imagen 1. Radiografía simple de tórax. En el LSI se observa una masa cavitada. El concepto de masa, por lo general, implica a una opacidad sólida o parcialmente sólida que puede ser objetivada por radiografía. Las masas pulmonares suelen formar ángulos agudos con la pared torácica, se centran en el pulmón, y retraen la vasculatura pulmonar. Una lesión pleural muestra ángulos obtusos con la pared lateral del tórax, desplaza la vasculatura pulmonar, se mueve durante la respiración y puede mostrar márgenes incompletos en la radiografía de tórax (136). Valoramos también si la masa pulmonar estaba o no cavitada (imagen 1). - Consolidación pulmonar: La consolidación se refiere a la presencia de un exudado u otro producto de la enfermedad que reemplaza el aire alveolar, compactando el parénquima pulmonar. En las radiografías, la consolidación consiste en opacidades de bordes mal definidos que borran los vasos pulmonares de su interior y que pueden contener estructuras tubulares aéreas (broncograma). Valoramos también si la consolidación pulmonar estaba o no cavitada. - Atelectasia: Consiste en un colapso o falta de expansión del pulmón y puede ser pulmonar, lobular, segmentario o subsegmentario. La representación radiográfica de la atelectasia depende de la cantidad de pulmón afectado (137). En este estudio se recogió la presencia o ausencia de atelectasia y no sus distintos tipos. - Patrón intersticial: Se trata de opacidades tenues que no impiden la visualización de los vasos pulmonares. Se da en enfermedades que afectan al intersticio (imagen 2). 41 Imagen 2. Radiografía simple de tórax. Patrón intersticial difuso bilateral. Se valoró si el patrón intersticial era focal o difuso en la radiografía de tórax. - Bronquiectasias: Dilatación localizada e irreversible del árbol bronquial. Se deben a múltiples causas, aunque la más frecuente es la infección aguda, crónica o recidivante. En radiografía simple de tórax se observa hallazgos directos como la dilatación bronquial y otros indirectos como engrosamiento de la pared bronquial, signo en vías de tranvía, imagen en anillo de sello, opacidades en “Y” o “V” y quistes con niveles hidroaéreos (137). 3.2.3.2. Hallazgos mediastínicos: - Masas mediastínicas: El aspecto del mediastino se debe valorar fundamentalmente en la proyección posteroanterior. Con la radiografía se puede detectar si existe un ensanchamiento mediastínico focal o difuso, además de valorar desplazamientos de las estructuras mediastínicas que sugieran la presencia de masa mediastínica. Otros aspectos a valorar son las líneas mediastínicas, pudiendo observarse un desplazamiento de la línea pleuroacigoesofágica en presencia de una hernia de hiato o un engrosamiento de la línea paratraqueal mayor de 4 mm, que suele ser sugestivo de adenopatías. El desplazamiento o engrosamiento de las líneas paravertebrales puede ser sugestivo de patología vertebral o paravertebral (137). - Agrandamiento de los hilios pulmonares: La posición de los hilios debe evaluarse en todas las radiografías de tórax. El izquierdo está unos 2 cm más alto que el derecho. Su desplazamiento puede ser sugestivo de atelectasia/colapso pulmonar. Uno de los hilios puede aparecer más grande o más radiopaco de lo normal, lo cual puede ser un indicio de la existencia de adenopatías. Cuando existen adenopatías 42 bilaterales, en la proyección lateral se observa un imagen nodular radiopaca con un centro radiolúcido, lo cual constituye el signo “del donut” (137). Debe valorarse también la presencia de un agrandamiento hiliar ya que puede sugerir la presencia de masas a dicho nivel. 3.2.3.3. Hallazgos pleurales: - Derrame pleural: El término derrame pleural se refiere a una acumulación de líquido en el espacio pleural. En esencia, se produce cuando existe cualquier proceso patológico que sobrepasa la capacidad de la pleura para reabsorber fluido. Aunque a veces el término derrame pleural se utiliza para incluir todo tipo de líquido que se puede acumular en la cavidad pleural, en la práctica clínica generalmente se excluye algunos tipos que no constituyen un trasudado de fluido, como son el empiema, el quilotórax y el hemotórax, pese a que en la radiografía de tórax suelen ser indiferenciables. Se necesita un derrame de al menos 250-600 ml para que sea evidente en una proyección posteroanterior en bipedestación. Aunque la proyección lateral puede ser más sensible para la detección de derrames pleurales de menor tamaño. Algunos de los hallazgos que sugieren la presencia de un derrame pleural en una radiografía de tórax son: pinzamiento de algún ángulo costofrénico o cardiofrénico, presencia de líquido en cisuras, signo del menisco o concavidad radiopaca, desplazamiento mediastínico hacia el pulmón sano (imagen 3). Imagen 3. Radiografía simple de tórax. Proyección PA. Derrame pleural derecho. Se registró la presencia o ausencia de derrame pleural, indistintamente de los hallazgos radiológicos con los que se manifestara. 43 3.2.4. Variables en tomografía computarizada 3.2.4.1. Hallazgos pulmonares: - Micronódulo pulmonar: En la TC, un micronódulo aparece como un aumento de densidad redondeada o irregular, bien o mal definida, que mide menos de 3 mm. La densidad del nódulo suele ser de tejidos blandos, aunque también puede presentar una atenuación en vidrio deslustrado. Las características de los micronódulos recogidas en este estudio son:  Número: Se clasificó a los micronódulos en únicos o múltiples, en función de la presencia de uno o más micronódulos por lóbulo pulmonar. Un caso puede ser considerado como micronódulo único cuando no existe más de un nódulo por lóbulo, incluso cuando hay nódulos aislados en varios lóbulos diferentes. La presencia de más de un nódulo en un lóbulo es suficiente para incluir el caso en la categoría de nódulo múltiple.  Localización: Pueden estar localizados en el lóbulo superior derecho, lóbulo medio o lóbulo inferior, en el pulmón derecho, y en el lóbulo superior izquierdo, língula y lóbulo inferior izquierdo, en el pulmón izquierdo. Establecemos un código numérico para diferenciar la localización de los micronódulos y así facilitar la recogida de datos relacionados con esta variable. A continuación se describe que localización traduce cada número: 1: lóbulo superior derecho 2: lóbulo medio 3: lóbulo inferior derecho 4: lóbulo superior izquierdo 5: língula 6: lóbulo inferior izquierdo En caso de encontrarse el micronódulo en más de una localización, se optó por escribir sus números de localización en orden secuencial.  Distribución: En el caso de los micronódulos múltiples, también describimos su distribución centrolobulillar, perilinfática o al azar. o Centrolobulillares: Centrolobulillar describe la región broncovascular central de un lobulillo pulmonar secundario. Se utiliza para referirse a la ubicación de las lesiones más allá del bronquiolo terminal o incluso los conductos alveolares. A diferencia de los nódulos al azar y de los perilinfáticos, evitan la 44 localización pleural. Además, este patrón suele asociar nódulos en "vidrio deslustrado". o Perilinfáticos: Este patrón se caracteriza por la presencia de nódulos alrededor de los vasos linfáticos en el pulmón. Dichos nódulos se encuentran en las rutas recorridas por los vasos linfáticos junto a los paquetes broncovasculares, en los septos interlobulillares, alrededor de las grandes venas pulmonares y en la pleura. Los alveolos no tienen linfáticos y por tanto son respetados por este patrón. o Al azar: Se distribuyen de modo irregular tanto a nivel pleural/subpleural como en el resto del pulmón. Es un patrón que se suele asociar a la propagación hematógena de la tuberculosis (patrón miliar) y de tumores. Se establece un sistema numérico para diferenciar en patrón de distribución de los micronódulos, según las equivalencias que se describen a continuación: 1: centrolobulillar 2: perilinfática 3: al azar (miliar) En caso de observarse más de un patrón de distribución, se registraron en orden secuencial de menor a mayor. - Nódulo pulmonar: A diferencia de los micronódulos, los nódulos pulmonares tienen un tamaño que oscila entre los 3 mm y los 3 cm. Entre las características del nódulo que recogimos para este estudio se encuentran: localización, halo, cavitación y calcificación.  Localización: Pueden estar localizados en el lóbulo superior derecho, lóbulo medio o lóbulo inferior, en el pulmón derecho, y en el lóbulo superior izquierdo, língula y lóbulo inferior izquierdo, en el pulmón izquierdo. Establecemos un código numérico para localizar los nódulos y así facilitar la recogida de datos relacionados con esta variable. A continuación se describe que localización traduce cada número: 1: lóbulo superior derecho 2: lóbulo medio 3: lóbulo inferior derecho 4: lóbulo superior izquierdo 5: língula 6: lóbulo inferior izquierdo 45 En caso de encontrarse el micronódulo en más de una localización, se optó por escribir sus números de localización en orden secuencial.  Cavitación: Se clasificó los nódulos en función de la presencia o ausencia de cavitación central.  Halo: Fueron identificados los nódulos que presentaban a su alrededor un halo en vidrio deslustrado.  Calcificación: Se registró la presencia o ausencia de la misma, independientemente del patrón de calcificación observado. - Masa pulmonar: La masa se define como una lesión pulmonar que mide más de 3 cm de diámetro. La TC permite una evaluación más exacta del tamaño, la ubicación, la atenuación y otras características, en comparación con otras técnicas como la radiografía. En este estudio registramos no sólo las masas y su localización, sino también la presencia de halo, calcificaciones o cavitaciones, determinando también si esta cavitación es ocupada por un micetoma o por líquido (sobreinfección).  Micetoma: Un micetoma es una masa constituida por hifas entrelazadas, generalmente de Aspergillus, que colonizan una cavidad pulmonar producida por una enfermedad fibrocavitaria previa (fundamentalmente tuberculosis, aunque también puede producirse en las sarcoidosis). Junto con estas hifas, se puede encontrar cantidades variables de moco, fibrina y restos celulares. Imagen 4. TC de tórax con CIV. En LSD se observa una cavitación ocupada por un micetoma (flecha blanca). También se objetiva un engrosamiento pleural bilateral de predominio izquierdo y algunas bronquiectasias en língula y LSI. 46 En TC, el micetoma se observa como una masa hiperdensa localizada en el interior de una cavidad. Puede mostrar un patrón morfológico espongiforme y focos de calcificación en su seno. Presenta como particularidad que se puede mover con el cambio de posición del paciente (imagen 4). - Consolidación pulmonar: En TC, la consolidación aparece como un aumento homogéneo en la atenuación del parénquima pulmonar que oculta los márgenes de los vasos y las paredes de las vías respiratorias. El broncograma aéreo, definido por la presencia de bronquios aireados (hipodensos) en el seno de una consolidación, puede estar presente. Imagen 5. TC de tórax con contraste. En el LII se observa una consolidación que presenta una cavitación en su seno. Áreas parcheadas en vidrio deslustrado en LM. En este trabajo fueron descritas las neumonías objetivadas en TC de tórax, especificando también si existía o no cavitación en su interior (imagen 5 y 6). - Tractos fibróticos cicatriciales: Las cicatrices fibróticas son un hallazgo común en las pruebas de imagen de tórax (138). Las cicatrices fibróticas se definen como lesiones en la radiografía de tórax de más de 5 mm y que sugieren la existencia de una tuberculosis previa no tratada. Generalmente se las describe como lesiones bien definidas e hiperdensas, consistentes en nódulos, imágenes lineales de fibrosis con o sin retracción y bronquiectasia, que usualmente se localizan en los lóbulos superiores, sin que exista evidencia de componente alveolar y / o cavitaciones. También pueden asociar un complejo primario calcificado, un engrosamiento pleural localizado y / o un ángulo costofrénico ocupado, pero estos hallazgos pueden considerarse menos significativos y están excluidos de algunas definiciones (139). 47 - Atelectasia: Se trata de una insuflación reducida de la totalidad o parte del pulmón. Esta alteración se caracteriza por una reducción del volumen pulmonar, acompañado por un aumento de la atenuación en la parte afecta del pulmón. La atelectasia se asocia a menudo con un desplazamiento anormal de cisuras, bronquios, vasos, diafragma, corazón, o el mediastino. La distribución puede ser lobular, segmentaria o subsegmentaria. Puede presentar una morfología lineal o redonda. En ocasiones presenta calcificaciones lineales, discoides o en forma de placa. - Engrosamiento septal: En la tomografía computarizada, cualquier enfermedad que afecta a uno de los componentes de los septos interlobulares puede ser responsable de su engrosamiento, permitiendo así que sean visibles en la TCAR. El engrosamiento septal puede ser de tipo liso o nodular. - Pérdida de volumen: Tras la infección tuberculosa se puede producir una pérdida de volumen pulmonar debido a la aparición de una reacción fibrótica, que se acompaña de retracción hiliar y de un desplazamiento mediastínico ipsilateral. Además, también puede detectarse una hiperinsuflación compensatoria del pulmón contralateral. Esta pérdida de volumen afecta con mayor frecuencia a los lóbulos superiores. Suele ser asintomática, sin embargo, en algunos pacientes puede asociarse a algunas manifestaciones como disnea y tos. - Enfisema: Se caracteriza por la presencia de espacios aéreos permanentemente dilatados, distales al bronquiolo terminal, con la destrucción de las paredes alveolares. La ausencia de "fibrosis aparente" históricamente fue considerada como un criterio adicional, pero la validez de este criterio ha sido cuestionada debido a que algunas fibrosis intersticiales pueden estar presentes en el enfisema secundario al consumo de cigarrillos. El enfisema se suele clasificar en función de la parte del acino predominantemente afectado: proximal (centroacinar), distal (enfisema paraseptal) o de todo el acino (panacinar). En TC, el enfisema se manifiesta como zonas focales de baja atenuación, por lo general, sin paredes visibles.  Enfisema centrolobular o centroacinar: es el tipo de enfisema más frecuente y está relacionado con el tabaquismo. El enfisema centrolobular de grado leve o moderado se caracteriza en la TC por la presencia de múltiples áreas redondas pequeñas con una atenuación anormalmente baja, de varios milímetros de diámetro, distribuidas por todo el pulmón, pero de predominio en los lóbulos superiores en la mayoría de las ocasiones. Las áreas de trasparencia rodean, generalmente, las arterias centrolobulares, en el centro de los lóbulos pulmonares. En la mayoría de las ocasiones, estas áreas de baja atenuación carecen de paredes visibles. 48 En los casos severos de enfisema centrolobular, las áreas de destrucción pulmonar pueden confluir, utilizándose en estos casos el término “enfisema centrolobular confluente”.  Enfisema paraseptal: se caracteriza por la afectación de la zona distal del lóbulo secundario y es, por tanto, más obvio en una localización subpleural. Las áreas de enfisema paraseptal subpleural tienen con frecuencia paredes visibles muy finas, que se corresponden con los septos interlobares. Cuando las áreas de enfisema paraseptal son mayores de 1 cm pasan a denominarse bullas.  Enfisema panlobular o panacinar: se asocia a un déficit de alfa1antitripsina. En este tipo de enfisema se produce una disminución de la atenuación más difusa, debido a una destrucción uniforme del lóbulo pulmonar. Aunque los cambios asociados al enfisema panlobular leve son difíciles de distinguir en ocasiones, no sucede así con la forma severa, en la que se aprecia una destrucción pulmonar extensa y una parquedad de trazos vasculares. Este tipo de enfisema suele predominar en los lóbulos inferiores.  Enfisema paracicatricial: se trata a una forma de enfisema irregular que afecta a cualquier parte del lóbulo y siempre va asociado a fibrosis. - Patrón de árbol en brote: Éste es un patrón de ramificación centrolobulillar que constituye una estructura que se asemeja a un árbol en ciernes. Refleja un espectro de trastornos endo y peribronquiolares, y que incluyen impactación mucoide, inflamación y / o fibrosis. Este patrón es más pronunciado en la periferia del pulmón y, por lo general, se asocia con anormalidades de las vías respiratorias más grandes. Es particularmente común en la panbronquiolitis difusa, en la propagación endobronquial de las infecciones por micobacterias y en la fibrosis quística (imagen 6). Imagen 6. TC de tórax con CIV. Micronódulos con distribución centrolobulillar en LM, língula y LII. En la periferia de dichos lóbulos se detecta un patrón de "árbol en brote". En el segmento posterior del LID se identifica una consolidación. 49 - Patrón en vidrio deslustrado: Este patrón es inespecífico y aparece tanto en enfermedades que afectan al espacio aéreo como en las que afectan al intersticio. Puede estar causado por un llenado parcial de los espacios aéreos, por un engrosamiento intersticial (debido al líquido, las células, y / o fibrosis), por un colapso parcial de los alvéolos, por un aumento de volumen sanguíneo capilar o por una combinación de éstos. El factor común a todas estas causas es el desplazamiento parcial de aire. En la tomografía computarizada, se manifiesta como una opacidad nebulosa en el parénquima pulmonar, que respeta los márgenes de los bronquios y de las estructuras vasculares. La opacidad en vidrio esmerilado es menos opaca que la consolidación, en la que están oscurecidos márgenes broncovasculares (imagen 6). - Bronquiectasias: Se define como una dilatación bronquial irreversible, localizada o difusa, que se produce, generalmente, como resultado de la infección crónica, obstrucción de la vía aérea proximal o de una malformación congénita bronquial. Existen también las bronquiectasias de tracción, producidas por una retracción pulmonar de naturaleza fibrótica. Para su diagnóstico radiológico existe una serie de criterios morfológicos que incluyen la dilatación del bronquio con respecto a la arteria pulmonar que lo acompaña (signo del anillo de sello), la falta de estrechamiento distal de los bronquios y la identificación de los bronquios a 1 cm o menos de la superficie pleural. En este estudio se recogió la presencia de bronquiectasia en TC, especificando si existían o no signos de sobreinfección (sugiere su sobreinfección el engrosamiento de la pared del bronquio y la presencia de niveles hidroaéreos en el interior de la bronquiectasia). La localización de las bronquiectasias se codificó numéricamente. A continuación se describe que localización traduce cada número: 1: lóbulo superior derecho 2: lóbulo medio 3: lóbulo inferior derecho 4: lóbulo superior izquierdo 5: língula 6: lóbulo inferior izquierdo En caso de encontrarse el micronódulo en más de una localización, se optó por escribir sus números de localización en orden secuencial. 50 3.2.4.2. Hallazgos mediastínicos: - Adenopatías: Se incluye la presencia de número anormal de los nodos linfáticos, o una alteración de su arquitectura interna (algunos criterios independientes del tamaño que conducen a pensar que una adenopatía puede ser patológica son la pérdida del hilio graso, la transformación quística o la existencia de focos de necrosis). Además, el tamaño de los ganglios aceptado como normal no es igual en todas las cadenas linfáticas del organismo. Las adenopatías mediastínicas pueden asociarse a enfermedad parenquimatosa pulmonar, aunque no necesariamente. En este trabajo, como criterio métrico para definir las adenopatías mediastínicas, se consideró 10 mm el límite superior para los nodos linfáticos normales (diámetro transverso medido en su eje corto). Una vez identificada la adenopatía, se procedió a especificar su cadena ganglionar de pertenencia. Para ello, recurrimos a la clasificación más utilizada en radiología torácica, que es la de Mountain-Dressler, la cual se basa en la sección transversal de TC de tórax y usa términos aprobados por la AJCCUICC (American Joint Comitee on Cancer y la Unión Internacional contra el cancer) (140, 141).  Ganglios dentro de la pleura mediastínica: o Ganglios mediastínicos altos: por encima de línea imaginaria horizontal que pasa por borde superior de la vena braquicefálica. o Ganglios paratraqueales superiores: por encima de línea tangencial que pasa por encima del arco aórtico y por debajo del límite de los ganglios mediastínicos altos. o Ganglios prevasculares y retrotraqueales: ubicados por delante de los grandes vasos torácicos y por detrás de la tráquea. o Ganglios paratraqueales inferiores: por debajo de las cadenas paratraqueales superiores y por encima de una línea que cruza el borde superior del bronquio lobar superior izquierdo, medial al ligamento arterioso. o Ganglios subaórticos: en la ventana aorto-pulmonar, laterales al ligamento arterioso y proximales a la primera división de la arteria pulmonar izquierda. o Ganglios paraaórticos: anteriores y laterales a la aorta ascendente y cayado aórtico. o Ganglios subcarinales: por debajo de la carina de la tráquea. o Ganglios paraesofágicos: adyacentes a la pared del esófago, situados a ambos lados de la línea media. Deben ser diferenciados de los ganglios subcarinales. o Ganglios del ligamento pulmonar: ubicados dentro del ligamento pulmonar. Forman parte de este grupo aquellos ganglios localizados en contacto con la pared posterior e inferior de la vena pulmonar inferior. 51  Ganglios dentro de la pleura visceral: o Ganglios hiliares: por fuera de la reflexión de la pleura mediastínica. o Ganglios interlobares: localizados entre los bronquios lobares. o Ganglios lobulares: próximos al bronquio local distal. o Ganglios segmentarios: alrededor del bronquio segmentario. o Ganglios subsegmentarios: adyacentes al bronquio subsegmentario. Para la recogida de datos en nuestro estudio, se codificaron las estaciones ganglionares más representativas con las letras del alfabeto, de la forma que se expone a continuación: A: mediastino anterosuperior (ganglios mediastínicos altos). B: paratraqueales C: prevasculares y retrotraqueales D: subaórtica E: paraaórticas F: subcarinales G: paraesofágicas H: hiliares I: interlobares y lobulares. 3.2.4.3. Hallazgos pleuropericárdicos: - Derrame pleural: El derrame pleural se define por la presencia de una colección de líquido en el interior del espacio pleural. La tomografía computarizada es una técnica excelente para detectar pequeñas cantidades de líquido y también es capaz de identificar las causas intratorácicas subyacentes (por ejemplo, lesiones pleurales malignas o neoplasias pulmonares primarias), así como enfermedades subdiafragmáticas (por ejemplo, absceso subdiafragmático). Además, la TC también puede ayudar a distinguir entre un derrame pleural y un empiema pleural. - Empiema: Se refiere a un derrame pleural infectado y purulento, con frecuencia tabicado y que causa una colección pleural unilateral. Es una afección potencialmente mortal que requiere un diagnóstico y tratamiento rápido y oportuno. Cualquiera de estas características en la TC sugiere un empiema:  Engrosamiento hipercaptante de las hojas pleurales. 52     Burbujas de gas en pacientes que no han sido sometidos a toracocentesis recientemente. Tabicaciones pleurales evidentes. Consolidación asociada a la colección pleural. Infección asociada en partes blandas adyacentes (empiema necessitatis, absceso subdiafragmático, etc.). - Engrosamiento pleural: Engrosamiento pleural es un término descriptivo utilizado para definir cualquier forma de engrosamiento que implica ya sea a la pleura parietal o la visceral. Puede ser originada tanto por enfermedades pleurales benignas como malignas. El engrosamiento pleural también se puede clasificar en varios subtipos de acuerdo con la morfología:    Engrosamiento pleural focal o placas pleurales  placas pleurales calcificadas  placas pleurales no calcificadas Engrosamiento pleural difuso o engrosamiento pleural circunferencial Engrosamiento pleural nodular Las placas pleurales son zonas bien delimitadas de engrosamiento pleural, que conforman lesiones planas o con elevaciones nodulares que a menudo contienen calcificaciones. Las placas son de un espesor variable, que va desde menos de 1 cm hasta los 5 cm de diámetro, y son más fácilmente identificables en la TC que en las radiografías de tórax. En este estudio registramos cualquier tipo de engrosamiento pleural detectado por TC, independientemente de su naturaleza. También identificamos si el engrosamiento pleural asocia calcificación (paquipleuritis cálcica). - Derrame pericárdico: Los derrames pericárdicos ocurren cuando se acumula un exceso de líquido en el espacio pericárdico (un saco pericárdico normal contiene aproximadamente 30-50 ml de líquido). El grosor generalmente aceptado de un pericardio normal, medido en TC suele ser de 2 mm (142). El diagnóstico de derrame pericárdico en TC no suele ser problemático, siendo un hallazgo incidental frecuente en pacientes hospitalizados. En TC, se observa como una densidad líquida que circunscribe el corazón. 3.2.5. Evaluación microbiológica Una vez recibidas las muestras por el Servicio de Microbiología, se introdujeron los datos de cada paciente en el programa SIL. Tras esto, dichas muestras fueron trasladadas a la Sección de Micobacterias donde al día siguiente de su recepción fueron procesadas en bloque. Previo a su cultivo, se realizó un procesamiento primario con el fin de eliminar flora acompañante que podría interferir en el crecimiento de la micobacteria, concentrando así el inóculo 53 micobacteriano. La mayoría de muestras fueron sometidas a un pretratamiento (homogeneización, descontaminación y concentración) basado en el método Kúbica (143). Esta técnica se basa en la combinación de dos reactivos: a) hidróxido sódico (NaOH) al 2% (descontaminante por sus propiedades alcalinas); y el b) N-acetil-L-cisteina (mucolítico imprescindible para la disgregación de la matriz orgánica de la muestra y liberación de los microorganismos). No se realizó este procesamiento previo en los líquidos estériles, en cuyo caso sólo se utilizó la centrifugación para su concentración. Tras la descontaminación y concentración de las muestras, del sedimento obtenido se introdujeron 0.2 ml en el medio sólido Lowënstein-Jensen (BBLTM Lowenstein-Jensen, Becton Dickinson) y 0.5 ml en el medio líquido Middlebrook 7H9 (BBL MGIT, Becton Dickinson) suplementado previamente con BD BACTECTM MGITTM 960 supplement kit (Becton Dickinson). Los tubos de Lowënstein-Jensen fueron incubados durante un periodo de 42 días en una estufa a 37 ºC, procediendo a su lectura una vez por semana para detectar la presencia de crecimiento. Los tubos MGIT se incubaron en el sistema automático para el cultivo y antibiograma de micobacterias BACTECTM MGITTM 960 System (Becton Dickinson). Este sistema realiza lecturas cada hora y detecta el crecimiento de las micobacterias a través de su consumo de oxígeno. Los tubos MGIT contienen una matriz que emite fluorescencia cuando no hay oxígeno o la concentración de éste disminuye. Después de detectar crecimiento en los medios de cultivo, se procedió a descartar la contaminación bacteriana mediante una extensión, con posterior tinción con Ziehl Neelsen (Tec-Laim S.A.) y resiembra en agar chocolate PolyVitex (Bio Merieux). En caso de observar ácido-alcohol resistencia la identificación de las micobacterias se realizó hasta finales del año 2009 con un kit comercial de hibridación de ADN (Accu Probe SYSTEM, GEN-PROBE), el cual utiliza una sonda de ADN que hibrida con el ARN ribosomal específico de M. tuberculosis complex. A partir de esa fecha, la identificación se realizó mediante detección del antígeno MPT64 (BD MGIT TBc Identification Test, Becton Dickinson). Si en este test con el antígeno MPT64 es positivo, se identificó al germen como Mycobacterium Tuberculosis Complex, y si el test fue negativo y no detectó el antígeno MPT64 en la muestra, se identificó al germen como micobacteria atípica. Tras la detección de micobacteria atípica, se envió el informe al médico peticionario para que valorara la situación clínica y para que obtuviera una nueva muestra. Si esta nueva muestra no conseguía aislar la micobacteria atípica, se consideró que se tratataba de una micobacteria no productora de enfermedad. Al contrario, si en esa nueva muestra se repitió el aislamiento de micobacteria atípica, se procedió a identificar la especie, utilizando el kit Genotype Mycobacterium CM (Hain lifescience gmbh). 3.2.6. Análisis estadístico Los análisis estadísticos se realizaron utilizando el programa R versión 3.3.2. Los datos continuos como la edad se presentaron como media ± desviación estándar (DE). Se utilizó la prueba t de student para comprobar si las medias de dos poblaciones distribuidas en forma normal son iguales. Los datos 54 categóricos se presentaron como el número con el porcentaje (%), y se utilizó la prueba de Chi-cuadrado de Pearson o la prueba exacta de Fisher para comparar las diferencias en la distribución de los datos categóricos entre dos grupos (G1 y G2, G3 y G4, G5 y G6). También se realizó la comparación de proporciones entre los grupos con cultivo positivo para Mycobacterium tuberculosis y los que lo tenían negativo, así como entre los grupos con cultivo positivo para tuberculosis y aquellos cuyo cultivo era positivo para micobacterias atípicas. Se practicaron análisis de regresión logística multivariante para determinar los predictores de tuberculosis pulmonar activa con cultivo positivo. Los potenciales predictores de cultivo positivo para tuberculosis fueron identificados en el análisis univariante. Para depurar el modelo resultante se utilizó un proceso de eliminación hacia atrás basado en el AIC, y las variables con p <0,05 se utilizaron para derivar un sistema de puntuación basado en las estimaciones obtenidas en el modelo. Como resultados del modelo de regresión logística multivariante, se presentaron los parámetros estimados (β) con su error estándar (EE) junto con un intervalo de confianza del 95% (IC) y los correspondientes Odds Ratio (OR). Además, se determinó una puntuación relativa usando el β estimado como base. La puntuación relativa se obtuvo redondeando el valor de β estimado al entero más próximo. Si las variables no fueron incluidas en el sistema de puntaje predictivo, se les asignó una puntuación de 0. Se determinó el área bajo la curva ROC, y se usó el índice de Youden (sensibilidad + especificidad - 1) para indicar el mejor punto de corte. Todos los análisis estadísticos se consideraron significativos a p <0,05. 55 56 4. RESULTADOS 57 58 4.1. ANÁLISIS DESCRIPTIVO DE LAS VARIABLES EPIDEMIOLÓGICAS DE TODOS LOS PACIENTES Y DE SUS CARACTERÍSTICAS EPIDEMIOLÓGICAS, CLÍNICAS Y RADIOLÓGICAS Se han incluido es este estudio a todos los pacientes que poseen una TC de tórax y un cultivo de tuberculosis realizados durante el periodo del 1 de enero de 2005 y el 31 de Diciembre de 2014 en Hospital Universitario General de Castellón. Se llevaron a cabo 1.549 estudios de TC de tórax en pacientes con sospecha de tuberculosis pulmonar. Se analizaron en este punto las variables epidemiológicas de todos los pacientes en conjunto así como las características clínicas y radiológicas individuales. 4.1.1. VARIABLES EPIDEMIOLÓGICAS  Edad: La edad media de los 1.549 pacientes en el momento de la realización de la TC fue de 54,44 + 19,90 años, con una mediana de 56 años. En la tabla 1a podemos observar la media de edad en los diferentes grupos. Edad G1 G2 G3 G4 G5 G6 G7 Media± DE 16 ± 0 14.39 ± 4.54 41.10 ± 10.58 43.35 ± 13.64 72.73 ± 7.49 75.47 ± 6.52 67.66 ± 13.43 Mediana 16 16.5 41 43 69 75 72 Tabla 1a. Media de edad en los diferentes grupos.  Grupos de edad: Los grupos con mayor número de pacientes fueron los correspondientes a pacientes con edad media o avanzada y que tuvieron un cultivo negativo (el grupo G4 y el G6, con 864 y 547 individuos, respectivamente). Con 84 pacientes, el G3 constituyó el grupo más poblado dentro de los que implicaban un cultivo positivo, seguido del grupo G5 con 15. Los grupos G1 y G2, con pacientes de edades comprendidas entre los 0 y los 18 años, tuvieron una representación muy discreta (2 y 28 pacientes, respectivamente). Algo similar sucedió con el grupo G7, conformado por pacientes con cultivo positivo para micobacterias atípicas y en el que sólo se incluyeron 9 personas (tabla 1b; figura 1). 59 Figura 1. Diagrama de sectores con porcentajes de cada grupo. Frecuencias (%) 2 (0.13%) 28 (1.81%) 84 (5.42%) 864 (55.78%) 15 (0.97%) 547 (35.31%) 9 (0.58%) 1549 G1 G2 G3 G4 G5 G6 G7 Total Tabla 1b. Número de pacientes según grupos.  Sexo: De todos los pacientes analizados, un 66.8 % fueron hombres, porcentaje que es significativamente mayor al 33.12 % conformado por las mujeres (pvalor<0.0001). En la tabla 2 y en las figura 2 se puede observar la distribución por grupos de los individuos de cada sexo. G1 G2 G3 G4 G5 G6 Hombres 0 15 (53.6%) 53 (63.1%) 564 (65.3%) 12 (80.0%) 386 (70.6%) 6 (66.7%) 1036 (66.88%) Mujeres 2 (100%) 13 (46.4%) 31 (36.9%) 300 (34.7%) 3 (20.0%) 161 (29.4%) 3 (33.3%) 513 (33.12%) G7 Total Tabla 2. Distribución de pacientes por sexos en función del grupo. 60 Figura 2. Gráfico de barras comparativo de sexos por grupo.  País de procedencia1: Grupo Grupo 1 2 3 4 5 6 Tot 1 2 3 4 5 6 Tot ALEMANIA 0 0 1 0 0 0 1 IRÁN 0 0 0 0 0 1 1 ARGELIA 0 0 1 1 0 0 2 ITALIA 0 0 0 2 0 0 2 0 0 2 1 0 1 4 JORDANIA 0 0 0 1 0 0 1 BOSNIA 0 0 0 1 0 0 1 LETONIA 0 0 0 0 0 1 1 BRASIL 0 0 1 3 0 0 4 LÍBANO 0 0 0 1 0 0 1 BULGARIA 0 0 0 2 0 0 2 MARRUECOS 0 2 0 22 0 4 28 CHILE 0 0 0 1 0 1 2 MEXICO 0 0 2 5 0 0 7 CHINA 0 0 1 3 0 0 4 MOLDAVIA 0 0 1 0 0 0 1 COLOMBIA 0 0 1 8 0 0 9 NIGERIA 0 0 0 1 0 0 1 EGIPTO 0 0 0 1 0 0 1 PAKISTÁN 0 0 0 2 0 0 2 ESCOCIA 0 0 0 1 0 0 1 PARAGUAY 0 0 0 1 0 0 1 ESPAÑA 2 24 59 714 19 527 1345 PERÚ 0 0 1 5 0 2 8 ESTONIA 0 0 0 1 0 0 1 POLONIA 0 0 0 1 0 0 1 ARGENTINA 1 Continúa en la siguiente página. 61 FILIPINAS 0 0 1 0 0 0 1 PORTUGAL 0 0 0 2 0 0 2 FRANCIA 0 0 0 5 0 0 5 RUMANÍA 0 1 13 66 1 4 85 GIBRALTAR 0 0 0 1 0 0 1 RUSIA 0 0 1 2 0 0 3 GRECIA 0 0 0 1 0 0 1 SENEGAL 0 0 2 1 0 0 3 GUINEA ECUATORIAL 0 1 0 1 0 0 2 UCRANIA 0 0 0 1 0 0 1 HAITI 0 0 0 1 0 0 1 USA 0 0 0 2 0 2 4 HUNGRÍA 0 0 0 1 0 0 1 VENEZUELA 0 0 0 0 0 1 1 INGLATERRA 0 0 1 1 0 3 5 ZAMBIA 0 0 1 0 0 0 1 Tabla 3a. Países de procedencia. El país de procedencia que más se repitió en este estudio fue España, observándose en 1345 de los 1549 analizados. A mucha distancia se encontraron otros países como Rumanía (85 pacientes) y Marruecos (28 pacientes), que ocuparon el segundo y tercer puesto, respectivamente, con una distribución por grupos como muestra la tabla 3b. G1 G2 G3 G4 G5 G6 Tot ESPAÑA 2 24 59 714 19 527 1345 (86.83%) RUMANÍA 0 1 13 66 1 4 85 (5.49%) MARRUECOS 0 2 0 22 0 4 28 (1.81%) Tabla 3b. Distribución por grupos de los pacientes pertenecientes a alguno de los tres países de procedencia más habituales.  Institucionalización en centros penitenciarios: En el momento de ser sometidos a estudio, sólo el 1.68% del total de pacientes estaba institucionalizado en centros penitenciarios. G1 G2 G3 G4 G5 G6 G7 Si 0 0 3 (3.4%) 20 (2.3%) 0 3 (0.5%) 0 26 (1.68%) No 2 (100%) 28 (100%) 81 (96.4%) 844 (97.7%) 15 (100%) 544 (99.5%) 9 (100%) 1523 (98.32%) Total Tabla 4. Pacientes institucionalizados en centros penitenciarios. 62 1000 900 800 700 600 500 400 300 200 100 0 SI NO G1 G2 G3 G4 G5 G6 G7 Figura 3. Gráfico de columnas apiladas que muestra los valores absolutos de pacientes institucionalizados en cárceles.  Anemia: G1 G2 G3 G4 G5 G6 Si No 0 3 (10.7%) 2 (2.2%) 123 (14.3%) 2 (10.0%) 96 (17.6%) 0 226 (14.59%) 2 (100%) 25 (89.3%) 82 (97.6%) 741 (85.8%) 13 (86.7 %) 451 (82.4%) 9 (100%) 1323 (85.41%) p-valor comparación proporciones 1 0.004 0.9634 G7 Total Tabla 5a. Comparación por grupos de las proporciones de pacientes con anemia. El porcentaje de pacientes con anemia en el grupo 4 fue significativamente mayor que el porcentaje de pacientes con anemia en el grupo 3. Por tanto, se obtuvo una evidencia estadística que explicaba una menor presencia de anemia en el grupo con cultivo positivo para Mycobacterium tuberculosis para la franja de edad entre los 18 y los 65 años. ANEMIA G1+G3+G5 G7 SI 4 (4%) 0 NO 97 (96%) 9 (100%) p-valor=1 Tabla 5b. Comparación de las proporciones de pacientes con anemia entre los grupos con cultivo positivo para M. tuberculosis y el grupo de M. atípicas. Se compara la proporción de pacientes con anemia entre aquellos grupos que tuvieron un cultivo positivo para micobacterias tuberculosas (G1+G3+G5) y 63 aquellos cuyo cultivo es positivo para micobacterias atípicas (G7), no obteniendo diferencias estadísticamente significativas. TBC No (G2+G4+G6) 222 (15.4%) 1217 (84.6%) p-valor= 0.002676. RR de tener TBC debido a tener anemia=0.2397591 I.C. 95%: [0.0891 – 0.6452] Odds ratio: 0.2347 (I.C. 95%) (0.0699 - 0.5683) p-val:0.0004 Anemia Si (G1+G3+G5) 4 (4%) 97 (96%) Si No Tabla 5c. Comparación de las proporciones de pacientes con anemia entre los grupos con y sin cultivo positivo. Se sumaron los valores de todos los grupos que implicaban un cultivo positivo para tuberculosis (G1+G3+G5), por una parte, y aquellos que no (G2+G4+G6), por la otra, y se compararon. Con ello se concluyó que el porcentaje de pacientes con anemia en el grupo con tuberculosis era significativamente menor que el porcentaje de anemia en el grupo sin tuberculosis.  Antecedente de tuberculosis pulmonar previa: G1 G2 G3 G4 G5 G6 G7 Si No 0 1 (3.6%) 8 (9.5%) 10 (1.2%) 3 (20.0%) 12 (2.2%) 0 34 (2.19%) 2 (100%) 27 (96.4%) 76 (90.5%) 854 (98.8%) 12 (80.0%) 535 (97.8%) 9 (100%) 1515 (97.81%) p-valor comparación proporciones 1 <0.0001 0.0006 Total Tabla 6a. Comparación por grupos de la proporción de pacientes con tuberculosis previa. El porcentaje de pacientes con antecedente de tuberculosis pulmonar en el grupo G3 fue significativamente mayor que el observado en el grupo G4. Algo similar sucede en la comparación entre los grupos G5 y G6, observándose un porcentaje de pacientes con antecedente de tuberculosis pulmonar significativamente mayor en el grupo G5 con respecto al G6. TBC previa G1+G3+G5 G7 SI NO 11 (10.9%) 90 (89.1%) 0 9 (100%) p-valor=0.6428 Tabla 6b. Comparación de las proporciones de pacientes con tuberculosis previa entre los grupos con cultivo positivo para M. tuberculosis y el grupo de M. atípicas. 64 TBC No (G2+G4+G6) 23 (1.6%) 1416 (98.4%) p-valor <0.0001 RR de tener TBC debido a tener TBC previa =5.4137 I.C. 95%: [3.2002-9.1581] Odds ratio: 7.55 (I.C. 95%) (3.4226 - 15.7423) p-val<0.0001 TBC previa Si (G1+G3+G5) 11 (10.9%) 90 (89.1%) Si No Tabla 6c. Comparación de la proporción de pacientes con antecedentes de tuberculosis entre los grupos con y sin cultivo positivo. Se compara las proporciones de antecedentes de tuberculosis entre los grupos con cultivo positivo (G1+G3+G5) y aquellos que no lo tenían (G2+G4+G6), observándose diferencias estadísticamente significativas. Lo cual implica que fue más probable detectar cultivo positivo en pacientes con antecedentes de tuberculosis pulmonar previa. No hay diferencias entre los grupos con cultivo positivo para micobacterias tuberculosas (G1+G3+G5) y el de micobacterias atípicas (G7).  Antecedente de cirugía: CAVIDAD BUCAL G1 0 G2 0 G3 1 G4 1 G5 0 G6 0 G7 0 COLORECTAL 0 0 0 2 0 3 0 ESÓFAGO ESTÓMAGO 0 0 0 0 0 0 1 0 0 0 0 1 0 0 HÍGADO 0 0 0 0 1 0 0 LARINGE 0 0 0 6 1 2 0 MAMA PRÓSTATA 0 0 0 0 0 0 3 1 0 0 3 2 0 1 PULMÓN 0 0 0 4 0 6 0 TIROIDES VEJIGA 0 0 0 0 0 0 1 1 0 0 0 1 0 0 SIN CIRUGÍA PREVIA 2 28 83 844 13 530 8 Tabla 7a. Pacientes con antecedentes de cirugía. Los pacientes con antecedente de cirugía, así como sus localizaciones, pueden observarse en la tabla 7a. Las cirugías más frecuentes registradas fueron las de pulmón (n=10) y de laringe (n=9). G1 G2 G3 G4 G5 G6 G7 Total Si No 0 0 1 (1.2%) 20 (2.3%) 2 (13.3%) 17 (3.1%) 1 (11.1%) 41 (2.65%) 2 (100%) 28 (100%) 83 (98.8%) 844 (97.7%) 13 (86.7%) 530 (96.9%) 8 (88.9%) 1508 (97.35%) p-valor comparación proporciones 1 0.0875 Tabla 7b. Comparación por grupos de las proporciones de pacientes con antecedente de cirugía. 65 Si analizamos únicamente la presencia de cirugía, independientemente de su localización, con una confianza del 95%, se puede decir que no hay diferencias significativas entre los porcentajes de pacientes con cirugías previas entre los grupos que comparamos. Cirugía previa G1+G3+G5 G7 SI NO 3 (3.0%) 98 (97.0%) 1 (11.1%) 8 (88.9%) p-valor=0.2928 (*) Tabla 7c. Comparación de las proporciones de pacientes con antecedente de cirugía entre los grupos con cultivo positivo para M. tuberculosis y el grupo de M. atípicas. (*) Test exacto de Fisher. TBC No (G2+G4+G6) 37 (2.6%) 1402 (97.4%) p-valor 0.7431 RR de tener TBC debido a cirugía previa = 1.1479I.C. 95%: [0.3802-3.4660] Odds ratio: 1.2142 (I.C. 95%) (0.2791-3.4506) p-val= 0.7594 Cirugía previa Si No Si (G1+G3+G5) 3 (3.0%) 98 (97.0%) Tabla 7d. Comparación de la proporción de pacientes con antecedentes de cirugía entre los grupos con y sin cultivo positivo. Al comparar las proporciones de antecedentes de cirugía entre los grupos con cultivo positivo (G1+G3+G5) y aquellos que no lo tenían (G2+G4+G6), no se detectó diferencias estadísticamente significativas. Tampoco fueron observadas diferencias entre los grupos con cultivo positivo para M. tuberculosis y el de micobacterias atípicas.  Antecedente de trasplante: G1 G2 G3 G4 G5 G6 G7 Total No 2 (100%) 28 (100%) 84 (100%) 858 (99.3%) 15 (100%) 545 (99.6%) 9 (100%) 1541 (99.48%) Hepático 0 0 0 2 (0.2%) 0 1 (0.2%) MO 0 0 0 0 0 1 (0.2%) Pulmón 0 0 0 2 (0.2%) 0 0 Renal 0 0 0 2 (0.2%) 0 0 3 (0.19%) 1 (0.06%) 2 (0.13%) 2 (0.13%) Tabla 8a. Antecedentes de trasplante por grupo. MO= Médula Ósea. Se agruparon las muestras, separando a los pacientes en función de la presencia o ausencia de antecedente de trasplante, con el objeto de buscar diferencias entre ambos grupos. 66 G1 G2 G3 G4 G5 G6 G7 Si No p-valor comparación proporciones (*) 0 0 0 6 (0.7%) 0 2 (0.4%) 0 8 (0.52%) 2 (100%) 28 (100%) 84 (100%) 858 (99.3%) 15 (100%) 545 (99.6%) 9 (100%) 1541 (99.48%) 1 1 Total Tabla 8b. Comparación por grupos de las proporciones de pacientes con antecedente de trasplante. (*) Test exacto de Fisher. Con una confianza del 95%, no se detectaron diferencias significativas entre los porcentajes de pacientes con trasplantes entre los grupos que comparamos. SI 0 0 Trasplante G1+G3+G5 G7 NO 101 (100%) 9 (100%) Tabla 8c. Comparación de las proporciones de pacientes con antecedente de trasplante entre los grupos con cultivo positivo para M. tuberculosis y el grupo de M. atípicas. (*) Test exacto de Fisher. TBC Trasplante Si (G1+G3+G5) 0 101 (100%) Si No p-valor No (G2+G4+G6) 8 (0.6%) 1431 (99.4%) 1 (*) Tabla 8d. Comparación de la proporción de trasplantes entre pacientes con y sin cultivo positivo. (*) Test exacto de Fisher. Tampoco se obtuvieron diferencias significativas entre los porcentajes de trasplante al comparar los grupos con cultivo positivo (G1+G3+G5) y aquellos que no lo tenían (G2+G4+G6). Tampoco fueron observadas diferencias entre los grupos con cultivo positivo para M. tuberculosis y el de micobacterias atípicas.  Diabetes: G1 G2 G3 G4 G5 G6 G7 Si No 0 2 (7.1%) 5 (6.0%) 44 (5.1%) 5 (33.3%) 97 (17.7%) 4 (44.4%) 157 (10.14%) 2 (100%) 26 (92.9%) 79 (94.0%) 820 (94.9%) 10 (66.7%) 450 (82.3%) 5 (55.6%) 1392 (89.86%) p-valor comparación proporciones 1 0.9349 0.2274 Total Tabla 9a. Comparación por grupos de las proporciones de pacientes con diabetes. 67 Con una confianza del 95% no se detectaron diferencias significativas entre los porcentajes de pacientes con diabetes entre los grupos que comparamos. SI DIABETES G1+G3+G5 G7 NO 10 (9.9%) 91 (90.1%) 4 (44.4%) 5 (55.6%) p-valor=0.014 Tabla 9b. Comparación de las proporciones de pacientes con diabetes entre los grupos con cultivo positivo para M. tuberculosis y el grupo de M. atípicas. Se compararon los porcentajes de pacientes con diabetes entre los pacientes con cultivo positivo para M. tuberculosis y aquellos que presentaron cultivo positivo para M. atípicas, obteniéndose diferencias estadísticamente significativas en favor de estas últimas. Ello sugiere que hay una mayor presencia de diabetes en pacientes con infección pulmonar por micobacterias atípicas en comparación con aquellos que presentan una infección por micobacterias tuberculosas. TBC No (G2+G4+G6) 143 (9.9%) 1296 (90.1%) p-valor= 1 RR de tener TBC debido a tener anemia=0.996 I.C. 95%: [0.5299 – 1.8728] Odds ratio: 1.0094 (I.C. 95%) (0.4812 – 1.8967) p-val:0.9784 Diabetes Si No Si (G1+G3+G5) 10 (9.9%) 91 (90.1%) Tabla 9c. Comparación de la proporción de pacientes con diabetes entre los grupos con y sin cultivo positivo. No se observa diferencias significativas entre los grupos con cultivo positivo y negativo para M tuberculosis en cuanto a la proporción de diabetes.  Alcoholismo: G1 G2 G3 G4 G5 G6 G7 Si No 0 1 (3.6%) 14(16.7%) 55 (6.4%) 2 (13.3%) 36 (6.6%) 1 (11.1%) 109 2 (100%) 27 (96.4%) 70 (83.3%) 809 (93.6%) 13 (86.7%) 511(93.4%) 8 (88.9%) 1440 p-valor comparación proporciones 1 0.0012 0.6126 Total Tabla 10a. Comparación por grupos de las proporciones de pacientes con alcoholismo. 68 El porcentaje de pacientes con alcoholismo en el grupo G3 fue significativamente mayor que el porcentaje de pacientes con alcoholismo en el grupo G4. ALCOHOLISMO G1+G3+G5 G7 SI 85 (84.2%) 8 (88.9%) NO 16 (15.8%) 1 (11.1%) p-valor=1 Tabla 10b. Comparación de las proporciones de pacientes con alcoholismo entre los grupos con cultivo positivo para M. tuberculosis y el grupo de M. atípicas. TBC No (G2+G4+G6) 92 (6.4%) 1347 (93.6%) p-valor= 0.00069 RR de tener TBC debido al alcoholismo=2.4959 I.C. 95%: [1.5183 – 4.1027] Odds ratio: 2.7726 (I.C. 95%) (1.5089-4.8169) p-val:0.0016 Alcoholismo Si No Si (G1+G3+G5) 16 (15.8%) 85 (84.2%) Tabla 10c. Comparación de la proporción de pacientes con alcoholismo entre los grupos con y sin cultivo positivo. No se observaron diferencias estadísticamente significativas entre los grupos con cultivo positivo para M. tuberculosis (G1+G3+G5) y el que implicaba un cultivo positivo para micobacterias atípicas (G7). Por el contrario, sí que se obtuvieron diferencias significativas entre los porcentajes de alcoholismo al comparar los grupos con cultivo positivo para M. tuberculosis (G1+G3+G5) y aquellos que no lo tenían (G2+G4+G6), con un riesgo relativo de tener tuberculosis debido al alcoholismo de 2.49.  Terapia con fármacos esteroideos: G1 G2 G3 G4 G5 G6 G7 Si No 0 0 1 (1.2%) 13 (1.5%) 1 (6.7%) 15 (2.7%) 1 (11.1%) 31 (2.0%) 2 (100%) 28 (100%) 83 (98.8%) 851(98.5%) 14 (93.3%) 532 (97.3%) 8 (88.9%) 1518 (98.0%) p-valor comparación proporciones (*) 1 0.3552 Total Tabla 11a. Comparación por grupos de las proporciones de pacientes con tratamiento esteroideo. (*) test exacto de Fisher. Con una confianza del 95% no se detectan diferencias significativas entre los porcentajes de pacientes con consumo de esteroides frente a los pacientes que no los consumían entre los grupos que comparamos. 69 ESTEROIDES G1+G3+G5 G7 SI NO 2 (2.0%) 99 (98.0%) 1 (11.1%) 8 (88.9%) p-valor=0.5867 Tabla 11b. Comparación de las proporciones de pacientes con tratamiento esteroideo entre los grupos con cultivo positivo para M. tuberculosis y el grupo de M. atípicas. TBC Esteroides Si No Si (G1+G3+G5) 2 (2%) 99 (98%) p-valor= RR de tener TBC debido al consumo de esteroides=1.0168 Odds ratio: 1.0894 (I.C. 95%) (0.1613 – 3.7112) No (G2+G4+G6) 28 (1.9%) 1411 (98.1%) 1 I.C. 95%: [0.1613 – 3.7112] p-val:0.9105 Tabla 11c. Comparación de las proporciones de pacientes con tratamiento esteroideo entre los grupos con y sin cultivo positivo. No se observaron diferencias estadísticamente significativas en cuanto al consumo de esteroides entre los grupos con cultivo positivo para M. tuberculosis (G1+G3+G5) y el que implicaba un cultivo positivo para micobacterias atípicas (G7). Tampoco se evidenciaron diferencias significativas entre los grupos con cultivo positivo para tuberculosis (G1+G3+G5) y los que no (G2+G4+G6).  Enfermedades hematológicas: G1 G2 G3 G4 G5 G6 G7 0 0 0 1 0 1 0 0 0 0 5 0 1 0 0 2 1 6 0 2 0 0 0 0 3 0 0 0 0 0 0 1 0 2 0 0 0 0 4 0 5 0 0 0 0 1 0 1 0 0 0 0 3 0 5 0 0 0 0 1 0 1 0 0 0 0 1 0 0 0 APLASIA MEDULAR LINFOMA HODGKIN LINFOMA NO HODGKIN LEUCEMIA LINFOIDE AGUDA LEUCEMIA LINFOIDE CRÓNICA LEUCEMIA MIELOIDE AGUDA LEUCEMIA MIELOIDE CRÓNICA MIELOMA MÚLTIPLE PANCITOPENIA MACROGLOBULINEMIA DE WALDESTROM SÍNDROME MIELODISPLÁSICO 0 0 0 1 0 0 0 Tabla 12a. Tipos de enfermedades hematológicas registradas en el estudio. Registramos en cada uno de los grupos el número de pacientes que ha padecido alguna enfermedad hematológica, independientemente de su tipo. Tras esto, comparamos las proporciones de anemia entre grupos de la misma franja de edad. 70 Si (alguna enf. Hematológica) 0 2 (7.1%) 1 (1.2%) 26 (3.0%) 0 20 (3.7%) 0 49 G1 G2 G3 G4 G5 G6 G7 No p-valor comparación proporciones (*) 1 2 (100%) 26 (92.9%) 83 (98.8%) 838 (97.0%) 15 (100%) 527 (96.3%) 9 (100%) 1500 0.5031 1 Total Tabla 12b. Comparación por grupos de las proporciones de pacientes con enfermedad hematológica. (*) test exacto de Fisher. Con una confianza del 95% no se detectaron diferencias significativas entre los porcentajes de pacientes con enfermedades hematológicas entre los grupos que comparamos. Enf. hem. G1+G3+G5 G7 SI 1 (1%) 0 p-valor=1 NO 100 (99%) 9 (100%) (*) Tabla 12c. Comparación de las proporciones de pacientes con enfermedad hematológica entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. TBC No (G2+G4+G6) 48 (3.3%) 1391 (96.7%) p-valor= 0.3149 RR de tener TBC debido Enf. hem.=0.3043 I.C. 95%: [0.0433 – 2.1367] Odds ratio: 0.3307 (I.C. 95%) (0.0141 – 1.5154) p-val:0.1895 Enf. hem. Si No Si (G1+G3+G5) 1 (1%) 100 (99%) Tabla 12d. Comparación de las proporciones de pacientes con enfermedad hematológica entre los grupos con y sin cultivo positivo. Enf. Hem. = Enfermedades Hematológicas. No se evidenciaron diferencias en cuanto al porcentaje de pacientes con antecedente de enfermedad hematológica entre los grupos con cultivo positivo (G1+G3+G5) y aquellos que tuvieron un cultivo negativo (G2+G4+G6). Tampoco fueron observadas diferencias significativas entre los grupos con cultivo positivo para tuberculosis (G1+G3+G5) al compararlos con el grupo de pacientes con cultivo positivo para micobacterias atípicas (G7). 71  Enfermedad renal crónica (ERC): G1 G2 G3 G4 G5 G6 G7 Si No 0 0 2 (2.4%) 18 (2.1%) 2 (13.3%) 46 (8.4%) 1 (11.1%) 69 (4.45%) 2 (100%) 28 (100%) 82(97.6%) 846 (97.9%) 13 (86.7%) 501 (91.6%) 8 (88.9%) 1480 (95.54%) p-valor comparación proporciones (*) 1 0.6952 0.3718 Total Tabla 13a. Comparación por grupos de las proporciones de pacientes con enfermedad renal crónica. (*) test exacto de Fisher. Con una confianza del 95%, no se observaron diferencias significativas entre los porcentajes de pacientes con ERC entre los grupos que comparamos. ERC G1+G3+G5 G7 p-valor Si 4 (4%) 1 (11.1%) NO 97 (96%) 8 (88.9%) 0.8793 Tabla 13b. Comparación de las proporciones de pacientes con enfermedad renal crónica entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. TBC ERC Si No Si (G1+G3+G5) 4 (4%) 97 (96%) No (G2+G4+G6) 64 (4.4%) 1375 (95.6%) 1 p-valor= RR de tener TBC debido ERC=0.892 I.C. 95%: [0.34 –2.35] Odds ratio: 0.92 (I.C. 95%) (0.27 – 2.29) p-val=0.87 Tabla 13c. Comparación de las proporciones de pacientes con enfermedad renal crónica entre los grupos con y sin cultivo positivo. Tampoco se detectaron diferencias significativas en cuanto a los porcentajes de pacientes con ERC entre los grupos con cultivo positivo (G1+G3+G5) y los que tuvieron un cultivo negativo (G2+G4+G6), ni entre los cultivos positivos para M. tuberculosis y micobacterias atípicas. 72  Colagenopatías: G1 G2 G3 G4 G5 G6 G7 Artritis reumatoide 0 0 0 3 0 5 1 Arteritis temporal 0 0 0 0 0 1 0 Enf. Inflamatoria Intestinal 0 0 0 2 0 0 0 Espondilitis Anquilopoyetica 0 0 0 1 0 0 0 Fiebre Reumática 0 0 0 1 0 0 0 Panarteritis Nodosa (PAN) 0 0 0 1 0 1 0 Poliartritis reumatoidea 0 0 0 0 0 1 0 Síndrome Churg-Strauss 0 0 0 1 0 0 0 Síndrome CREST 0 0 0 1 0 0 0 Tabla 14a. Lista de colagenopatías observadas en el estudio. G1 G2 G3 G4 G5 G6 G7 Si No 0 0 0 10 (1.2%) 0 8 (1.5%) 1 (11.1%) 19 (1.23%) 2 28 84 854 (98.8%) 15 539 (98.5%) 8 (88.9%) 1530 (98.77%) p-valor comparación proporciones 1 1 1 Total Tabla 14b. Comparación por grupos de las proporciones de pacientes con colagenopatía. Con una confianza del 95%, no se observaron diferencias significativas entre los porcentajes de pacientes con colagenopatías entre los grupos comparados. Colagenopatías G1+G3+G5 G7 p-valor Si 0 1 (11.1%) NO 101 8 (88.9%) 0.082 Tabla 14c. Comparación de las proporciones de pacientes con colagenopatía entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. 73 TBC Colagenopatías Si (G1+G3+G5) 0 101 Si No No (G2+G4+G6) 18 (1.3%) 1421 (98.7%) p-valor= 0.5146 Tabla 14d. Comparación de las proporciones de pacientes con colagenopatía entre los grupos con y sin cultivo positivo. No se evidenciaron diferencias significativas en función de que los grupos presentaran o no un cultivo positivo para tuberculosis. No se apreciaron diferencias significativas entre los grupos con cultivo positivo para M. tuberculosis y aquellos que presentaban un cultivo positivo para micobacterias atípicas.  EPOC: G1 G2 G3 G4 G5 G6 G7 Si No 0 0 11 (13.1%) 62 (7.2%) 6 (40.0%) 85 (15.5%) 7 (77.8%) 172 2 (100%) 28 (100%) 73 (86.9%) 802 (92.8%) 9 (60.0%) 462 (84.5%) 2 (22.2%) 1377 p-valor comparación proporciones 1 0.0839 0.0223 Total Tabla 15a. Comparación por grupos de las proporciones de pacientes con EPOC. En la comparación entre el grupo G1 y el G2, así como al comparar los grupos G3 y G4, no se obtuvieron diferencias estadísticamente significativas en cuanto al porcentaje de pacientes con EPOC. Esta diferencia sí que fue significativa entre los grupos G5 y G6, siendo mayor el porcentaje de pacientes con EPOC en el G5 en comparación con el G6. EPOC G1+G3+G5 G7 SI NO 17 (16.8%) 84 (83.2%) 7 (77.8%) 2 (22.2%) p-valor=0.0001 Tabla 15b. Comparación de las proporciones de pacientes con EPOC entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. Fueron comparadas las proporciones de pacientes con EPOC entre los grupos con cultivo positivo para Mycobacterium tuberculosis (G1+G3+G5) y el grupo que representaba a los pacientes con un cultivo positivo para micobacterias atípicas (G7), obteniéndose una diferencia estadísticamente significativa en favor de este último. 74 TBC No (G2+G4+G6) 147 (10.2%) 1292 (89.8%) p-valor= 0.0553 RR de tener TBC debido al EPOC=1.6980 I.C. 95%: [1.0346 – 2.7869] Odds ratio: 1.7902 (I.C. 95%) (1.0011 – 3.0301) p-val:0.0496 EPOC Si (G1+G3+G5) 17 (16.8%) 84 (83.2%) Si No Tabla 15c. Comparación de las proporciones de pacientes con EPOC entre los grupos con y sin cultivo positivo. Al comparar las proporciones de pacientes con cultivo positivo para tuberculosis (G1+G3+G5) y en aquellos que mostraban un cultivo negativo, se observó un valor de p muy próximo al 0.05, en el límite de la significación estadística.  VIH: G1 G2 G3 G4 G5 G6 G7 Si No 0 0 7 (8.3%) 23 (2.7%) 0 1 (0.2%) 0 31 2 (100%) 28 (100%) 77 (91.7%) 841 (97.3%) 15 (100%) 546 (99.8%) 9 (100%) 1518 p-valor comparación proporciones (*) 0.0126 1 Total Tabla 16a. Comparación por grupos de las proporciones de pacientes con VIH. (*) test exacto de Fisher. Al comparar los grupos G3 y G4, observamos que el porcentaje de pacientes con VIH es significativamente mayor en el G3 en comparación con el G4. VIH G1+G3+G5 G7 SI 7 (6.9%) 0 NO 94 (93.1%) 9 (100%) p-valor=1 (*) Tabla 16b. Comparación de las proporciones de pacientes con VIH entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. (*) test exacto de Fisher. No se detectaron diferencias estadísticamente significativas entre los grupos con cultivo positivo para Mycobacterium tuberculosis y el grupo de pacientes con cultivo positivo para micobacterias atípicas. TBC No (G2+G4+G6) 24 (1.7%) 1415 (98.3%) p-valor= 0.0011 RR de tener TBC debido al VIH= 3.6249I.C. 95%: [1.8354 – 7.1592] Odds ratio: 4.4498 (I.C. 95%) (1.7133 – 10.1517) p-val:0.0036 VIH Si No Si (G1+G3+G5) 7 (6.9%) 94 (93.1%) Tabla 16c. Comparación de las proporciones de pacientes con VIH entre los grupos con y sin cultivo positivo. 75 Al contrario, sí que se apreciaron diferencias significativas entre los grupos con cultivos positivos para tuberculosis y los grupos con cultivo negativo, obteniéndose un riesgo relativo de tener tuberculosis debida al VIH de 3.6.  Tabaquismo: G1 G2 G3 G4 G5 G6 G7 Ex Si No 0 1 (3.6%) 4 (4.8%) 165 (19.1%) 1 (6.7%) 144 (26.3%) 1 (11.1%) 316 (20.40%) 0 2 (7.1%) 24 (28.6%) 247 (28.6%) 2 (13.3%) 8 (1.5%) 0 283 (18.27%) 2 (100%) 25 (89.3%) 56 (66.7%) 452 (52.3%) 12 (80.0%) 395 (72.2%) 8 (88.9%) 950 (61.33%) p-valor comparación proporciones (*) 1 0.0008 0.0079 Total Tabla 17a. Comparación por grupos de las proporciones de pacientes con hábito tabáquico. (*) test exacto de Fisher. Al comparar los grupos G1 y G2, no se observaron diferencias significativas de porcentajes entre los fumadores, no fumadores y exfumadores. Sí que se obtuvo una diferencia significativa al comparar el grupo G3 con el G4 y al comparar el grupo G5 con el G6, demostrándose un porcentaje de exfumadores significativamente mayor en el grupo G4, respecto al G3, y en el G6, respecto al G5. Dichos hallazgos sugieren que el abandono del hábito tabáquico puede ser un factor protector contra la infección por M. tuberculosis. TBC Fumador Ex Si No Si (G1+G3+G5) 5 (5.0%) 26 (25.7%) 70 (69.3%) p-valor= No (G2+G4+G6) 310 (21.5%) 257 (17.9%) 872 (60.6%) 0.0002 Tabla 17b. Comparación de las proporciones de pacientes con hábito tabáquico entre grupos con cultivo positivo y negativo para M. tuberculosis. G1+G3+G5 G7 p-valor Ex 5 (5%) 1 (11.1%) Si 26 (25.7%) 0 0.1884 No 70 (69.3%) 8 (88.9%) Tabla 17c. Comparación de las proporciones de pacientes con hábito tabáquico entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. Cuando se compararon los grupos de pacientes con cultivo positivo para tuberculosis con aquellos en los que no creció el cultivo, se observa diferencias estadísticamente significativas, con una mayor proporción de pacientes exfumadores en los grupos con cultivo negativo y una mayor proporción de pacientes con un hábito tabáquico activo en los grupos con cultivo positivo. No 76 se observaron diferencias significativas al comparar los grupos de pacientes con tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. 4.1.2. VARIABLES CLÍNICAS  Fiebre: G1 G2 G3 G4 G5 G6 G7 Si No p-valor comparación proporciones (*) 0 0 6 (7.1%) 44 (5.1%) 2 (13.3%) 22 (4.0%) 0 74 (4.78%) 2 (100%) 28 (100%) 78 (92.9%) 820 (94.9%) 13 (86.7%) 525 (96.0%) 9 (100%) 1475 (95.22%) 0.4385 0.1308 Total Tabla 18a. Comparación por grupos de las proporciones de pacientes con fiebre. (*) test exacto de Fisher. Con una confianza del 95% no se detectaron diferencias significativas entre los porcentajes de pacientes que presentaron fiebre entre los grupos que comparamos. Fiebre G1+G3+G5 G7 SI 8 (7.9%) 0 p-valor=1 (*) NO 93 (92.1%) 9 (100%) Tabla 18b. Comparación de las proporciones de pacientes con fiebre entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. (*) test exacto de Fisher. TBC No (G2+G4+G6) 66 (4.6%) 1373 (95.4%) p-valor= 0.1436 RR de tener TBC debido a fiebre=1.7041 I.C. 95%: [0.8605– 3.3750] Odds ratio: 1.8180 (I.C. 95%) (0.78012– 3.6995) p-val:0.1542 Fiebre Si No Si (G1+G3+G5) 8 (7.9%) 93 (92.1%) Tabla 18c. Comparación de las proporciones de pacientes con fiebre entre los grupos con y sin cultivo positivo. No se encontraron diferencias significativas en cuanto al porcentaje de pacientes con fiebre, al comparar los grupos con cultivo positivo para tuberculosis con el grupo de cultivo positivo para micobacterias atípicas ni tampoco al comparar los grupos con cultivo positivo para tuberculosis con los grupos con cultivo negativo. 77  Febrícula: G1 G2 G3 G4 G5 G6 G7 Si No p-valor comparación proporciones (*) 0 0 0 6 (0.7%) 0 2 (0.4%) 0 8 (0.52%) 2 (100%) 28 (100%) 84 (100%) 858 (99.3%) 15(100%) 545 (99.6%) 9 (100%) 1541 (99.48%) 1 1 Total Tabla 19. Comparación por grupos de las proporciones de pacientes con febrícula. (*) test exacto de Fisher. Con una confianza del 95% no se detectaron diferencias significativas entre los porcentajes de pacientes con febrícula entre los grupos que comparamos.  Pérdida de peso: G1 G2 G3 G4 G5 G6 G7 Si No p-valor comparación proporciones (*) 0 0 1 (1.2%) 6 (0.7%) 0 4 (0.7%) 1 (11.1%) 12 (0.77%) 2 28 83 (98.8%) 858 (99.3%) 15 (100.0%) 543 (99.3%) 8 (88.9%) 1537 (99.23%) 0.4788 1 Total Tabla 20a. Comparación por grupos de las proporciones de pacientes con pérdida de peso. (*) test exacto de Fisher. Con una confianza del 95% no se detectaron diferencias significativas entre los porcentajes de pacientes que presentaron pérdida de peso entre los grupos que comparamos. Pérdida peso G1+G3+G5 G7 SI NO 1 (1.0%) 100 (99.0%) 1 (11.1%) 8 (88.9%) p-valor=0.1576 (*) Tabla 20b. Comparación de las proporciones de pacientes con pérdida de peso entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. (*) test exacto de Fisher. TBC No (G2+G4+G6) 10 (0.7%) 1429 (99.3%) p-valor= 0.527 RR de tener TBC debido a la perdida peso=1.39 I.C. 95%: [0.2124 – 9.0942] Odds ratio: 1.6094 (I.C. 95%) (0.0647 – 8.6429) p-val:0.6863 Pérdida peso Si No Si (G1+G3+G5) 1 (1.0%) 100 (99.0%) Tabla 20c. Comparación de las proporciones de pacientes con pérdida de peso entre los grupos con y sin cultivo positivo para tuberculosis. 78 Tampoco se observaron diferencias significativas en cuanto al porcentaje de pacientes con pérdida de peso al comparar los grupos con tuberculosis (G1+G3+G5) con aquellos que no tenían (G2+G4+G6), ni al comparar los grupos con cultivo positivo (G1+G3+G5) con el grupo con cultivo positivo para M. atípicas (G7).  Tos: G1 G2 G3 G4 G5 G6 G7 Si No p-valor comparación proporciones 1 0 1 (3.6%) 5 (6.0%) 61 (7.1%) 2 (13.3%) 57 (10.4%) 0 126 (8.13%) 2 (100%) 27 (96.4%) 79 (94.0%) 803 (92.9%) 13 (86.7%) 490 (89.6%) 9 (100%) 1423 (91.87%) Con expectoración Sin expectoración No 0 1 (3.6%) 4 (4.8%) 37 (4.3%) 2 (13.3%) 44 (8.1%) 0 88 (5.68%) 0 0 1 (1.2%) 24 (2.8%) 0 13(2.4%) 0 38 (2.45%) 2 (100%) 27 (96.4%) 79 (94.0%) 803 (92.9%) 13 (86.7%) 490 (89.6%) 9 (100%) 1423 (91.87%) 1 0.667 Total Tabla 21a. Comparación por grupos de las proporciones de pacientes con tos. G1 G2 G3 G4 G5 G6 Total p-valor comparación proporciones (*) 1 0.8518 0.559 Tabla 21b. Comparación por grupos de las proporciones de pacientes con tos, según los tipos de tos (con expectoración o sin expectoración). (*) test exacto de Fisher. Con una confianza del 95% no se detectaron diferencias significativas entre los porcentajes de pacientes que presentaron tos entre los grupos que comparamos, ni siquiera al subdividir los grupos en función del tipo de tos (con expectoración/ sin expectoración). TOS G1+G3+G5 G7 p-valor Si 7 (6.9%) 0 NO 94 (93.1%) 9 0.9175 Tabla 21c. Comparación de las proporciones de pacientes con tos entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. 79 TBC TOS Si (G1+G3+G5) 7 (6.9%) 94 (93.1%) Si No No (G2+G4+G6) 119 (8.3%) 1320 (91.7%) 0.7743 p-valor= RR de tener TBC debido TOS=0.835 I.C. 95%: [0.39 –1.76] Odds ratio: 0.84 (I.C. 95%) (0.34 – 1.74) p-val=0.67 Tabla 21d. Comparación de las proporciones de pacientes con tos entre los grupos con y sin cultivo positivo para tuberculosis. No se encontraron diferencias significativas en cuanto al porcentaje de pacientes con fiebre, al comparar los grupos con cultivo positivo para tuberculosis con el grupo de cultivo positivo para micobacterias atípicas ni tampoco al comparar los grupos con cultivo positivo para tuberculosis con los grupos con cultivo negativo.  Dolor torácico: G1 G2 G3 G4 G5 G6 Si No 0 0 0 25 (2.9%) 1 (6.7%) 11 (2.0%) 0 37 (2.39%) 2 (100%) 28 (100%) 84 (100%) 839 (97.1%) 14 (93.3%) 536 (98.0%) 9 (100%) 1512 (97.61%) p-valor comparación proporciones (*) 1 0.1577 0.2796 G7 Total Tabla 22a. Comparación por grupos de las proporciones de pacientes con dolor torácico. (*) test exacto de Fisher. Con una confianza del 95%, no se detectaron tampoco diferencias significativas entre los porcentajes de pacientes que presentaron dolor torácico entre los grupos que comparamos. Dolor torácico G1+G3+G5 G7 SI 1 (1%) 0 NO 100 (99%) 9 (100%) p-valor=1 (*) Tabla 22b. Comparación de las proporciones de pacientes con dolor torácico entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. (*) test exacto de Fisher. D. torácico = dolor torácico. TBC Si (G1+G3+G5) 1 (1%) 100 (99%) No (G2+G4+G6) Dolor Si 36 (2.5%) torácico No 1403 (97.5%) p-valor= 0.5096 RR de tener TBC debido a Dolor torácico=0.41 I.C. 95%: [0.0582 – 2.8339] Odds ratio: 0.4442 (I.C. 95%) (0.0188 – 2.0638) p-val:0.3675 Tabla 22c. Comparación de las proporciones de pacientes con dolor torácico entre los grupos con y sin cultivo positivo para tuberculosis. D. torácico = dolor torácico. 80 No se detectaron diferencias estadísticamente significativas al agrupar a los pacientes en función del resultado de su cultivo.  Sudoración nocturna: G1 G2 G3 G4 G5 G6 G7 Si No 0 0 0 3 (0.3%) 0 1 (0.2%) 0 4 2 (100%) 28 (100%) 84 (100%) 861 (99.7%) 15 (100%) 546 (99.8%) 9 1545 p-valor comparación proporciones (*) 1 1 1 Total Tabla 23. Comparación por grupos de las proporciones de pacientes con sudoración nocturna. (*) test exacto de Fisher. Con una confianza del 95% no se detectaron diferencias significativas entre los porcentajes de pacientes con sudoración nocturna entre los grupos que comparamos.  Disnea: G1 G2 G3 G4 G5 G6 G7 Si 0 1 (3.6%) 2 (2.4%) 40 (4.6%) 1 (6.7%) 40 (7.3%) 1 (11.1%) 85 (5.49%) No 2 (100%) 27 (96.4%) 82 (97.6%) 824 (95.4%) 14 (93.3%) 507 (92.7%) 8 (88.9%) 1464 (94.51%) p-valor comparación proporciones (*) 1 0.57746 1 Total Tabla 24a. Comparación por grupos de las proporciones de pacientes con disnea. (*) test exacto de Fisher. Con una confianza del 95%, no se observaron diferencias significativas entre los porcentajes de pacientes que mostraron disnea entre los grupos que comparamos. Disnea G1+G3+G5 G7 SI NO 3 (3.0%) 98 (97.0%) 1 (11.1%) 8 (88.9%) p-valor=0.2928 (*) Tabla 24b. Comparación de las proporciones de pacientes con disnea entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. (*) test exacto de Fisher. 81 TBC No (G2+G4+G6) 81 (5.6%) 1358 (94.4%) p-valor= 0.3625 RR de tener TBC debido a la disnea=0.5306 I.C. 95%: [0.1718 – 1.6385] Odds ratio: 0.5387 (I.C. 95%) (0.1261 – 1.4765) p-val:0.2584 Disnea Si (G1+G3+G5) 3 (3.0%) 98 (97.0%) Si No Tabla 24c. Comparación de las proporciones de pacientes con disnea entre los grupos con y sin cultivo positivo para tuberculosis. No se observaron diferencias al dividir a los pacientes en función del resultado de su cultivo ni al distinguir entre pacientes con micobacterias tuberculosas y atípicas.  Hemoptisis: G1 G2 G3 G4 G5 G6 G7 Total Si No 0 0 5 (6.0%) 107 (12.4%) 3 (20.0%) 55 (10.1%) 2 (22.2%) 172 (11.10%) 2 (100%) 28 (100%) 79 (94.0%) 757 (87.6%) 12 (80.0%) 492 (89.9%) 7 (77.8%) 1377 (8.90%) p-valor comparación proporciones 0.1085 0.1945 Tabla 25a. Comparación por grupos de las proporciones de pacientes con hemoptisis. (*) test exacto de Fisher. Con una confianza del 95%, no se observaron diferencias significativas entre los porcentajes de pacientes que presentaron hemoptisis entre los grupos que comparamos. Hemoptisis G1+G3+G5 G7 SI NO 8 (7.9%) 93 (92.1%) 2 (22.2%) 7 (77.8%) p-valor=0.1896 (*) Tabla 25b. Comparación de las proporciones de pacientes con hemoptisis entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. (*) test exacto de Fisher. No se encontraron diferencias significativas en cuanto al porcentaje de pacientes con hemoptisis, al comparar los grupos con cultivo positivo para tuberculosis con el grupo con cultivo positivo para micobacterias atípicas. 82 TBC No (G2+G4+G6) 162 (11.3%) 1277 (88.7%) p-valor= 0.3842 RR de tener TBC debido a la hemoptisis=0.6932 I.C. 95%: [0.3428 – 1.4021] Odds ratio: 0.6902 (I.C. 95%) (0.3019 – 1.3648) p-val:0.3056 Hemoptisis Si No Si (G1+G3+G5) 8 (7.9%) 93 (92.1%) Tabla 25c. Comparación de las proporciones de pacientes con hemoptisis entre los grupos con y sin cultivo positivo para tuberculosis. Tampoco había diferencias significativas al comparar los grupos con cultivo positivo para tuberculosis con los grupos con cultivo negativo.  MEG (Mal Estado General): G1 G2 G3 G4 G5 G6 G7 Total Si No 0 0 0 6 (0.7%) 0 5 (0.9%) 0 11 (0.71%) 2 (100%) 28 (100%) 84 (100%) 858 (99.3%) 15 (100%) 542 (99.1%) 9 (100%) 1538 (99.29%) p-valor comparación proporciones 1 1 1 Tabla 26. Comparación por grupos de las proporciones de pacientes con MEG. No se objetivaron diferencias estadísticamente significativas entre grupos en cuanto al porcentaje de pacientes con mal estado general.  Síndrome constitucional: G1 G2 G3 G4 G5 G6 G7 Total Si No 0 0 3 (3.6%) 11 (1.3%) 1 (6.7%) 30 (5.5%) 1 (11.1%) 46 (2.97%) 2 (100%) 28 (100%) 81 (96.4%) 853 (98.7%) 14 (93.3%) 517 (94.5%) 8 (88.9%) 1503 (97.03%) p-valor comparación proporciones 1 0.1198 0.5778 Tabla 27a. Comparación por grupos de las proporciones de pacientes con síndrome constitucional. Con una confianza del 95%, no se detectaron diferencias significativas entre los porcentajes de pacientes que mostraron síndrome constitucional entre los grupos que comparamos. 83 SD C. G1+G3+G5 G7 SI NO 4 (4.0%) 97 (96.0%) 1 (11.1%) 8 (88.9%) p-valor=0.3528 (*) Tabla 27b. Comparación de las proporciones de pacientes con síndrome constitucional entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. (*) test exacto de Fisher. SD. C. = Síndrome Constitucional. TBC No (G2+G4+G6) 41 (2.8%) 1398 (97.2%) p-valor= 0.5331 RR de tener TBC debido a SD C.=1.3699 I.C. 95%: [0.5272 – 3.5601] Odds ratio: 1.4543 (I.C. 95%) (0.4209 – 3.7114) p-val:0.5088 SD C. Si No Si (G1+G3+G5) 4 (4.0%) 97 (96.0%) Tabla 27c. Comparación de las proporciones de pacientes con síndrome constitucional entre los grupos con y sin cultivo positivo para tuberculosis. SD. C. = Síndrome Constitucional. No se encontraron diferencias estadísticamente significativas en cuanto al porcentaje de pacientes con síndrome constitucional, al comparar los grupos con cultivo positivo para tuberculosis con el grupo con cultivo positivo para micobacterias atípicas ni al comparar los grupos con cultivo positivo para tuberculosis con los grupos con cultivo negativo. 4.1.3. VARIABLES DE RADIOGRAFÍA SIMPLE DE TÓRAX: 4.1.3.1. Hallazgos pulmonares:  G1 G2 G3 G4 G5 G6 G7 Total Nódulo pulmonar: Sin nódulos Nódulo único 2 (100%) 28 (100%) 71 (84.5%) 822 (95.1%) 13 (86.7%) 503 (92.0%) 9 (100%) 1448 (93.48%) 0 0 11 (13.1%) 34 (3.9%) 2 (13.3%) 39 (7.1%) 0 86 (5.55%) Nódulos múltiples 0 0 2 (2.4%) 8 (0.9%) 0 5 (0.9%) 0 15 (0.97%) p-valor comparación proporciones 0.0003 0.6213 Tabla 28a. Comparación por grupos de las proporciones de pacientes con nódulos pulmonares y clasificándolos en función de si estos son únicos o múltiples. Con una confianza del 95%, se puede decir que existieron diferencias estadísticamente significativas entre el grupo G3 y G4, en favor de este primero, en cuanto a la proporción de pacientes con nódulos únicos. No fue así en el resto de grupos comparados. 84 G1 G2 G3 G4 G5 G6 G7 Si No 0 0 13 (15.5%) 42 (4.9%) 2 (13.3%) 44 (8.0%) 0 101 (6.52%) 2 28 71 (84.5%) 822 (95.1%) 13 (86.7%) 503 (92.0%) 9 1448 (93.48%) p-valor comparación proporciones 1 0.0005 0.3515 Total Tabla 28b. Comparación por grupos de las proporciones de pacientes con nódulos pulmonares. TBC No (G2+G4+G6) 86 (6%) 94 (94%) p-valor= 0.00106 RR de tener TBC debido Nódulo.RX=2.48 I.C. 95%: [1.49 –4.14] Odds ratio: 2.76 (I.C. 95%) (1.47 – 4.87) p-val=0.0022 Nódulo.RX Si (G1+G3+G5) 15 (14.9%) 86 (85.1%) Si No Tabla 28c. Comparación de las proporciones de pacientes con nódulos pulmonares entre los grupos con y sin cultivo positivo para tuberculosis. La diferencia observada entre los grupos G3 y G4 también es estadísticamente significativa si agrupamos a los pacientes con nódulos (independientemente de su número) y los comparamos con los pacientes sin nódulos. Como se aprecia en la tabla 28b, se detectó una mayor proporción de pacientes con nódulos en radiografía de tórax en el grupo con cultivo positivo para tuberculosis en este grupo de edad (G3). También es significativa la diferencia observada en el conjunto de grupos con tuberculosis demostrada (G1+G3+G5) con respecto a los que tienen un cultivo negativo (tabla 28 c). Nódulo.RX G1+G3+G5 G7 p-valor Si 15 (14.9%) 0 NO 86 (85.1%) 9 0.461 Tabla 28d. Comparación de las proporciones de pacientes con nódulos pulmonares entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. No se obtuvieron diferencias al comparar los grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para micobacterias atípicas, en cuanto a la proporción de pacientes con nódulos pulmonares.  Masa pulmonar: G1 G2 G3 G4 G5 G6 G7 Total SI NO 0 2 (7.1%) 14 (16.7%) 29 (3.4%) 3 (20%) 14 (2.6%) 0 62 (4%) 2 (100%) 26 (92.9%) 70 (83.3%) 835 (96.6%) 12 (80.0%) 533 (97.4%) 9 (100%) 1487 (96%) p-valor comparación proporciones 1 <0.0001 0.008 Tabla 29a. Comparación por grupos de las proporciones de pacientes con masas pulmonares. 85 Con una confianza del 95%, se puede afirmar que la proporción de masas pulmonares fue significativamente mayor en el grupo G3 con respecto al G4 y en el G5 con respecto al G6. Masa en RX G1+G3+G5 G7 p-valor Si 17 (16.8%) 0 NO 84 (83.2%) 9 0.3912 Tabla 29b. Comparación de las proporciones de pacientes con masas pulmonares entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. TBC Masa en RX Si No Si (G1+G3+G5) 17 (16.8%) 84 (83.2%) No (G2+G4+G6) 45 (3.1%) 1394 (96.9%) p-valor<0.0001 RR de tener TBC debido Masa en RX=4.82 I.C. 95%: [3.06 – 7.60] Odds ratio: 6.28 (I.C. 95%) (3.36 – 11.30) p-valor<0.0001 Tabla 29c. Comparación de las proporciones de pacientes con masas pulmonares entre los grupos con y sin cultivo positivo para tuberculosis. No se encontraron diferencias significativas en cuanto al porcentaje de pacientes con masas pulmonares, al comparar los grupos con cultivo positivo para tuberculosis con el grupo con cultivo positivo para micobacterias atípicas. Sí que había diferencias significativas al comparar los grupos con cultivo positivo para tuberculosis con los grupos con cultivo negativo. Tras el análisis preliminar en el que se detectó la presencia o ausencia de masas pulmonares en radiografía simple de tórax, se procedió a estudiar la existencia o no de cavitaciones en dichas masas. G1 G2 G3 G4 G5 G6 G7 Cavitada No cavitada 0 0 7 (50%) 7 (24.1%) 1 (33.3%) 0 0 15 (24.19%) 0 2 7 (50%) 22 (75.9%) 2 (66.7%) 14 0 47 (75.81%) p-valor comparación proporciones 1 0.1633 0.1765 Total Tabla 29d. Comparación por grupos de las proporciones de pacientes con masas cavitadas. No se detectaron diferencias significativas entre los porcentajes de pacientes que presentaron masas cavitadas y no cavitadas entre los grupos que comparamos. 86 TBC Masa.RX Cavitada No cavitada Si (G1+G3+G5) 8 (47.1%) 9 (52.9%) No (G2+G4+G6) 7 (15.6%) 38 (84.4%) p-valor= 0.024 RR de tener TBC debido a SI (C) =2.78 I.C. 95%: [1.31 – 5.92] Odds ratio: 4.65 (I.C. 95%) (1.32 – 17.23) p-val=0.017 Tabla 29f. Comparación de las proporciones de pacientes con masas pulmonares cavitadas entre los grupos con y sin cultivo positivo para tuberculosis. SI (C) = cavitación. La presencia de cavitaciones fue significativamente mayor en los pacientes con masas pulmonares en los grupos con cultivo positivo para M. tuberculosis.  Consolidación pulmonar: G1 G2 G3 G4 G5 G6 G7 Total Si No 2 (100%) 6 (21.4%) 47(56.0%) 226 (26.2%) 4 (26.7%) 161 (29.4%) 2 (22.2%) 448 (28.92%) 0 22 (78.6%) 37 (44.0%) 638 (73.8%) 11 (73.3%) 386 (70.6%) 7 (77.8%) 1101 (71.08%) p-valor comparación proporciones 0.06437 <0.0001 1 Tabla 30a. Comparación por grupos de las proporciones de pacientes con consolidaciones pulmonares. Con una confianza del 95%, se puede afirmar que el porcentaje de pacientes que presentaron una consolidación pulmonar fue significativamente mayor en el grupo G3 que en el G4. No se detectan diferencias significativas entre los porcentajes de los grupos G1 y G2 ni entre los grupos G5 y G6. Consolidación en RX G1+G3+G5 G7 p-valor Si 53 (52.5%) 2 (22.2%) NO 48 (47.5%) 7 (77.8%) 0.1614 Tabla 30b. Comparación de las proporciones de pacientes con consolidaciones pulmonares entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. No existieron diferencias significativas en cuanto a la presencia de consolidaciones pulmonares en radiografía de tórax al comparar los grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. 87 TBC No (G2+G4+G6) 393 (27.3%) 1046 (72.7%) p-valor<0.0001 RR de tener TBC debido a Consolidación en RX=2.71 I.C. 95%: [1.86 – 3.94] Odds ratio: 2.94 (I.C. 95%) (1.95 – 4.42) p-valor<0.0001 Consolidación en RX Si No Si (G1+G3+G5) 53 (52.5%) 48 (47.5%) Tabla 30c. Comparación de las proporciones de pacientes con consolidaciones pulmonares entre los grupos con y sin cultivo positivo para tuberculosis. De forma significativa, se detectó una mayor proporción de pacientes con consolidación en radiografía de tórax entre los grupos con cultivo positivo para M. tuberculosis. Tras una primera observación de las proporciones de consolidación, se separaron los pacientes con consolidaciones en radiografía y se estudió el porcentaje de ellos que presentaron cavitaciones en su seno. G1 G2 G3 G4 G5 G6 G7 Consolidación cavitada 0 0 5 (10.6%) 8 (3.5%) 1 (25%) 1 (0.6%) 0 15 (3.35%) No cavitada 2 6 42 (89.4%) 218 (96.5%) 3 (75%) 160 (99.4%) 2 433 (96.65%) p-valor comparación proporciones 1 0.053 0.048 Total Tabla 30d. Comparación por grupos de las proporciones de pacientes con consolidaciones pulmonares cavitadas. Fueron detectadas diferencias estadísticamente significativas en la proporción de pacientes con consolidación cavitada entre los grupos G5 y G6, siendo más probable encontrar cavitación en las neumonías del grupo G5. Consolidación en RX G1+G3+G5 G7 p-valor Cavitación 6 (11.3%) 0 No cavitación 47 (88.7%) 2 1 Tabla 30e. Comparación de las proporciones de pacientes con consolidaciones pulmonares cavitadas entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. No se observaron diferencias significativas entre los pacientes con cultivo positivo para M. tuberculosis y aquellos con cultivo positivo para micobacterias atípicas. 88 TBC Consolidación.RX Si (G1+G3+G5) 6 (11.3%) 47 (88.7%) Cavitación No cavitación No (G2+G4+G6) 9 (2.3%) 384 (97.7%) p-valor= 0.0025 RR de tener TBC debido s SI(C )=3.66 I.C. 95%: [1.86 – 7.21] Odds ratio: 5.45 (I.C. 95%) (1.72 – 16.08) p-val=0.005 Tabla 30f. Comparación de las proporciones de pacientes con consolidaciones pulmonares cavitadas entre los grupos con y sin cultivo positivo para tuberculosis. SI (C) = consolidación cavitada. Como demuestra la tabla 30f, fue más probable encontrar cavitación en las consolidaciones de los pacientes con cultivo positivo para M. tuberculosis.  Atelectasias: G1 G2 G3 G4 G5 G6 G7 Total Si No 0 2 (7.1%) 15 (17.9%) 99 (11.5%) 2 (13.3%) 98 (17.9%) 0 216 (13.94%) 2 (100%) 26 (92.9%) 69 (82.1%) 765 (88.5%) 13 (86.7%) 449 (82.1%) 9 (100%) 1333 (86.06%) p-valor comparación proporciones 1 0.1222 1 Tabla 31a. Comparación por grupos de las proporciones de pacientes con atelectasias. Con una confianza del 95%, no se detectan diferencias significativas en los porcentajes de pacientes que presentaron atelectasia en su radiografía entre los grupos que comparamos. Atelectasia G1+G3+G5 G7 SI NO 17 (16.8%) 84 (83.2%) 0 9 (100%) p-valor=0.3497 (*) Tabla 31b. Comparación de las proporciones de pacientes con atelectasias entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. (*) test exacto de Fisher. TBC No (G2+G4+G6) 199 (13.8%) 1240 (86.2%) p-valor= 0.3763 RR de tener TBC debido a atelectasia=1.2405 I.C. 95%: [0.7517 – 2.0473] Odds ratio: 1.2697 (I.C. 95%) (0.7139 – 2.1343) p-val:0.4 Atelectasia Si No Si (G1+G3+G5) 17 (16.8%) 84 (83.2%) Figura 31c. Comparación de las proporciones de pacientes con atelectasias entre los grupos con y sin cultivo positivo para tuberculosis. 89 Tampoco se observaron diferencias estadísticamente significativas al separar a los pacientes en función del resultado de su cultivo y comparar sus proporciones de atelectasia en radiografía.  Patrón intersticial: G1 G2 G3 G4 G5 G6 G7 Total Sí No 0 0 8 (9.5%) 37 (4.3%) 3 (20%) 35 (6.4%) 1 (11.1%) 84 (5.42%) 2 (100%) 28 (100%) 76 (90.5%) 827 (95.7%) 12 (80.0%) 512 (93.6%) 8 (88.9%) 1465 (94.58%) p-valor comparación proporciones 1 0.052 0.074 Tabla 32a. Comparación por grupos de las proporciones de pacientes con patrón intersticial. Con una confianza del 95%, no se obtuvieron diferencias estadísticamente significativas al comparar los porcentajes de pacientes con patrón intersticial en radiografía de tórax. Patrón. intersticial G1+G3+G5 G7 p-valor Si 11 (10.9%) 1 (11.1%) NO 90 (89.1%) 8 (88.9%) 1 Tabla 32b. Comparación de las proporciones de pacientes con patrón intersticial entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. No se detectaron diferencias en cuanto a la presencia de pacientes con patrón intersticial en radiografía de tórax entre los grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. TBC Patrón. intersticial Si No Si (G1+G3+G5) 11 (10.9%) 90 (89.1%) No (G2+G4+G6) 72 (5%) 1367 (95%) p-valor=0.021 RR de tener TBC debido a Patrón. intersticial=2.14 I.C. 95%: [1.19 – 3.85] Odds ratio: 2.34 (I.C. 95%) (1.13 – 4.42) p-valor=0.023 Tabla 32c. Comparación de las proporciones de pacientes con patrón intersticial entre los grupos con y sin cultivo positivo para tuberculosis. De forma significativa, se observó una mayor proporción de pacientes con patrón intersticial en los grupos con cultivo positivo para tuberculosis. Se revisaron los 84 pacientes que habían presentado un patrón intersticial en la radiografía de tórax, analizando en cada caso si este patrón era focal o difuso. 90 G1 G2 G3 G4 G5 G6 G7 DIFUSO FOCAL 0 0 4 (50%) 27 (73%) 3 25 (71.4%) 1 60 (71.43%) 0 0 4 (50%) 10 (27%) 0 10 (28.6%) 0 24 (28.57%) p-valor comparación proporciones 1 0.2315 0.5519 Total Tabla 32d. Comparación por grupos de las proporciones de pacientes con los diferentes tipos de patrón intersticial. No se observaron diferencias significativas en cuanto a la proporción de los dos tipos de patrón intersticial en radiografía de tórax, entre los grupos estudiados. Patrón. intersticial G1+G3+G5 G7 p-valor DIFUSO 7 (63.6%) 1 FOCAL 4 (36.4%) 0 1 Tabla 32e. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas, de las proporciones de pacientes con los diferentes tipos de patrón intersticial. TBC Si (G1+G3+G5) 7 (63.6%) 4 (36.4%) No (G2+G4+G6) Patrón. DIFUSO 52 (72.2%) intersticial FOCAL 20 (27.8%) p-valor= 0.8197 RR de tener TBC debido a DIFUSO =0.71 I.C. 95%: [0.23 – 2.21] Odds ratio: 0.67 (I.C. 95%) (0.17 – 2.89) p-val=0.568 Tabla 32f. Comparación entre los grupos con y sin cultivo positivo para tuberculosis, de las proporciones de pacientes con los diferentes tipos de patrón intersticial Al comparar los grupos con cultivo positivo para tuberculosis y el que tenía un cultivo positivo para micobacterias atípicas, no fueron significativas las diferencias en cuanto a la proporción de pacientes con cada tipo de patrón intersticial. Tampoco fue significativa la diferencia entre los grupos con cultivo positivo para tuberculosis y los grupos con cultivo negativo en cuanto a la presencia de patrón intersticial focal o difuso.  Bronquiectasias: G1 G2 G3 G4 G5 G6 G7 Total Si No 0 2 (7.1%) 4 (4.8%) 43 (5.0%) 0 43 (7.9%) 4 (44.4%) 96 (6.20%) 2 (100%) 26 (92.9%) 80 (95.2%) 821 (95.0%) 15 (100.0%) 504 (92.1%) 5 (55.6%) 1453 (9.80%) p-valor comparación proporciones 1 1 0.619 Tabla 33a. Comparación por grupos de las proporciones de pacientes con bronquiectasias. 91 Con una confianza del 95%, se puede decir que no se detectaron diferencias significativas entre los porcentajes de pacientes con bronquiectasias en su radiografía de tórax, en los grupos que comparamos. Bronquiectasia G1+G3+G5 G7 % NO 4 (4.0%) 97 (96.0%) 4 (44.5%) 5 (55.6%) p-valor=0.0551 (*) Tabla 33b. Comparación de las proporciones de pacientes con bronquiectasias entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. (*) test exacto de Fisher. TBC Si (G1+G3+G5) 4 (4%) 97 (96%) No (G2+G4+G6) Bronquiectasia Si 88 (6.1%) No 1351 (93.6%) p-valor= 0.5146 RR de tener TBC debido a Bronquiectasia =0.6490I.C. 95%: [0.2442– 1.7250] Odds ratio: 0.6564(I.C. 95%) (0.1937 – 1.6185) p-val:0.3946 Figura 33c. Comparación de las proporciones de pacientes con bronquiectasia entre los grupos con y sin cultivo positivo para tuberculosis. Tampoco se observaron diferencias estadísticamente significativas al agrupar a los pacientes en función del resultado de su cultivo y comparar sus proporciones de bronquiectasias en radiografía. No se detectaron diferencias significativas entre los grupos con cultivo positivo para M. tuberculosis y el grupo de cultivo positivo para micobacterias atípicas. 4.1.3.2. Hallazgos mediastínicos:  Masas mediastínicas: G1 G2 G3 G4 G5 G6 G7 Total Si No 0 0 7 (8.3%) 21 (2.4%) 1 (6.7%) 14 (2.6%) 0 43 (2.78%) 2 (100%) 28 (100%) 77 (91.7%) 843 (97.6%) 14 (93.3%) 533 (97.4%) 9 (100%) 1506 (97.22%) p-valor comparación proporciones 1 0.0067 0.337 Tabla 34a. Comparación por grupos de las proporciones de pacientes con masa mediastínica. Con una confianza del 95%, podemos afirmar que el porcentaje de pacientes que han presentado en la radiografía de tórax una masa mediastínica es significativamente mayor en el grupo G3 que en el G4. No se detectan diferencias significativas entre los porcentajes en los grupos G5 y G6. 92 Masa.med G1+G3+G5 G7 SI 8 (7.9%) 0 NO 93 (92.1%) 9 p-valor=1 (*) Tabla 34b. Comparación de las proporciones de pacientes con masa mediastínica entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. (*) test exacto de Fisher. Masa.med = Masa mediastínica. TBC No (G2+G4+G6) 35 (2.4%) 1404 (97.6%) p-valor= 0.0.005 RR de tener TBC debido a Masa.med =2.9948I.C. 95%: [1.5549 – 5.7678] Odds ratio: 3.4959 (I.C. 95%) (1.4596 – 7.4231) p-val:0.0068 Masa.med Si (G1+G3+G5) 8 (7.9%) 93 (92.1%) Si No Tabla 34c. Comparación de las proporciones de pacientes con masa mediastínica entre los grupos con y sin cultivo positivo para tuberculosis. Masa.med = Masa mediastínica. Se observaron diferencias estadísticamente significativas al comparar los grupos con cultivo positivo para tuberculosis y aquellos que no lo tenían, en relación al porcentaje de pacientes que presentaron masas mediastínicas en la radiografía. Se demostró que el conjunto de grupos con cultivo positivo (G1+G3+G5) tuvo una mayor proporción de pacientes con masa mediastínica que el conjunto constituido por los grupos con cultivo negativo (G2+G4+G6). No hubo diferencias entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para micobacterias atípicas (G7).  Agrandamiento hiliar: G1 G2 G3 G4 G5 G6 G7 Total Si No 0 0 1 (1.2%) 6 (0.7%) 0 11 (2.0%) 0 18 (1.16%) 2 (100%) 28 (100%) 88 (98.8%) 858 (99.3%) 15 (100%) 536 (98.0%) 9 (100%) 1531 (98.94%) p-valor comparación proporciones 1 0.4788 1 Tabla 35a. Comparación por grupos de las proporciones de pacientes con agrandamiento hiliar . Con una confianza del 95%, no se detectaron diferencias significativas en los porcentajes de pacientes que presentaron en su radiografía un agrandamiento hiliar, entre los grupos que comparamos. Masa hiliar G1+G3+G5 G7 % 1 (1%) 0 NO 100 (99%) 9 (100%) p-valor=1 (*) Tabla 35b. Comparación de las proporciones de pacientes con agrandamiento hiliar entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. (*) test exacto de Fisher. 93 TBC No (G2+G4+G6) 17 (1.2%) 1422 (98.8%) p-valor= 1 RR de tener TBC debido a masa hiliar =0.8456I.C. 95%: [0.1247 – 5.7338] Odds ratio: 0.9484(I.C. 95%) (0.0393 – 4.6880) p-val:0.9601 Masa hiliar Si No Si (G1+G3+G5) 1 (1%) 100 (99%) Tabla 35c. Comparación de las proporciones de pacientes con agrandamiento hiliar entre los grupos con y sin cultivo positivo para tuberculosis. No se encontraron diferencias estadísticamente significativas en cuanto al porcentaje de pacientes con agrandamiento hiliar, al comparar los grupos con cultivo positivo para tuberculosis con el grupo con cultivo positivo para micobacterias atípicas ni al comparar los grupos con cultivo positivo para tuberculosis con los grupos con cultivo negativo. 4.1.3.3. Hallazgos pleurales:  Derrame pleural: G1 G2 G3 G4 G5 G6 G7 Total Si 0 2 (7.1%) 7 (8.3%) 91 (10.5%) 0 81 (14.8%) 1 (11.1%) 182 (11.75%) No 2 (100%) 26 (92.9%) 77 (91.7%) 773 (89.5%) 15 (100.0%) 466 (85.2%) 8 (88.9%) 1367 (88.25%) p-valor comparación proporciones 1 0.6568 0.1447 Tabla 36a. Comparación por grupos de las proporciones de pacientes con derrame pleural. Con una confianza del 95%, no se detectaron diferencias significativas en los porcentajes de pacientes que presentaron en su radiografía un derrame pleural, entre los grupos que comparamos. Derrame Pl G1+G3+G5 G7 SI NO 7 (6.9%) 94 (93.1%) 1 (11.1%) 8 (88.9%) p-valor=0.5067 (*) Tabla 36b. Comparación de las proporciones de pacientes con derrame pleural entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. (*) test exacto de Fisher. Derrame Pl = Derrame Pleural. TBC No (G2+G4+G6) 174 (12.1%) 1265 (87.9%) p-valor= 0.1489 RR de tener TBC debido a derrame pl =0.5591 I.C. 95%: [0.2636 – 1.1861] Odds ratio: 0.5527 (I.C. 95%) (0.2279 – 1.1305) p-val:0.11 Derrame Pl Si No Si (G1+G3+G5) 7 (6.9%) 94 (93.1%) Tabla 36c. Comparación de las proporciones de pacientes con derrame pleural entre los grupos con y sin cultivo positivo para tuberculosis. Derrame Pl = Derrame Pleural. 94 No se observaron diferencias significativas en cuanto a los porcentajes de derrame pleural al dividir a los pacientes en función del resultado de su cultivo (positivo-negativo; M. tuberculosis- M. atípicas), sin tener en cuenta el grupo de edad de pertenencia. 4.1.4. VARIABLES EN TOMOGRAFÍA COMPUTARIZADA DE TÓRAX: 4.1.4.1. Hallazgos pulmonares:  Micronódulo pulmonar: G1 G2 G3 G4 G5 G6 G7 Total Si No 0 5 (17.9%) 45 (53.6%) 219 (25.3%) 8 (53.3%) 141 (25.8%) 2 (22.2%) 420 (27.11%) 2 232 (82.1%) 39 (46.4%) 645 (74.7%) 7 (46.7%) 406 (74.2%) 7 (77.8%) 1129 (72.89%) p-valor comparación proporciones 1 <0.0001 0.0367 Tabla 37a. Comparación por grupos de las proporciones de pacientes con micronódulos pulmonares. Con una confianza del 95%, se obtuvieron diferencias estadísticamente significativas en las proporciones de micronódulos entre los grupos G3 y G4, en favor del primero. Lo mismo sucedió entre el grupo G5 y G6 observándose también una mayor proporción de micronódulos en el grupo con cultivo positivo (G5). Micronódulos G1+G3+G5 G7 p-valor Si 53 (52.5%) 2 (22.2%) NO 48 (47.5%) 7 (77.8%) 0.1641 Tabla 37b. Comparación de las proporciones de pacientes con micronódulos entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. No se detectaron diferencias significativas en cuanto a la presencia de micronódulos únicos o múltiples entre los grupos con cultivo positivo para M. tuberculosis (G1+G3+G5) y aquellos que presentaron un cultivo positivo para micobacterias atípicas (G7). 95 TBC No (G2+G4+G6) 365 (25.4%) 1074 (74.6%) p-valor<0.0001 RR de tener TBC debido a micronódulos=2.96 I.C. 95%: [2.03 – 4.31] Odds ratio: 3.24 (I.C. 95%) (2.15 – 4.89) p-valor<0.0001 Micronódulos Si No Si (G1+G3+G5) 53 (52.5%) 48 (47.5%) Tabla 37c. Comparación de las proporciones de pacientes con micronódulos entre los grupos con y sin cultivo positivo para tuberculosis. Se obtuvo una diferencia estadísticamente significativa entre los grupos con cultivo positivo para M. tuberculosis (G1+G3+G5) y los que presentaron un cultivo negativo (G2+G4+G6), en relación a la presencia de micronódulos, independientemente de que éstos fueran únicos o múltiples. Centrándonos en los 420 pacientes que tienen micronódulos, éstos fueron divididos en únicos y múltiples, obteniendo las siguientes tablas: G1 G2 G3 G4 G5 G6 G7 Únicos Múltiples 0 3 (60%) 12 (26.7%) 136 (62.1%) 0 106 (75.2%) 1 (50%) 258 (61.43%) 0 2 (40%) 33 (73.3%) 83 (37.9%) 8 (100%) 35 (24.8%) 1 (50%) 162 (38.57%) p-valor comparación proporciones 1 <0.0001 <0.0001 Total Tabla 37d. Comparación por grupos de las proporciones de pacientes con micronódulos pulmonares de cada tipo (únicos/múltiples). Se detectó una mayor proporción de micronódulos múltiples en el grupo G3 con respecto al G4 y en el grupo G5 en comparación con el G6. Micronódulos G1+G3+G5 G7 p-valor Únicos 12 (22.6%) 1 (50%) Múltiples 41 (77.4%) 1 (50%) 0.9631 Tabla 37e. Comparación de las proporciones de pacientes con micronódulos de cada tipo (únicos/ múltiples) entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. No se obtuvo diferencias estadísticamente significativas al comparar los grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. 96 TBC No (G2+G4+G6) 245 (67.1%) 120(32.9%) p-valor<0.0001 RR de tener TBC debido a tipo micronódulos únicos =0.18 I.C. 95%: [0.09 – 0.34] Odds ratio: 0.14 (I.C. 95%) (0.07 – 0.28) p-val<0.0001 Micronódulos Si (G1+G3+G5) 12(22.6%) 41 (77.4%) Únicos Múltiples Tabla 37f. Comparación de las proporciones de pacientes con micronódulos de cada tipo entre los grupos con y sin cultivo positivo para tuberculosis. Se objetivó una mayor proporción de nódulos múltiples en los grupos con cultivo positivo para tuberculosis en comparación con aquellos que mostraron un cultivo negativo para dicho microorganismo. LOCALIZACIÓN DE MICRONÓDULOS ÚNICOS2 Establecemos un código numérico para diferenciar la localización de los micronódulos y así facilitar la recogida de datos relacionados con esta variable. A continuación se describe que localización traduce cada número: 1: lóbulo superior derecho 2: lóbulo medio 3: lóbulo inferior derecho 4: lóbulo superior izquierdo 5: língula 6: lóbulo inferior izquierdo En caso de encontrarse el micronódulo en más de una localización, se optó por escribir sus números de localización en orden secuencial. 1 12 123 123456 124 1245 126 13 134 136 14 146 15 16 2 23 2 G1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G2 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 G3 3 0 0 0 0 0 0 1 0 0 2 0 0 0 0 0 G4 47 3 2 1 0 1 2 1 0 1 11 0 1 4 6 1 Continúa en la siguiente página. 97 G5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G6 18 3 1 4 1 1 0 6 1 0 7 1 1 1 11 4 G7 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 235 2356 24 2356 24 25 3 35 36 4 46 5 56 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 1 0 3 0 1 0 0 0 0 1 0 1 1 24 1 2 10 2 2 0 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 16 0 4 5 0 3 2 12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Tabla 37g. Clasificación por grupos de las diferentes localizaciones de micronódulos únicos observadas en este estudio. Tras esto, se procedió a estudiar la presencia de micronódulos únicos en cada lóbulo por separado, obteniéndose la siguiente tabla: 1 2 3 4 5 6 pvalor G1 0 0 0 0 0 0 G2 2 (40%) 0 1 (20%) 1 (20%) 0 1 (20%) G3 G4 6 (37.5%) 74 (40.9%) 0 18 (9.9%) 3 (18.8%) 33 (18.2%) 5 (31.2%) 26 (14.4%) 2 (12.5%) 7 (3.9%) 0 23 (12.7%) 0.1204 G5 0 0 0 0 0 0 G6 45 (26.2%) 29 (16.9%) 38 (22.1%) 21 (12.2%) 14 (8.1%) 25 (14.5%) G7 1 (50%) 0 0 1 (50) 0 0 Tabla 37h. Comparación por grupos de las proporciones de pacientes según la localización de los micronódulos únicos. Con una confianza del 95%, no fueron observadas diferencias estadísticamente significativas entre los grupos en cuanto a la localización de los micronódulos solitarios. 40 60 lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 Porcentaje 80 100 Localización G2 G3 G4 G6 G7 Figura 4. Gráfico de barras apiladas con los porcentajes de pacientes con micronódulos únicos en diferentes localizaciones por grupo. 98 LOCALIZACIÓN DE MICRONÓDULOS MÚLTIPLES 3 G1 1 12 123 1234 123456 124 1245 1246 125 1256 13 136 14 145 1456 146 2 23 2346 235 2356 236 25 3 36 4 456 46 5 6 G2 G3 1 1 1 G4 4 2 2 11 33 1 1 G5 1 1 G6 6 G7 1 1 3 13 1 1 1 1 1 6 1 1 2 9 4 1 2 1 1 1 3 1 2 3 8 3 1 2 2 1 1 1 1 2 3 1 1 1 3 2 1 2 Tabla 37i. Clasificación por grupos de las diferentes localizaciones de micronódulos múltiples observadas en este estudio. Tras esto, se procedió a estudiar la presencia de micronódulos múltiples en cada lóbulo por separado, obteniéndose la siguiente tabla: 3 1 G1 0 G2 1 (10%) 2 0 2 (20%) 3 0 2 (20%) 4 0 1 (10%) G3 26 (22.6%) 13 (11.3%) 15 (13.0%) 25 (21,7%) G4 54 (18.0%) 52 (17.3%) 53 (17.7%) 48 (16.0%) Continúa en la siguiente página 99 G5 7 (21.9%) 5 (15.6%) 4 (12.5%) 6 (18.8%) G6 24 (21.9%) 17 (15.6%) 21 (18.6%) 17 (15.0%) G7 0 0 1 (50%) 0 5 0 2 (20%) 6 0 2 (20%) 15 43 (13.0%) (14.3%) 21 50 (18.3%) (16.7%) 0.1204 pvalor 5 (15.6%) 15 (13.3%) 5 (15.6%) 19 (16.8%) 0.5653 0 1 (50%) Tabla 37j. Comparación por grupos de las proporciones de pacientes según la localización de los micronódulos múltiples. No se detectaron diferencias significativas en la localización de micronódulos múltiples entre los grupos estudiados. 40 60 lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 Porcentaje 80 100 Localización G2 G3 G4 G5 G6 G7 Figura 5. Gráfico de barras apiladas con los porcentajes de pacientes con micronódulos múltiples en diferentes localizaciones por grupo. DISTRIBUCION DE MICRONÓDULOS MÚLTIPLES Nuevamente, se establece un sistema numérico para diferenciar el patrón de distribución de los micronódulos, según las equivalencias que se describen a continuación: 1: centrolobulillar 2: perilinfática 3: al azar (miliar) En caso de observarse más de un patrón de distribución, se registraron en orden secuencial de menor a mayor. 1 2 12 3 G1 0 0 0 0 G2 2 0 0 0 G3 15 0 1 17 G4 59 12 2 9 G5 5 0 0 2 G6 22 10 1 2 G7 0 0 0 1 Tabla 37k. Clasificación por grupos de las diferentes distribuciones de micronódulos múltiples observadas en este estudio. 100 1 G1 0 G2 2 G3 16 (47.0%) G4 61 (72.6%) 2 0 0 1 (3.0%) 14 (16.7%) 3 0 0 17 (50.0%) 9 (10.7%) <0.0001 G5 5 (71.4%) G6 23 (63.9%) 0 11 (30.6%) 2 (28.6%) 2 (5.6%) 0.0609 G7 0 0 1 Tabla 37l. Comparación por grupos de las proporciones de pacientes con cada patrón de distribución de los micronódulos múltiples. Se obtuvieron diferencias estadísticamente significativas en relación con la distribución de los micronódulos pulmonares múltiples entre los grupos G3 y G4, observándose una mayor proporción de patrón centrolobulillar en los grupos con cultivo negativo para M. tuberculosis (G4 y G6). Se detectó una mayor proporción de patrón “al azar” en los grupos G3 y G5 frente a los grupos G4 y G6. 40 60 centrolobulillar perilinfática al azar (miliar) 0 20 Porcentaje 80 100 Distribución G2 G3 G4 G5 G6 G7 Figura 6. Gráfico de barras apiladas con los porcentajes de pacientes con micronódulos múltiples según su distribución por grupo. A continuación, dividimos los micronódulos en función de si presentaron distribución “al azar” o no, obteniendo la siguiente tabla: G1 G2 G3 G4 G5 G6 G7 Total Si No 0 0 17 (51.5%) 9 (11%) 2 (28.6%) 2 (5.7%) 1 31 (19.37%) 0 2 16 (48.5%) 73 (89%) 5 (71.4%) 33 (94.3%) 0 129 (80.62%) p-valor comparación proporciones 1 <0.0001 0.1229 Tabla 37m. Comparación por grupos de las proporciones de pacientes con patrón “al azar” entre todos aquellos que presentaron micronódulos. 101 Se obtuvieron diferencias significativas entre los grupos G3 y G4 en cuanto a la proporción de pacientes cuya distribución de los micronódulos siguió un patrón “al azar”, estando dicho patrón más presente en el grupo G3. Distribución al azar G1+G3+G5 G7 p-valor Si 19 (47.5%) 1 NO 21 (52.5%) 0 0.9803 Tabla 37n. Comparación de las proporciones de pacientes con distribución “al azar” entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. TBC Si (G1+G3+G5) 19 (47.5%) 21 (52.5%) No (G2+G4+G6) Distribución Si 11 (9.2%) al azar No 109 (90.8%) p-valor<0.0001 RR de tener TBC debido a Distribución al azar =3.92 I.C. 95%: [2.43 – 6.32] Odds ratio: 8.74 (I.C. 95%) (3.68– 21.85) p-valor<0.0001 Tabla 37ñ. Comparación de las proporciones de pacientes con micronódulos de distribución al azar entre los grupos con y sin cultivo positivo para tuberculosis. No se detectaron diferencias significativas en cuanto a la proporción de patrón de distribución al azar entre los grupos con cultivo positivo para M. tuberculosis (G1+G3+G5) y aquellos que presentaron un cultivo positivo para micobacterias atípicas (G7). No obstante, sí que se detectó una mayor proporción de este patrón entre los grupos con cultivo positivo para tuberculosis (G1+G3+G5) al compararlos con los que tenían un cultivo negativo para dicho microorganismo (G2 + G4 + G6). o Calcificación de los micronódulos: Se agruparon los 420 pacientes con micronódulos pulmonares y se estudió la proporción de ellos que presentaran calcificación de los mismos. G1 G2 G3 G4 G5 G6 G7 Calcificación No calcificación 0 1 (20%) 5 (11.1%) 38 (17.4%) 0 43 (30.5%) 0 87 (20.71%) 0 4 (80%) 40 (88.9%) 181 (82.6%) 8 (100%) 98 (69.5%) 2 (100%) 333 (72.29%) p-valor comparación proporciones (*) 0.417 0.146 Total Tabla 37m. Comparación por grupos de las proporciones de pacientes con calcificación en los micronódulos pulmonares. 102 No se detectaron diferencias estadísticamente significativas en cuanto a las proporciones de pacientes con calcificaciones en los micronódulos pulmonares. CALCIFICACIÓN G1+G3+G5 G7 p-valor Si 5 (9.4%) 0 NO 48 (90.6%) 2 (100%) 1 Tabla 37n. Comparación de las proporciones de pacientes con micronódulos entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. Al comparar los grupos de pacientes con cultivo positivo para tuberculosis con el grupo con cultivo positivo para micobacterias atípicas, no se observaron diferencias significativas en relación a la proporción de pacientes con calcificación en sus micronódulos pulmonares. TBC No (G2+G4+G6) 82 (22.5%) 283 (77.5%) p-valor<0.04521 RR de tener TBC debido Calcif micronódulos=0.396 I.C. 95%: [0.16 – 0.96] Odds ratio: 0.37 (I.C. 95%) (0.12 – 0.88) p-val=0.227 Calcif micronódulos Si (G1+G3+G5) 5 (9.4%) 48 (90.6%) Si No Tabla 37ñ. Comparación de las proporciones de pacientes con micronódulos calcificados entre los grupos con y sin cultivo positivo para tuberculosis. Calcif = calcificación. Se objetivó una menor proporción de pacientes con micronódulos calcificados en los grupos con cultivo positivo para tuberculosis. LOCALIZACION CALCIFICACION MICRONÓDULOS 1 123 123456 14 145 146 16 2 23 2356 24 3 36 4 46 5 6 G1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G2 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 G3 0 0 0 0 0 0 0 0 0 0 0 2 0 2 0 1 0 G4 14 1 2 8 1 0 1 0 0 0 0 5 0 3 1 1 1 G5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G6 8 0 2 7 0 1 0 5 1 1 1 3 1 4 0 2 7 G7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Tabla 37o. Clasificación por grupos de las diferentes localizaciones de micronódulos calcificados observadas en este estudio. 103 Tras esto, se procedió a estudiar la presencia de micronódulos calcificados en cada lóbulo por separado, obteniéndose la siguiente tabla: 1 2 3 4 5 6 pvalor G1 0 0 0 0 0 0 G2 1 0 0 1 0 0 G3 0 0 2 2 1 0 G4 27 3 8 15 4 5 G5 0 0 0 0 0 0 G6 18 10 8 15 5 12 G7 0 0 0 0 0 0 0.1152 Tabla 37p. Comparación por grupos de las proporciones de pacientes según la localización de los micronódulos múltiples. No se detectaron diferencias significativas en cuanto a la localización de los micronódulos calcificados entre los grupos estudiados. 40 60 Todos los lobulos lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 Porcentaje 80 100 Localización G2 G3 G4 G6 Figura 7. Gráfico de barras apiladas con los porcentajes de pacientes con calcificación en los micronódulos calcificados en diferentes localizaciones por grupo.  Nódulo pulmonar: G1 G2 G3 G4 G5 G6 G7 Si No 1 (50%) 0 24 (28.6%) 65 (7.5%) 5 (33.3%) 41 (7.5%) 2 (22.2%) 138 (8.91%) 1 (50%) 28 (100%) 60 (71.4%) 799 (92.5%) 10 (66.7%) 506 (92.5%) 7 (77.8%) 1411 (91.09%) p-valor comparación proporciones (*) <0.0001 <0.0001 0.0018 Total Tabla 38a. Comparación por grupos de las proporciones de pacientes con nódulos pulmonares. 104 Con una confianza del 95%, se observó una mayor proporción de pacientes con nódulos pulmonares en los grupos con cultivo positivo en todos los intervalos de edad (G1, G3 y G5). NODULOS G1+G3+G5 G7 p-valor Si 30 (29.7%) 2 (22.2%) NO 71 (70.3%) 7 (77.8%) 0.9279 Tabla 38b. Comparación de las proporciones de pacientes con nódulos pulmonares entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. TBC Nódulos Si (G1+G3+G5) 30 (29.7%) 71 (70.3%) Si No No (G2+G4+G6) 106 (7.4%) 1333 (92.6%) p-valor<0.0001 RR de tener TBC debido a Nódulos=4.3620 I.C. 95%: [2.95 – 6.43] Odds ratio: 5.316 (I.C. 95%) (3.28 – 8.45) p-val<0.0001 Tabla 38c. Comparación de las proporciones de pacientes con nódulos pulmonares entre los grupos con y sin cultivo positivo para tuberculosis. Se unieron los pacientes de los diferentes grupos con cultivo positivo para tuberculosis y se compararon con los pacientes con cultivo negativo para este microorganismo, detectándose una mayor proporción de nódulos en los grupos con cultivo positivo (tabla 39c). Por el contrario, no se identificaron diferencias significativas entre los grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para micobacterias atípicas (tabla 39b). LOCALIZACIÓN DE LOS NÓDULOS PULMONARES4 1 12 123 12345 123456 13 1346 136 14 146 16 2 23 2356 25 26 3 34 4 G1 1 G2 G3 3 G4 11 G5 1 1 1 1 4 1 1 4 G6 11 G7 5 1 1 6 1 1 5 1 1 2 1 15 3 Continúa en la siguiente página 105 2 2 2 1 2 5 1 1 1 35 36 4 456 5 6 1 6 1 1 3 5 1 4 1 1 8 6 Tabla 38d. Clasificación por grupos de las diferentes localizaciones de nódulos pulmonares observadas en este estudio. A continuación, se procedió a estudiar la presencia de nódulos en cada lóbulo por separado, obteniéndose la siguiente tabla: 1 G1 1 G2 0 2 0 0 3 0 0 4 0 0 5 6 0 0 0 0 G3 9 (25.0%) G4 26 (22.8%) 0 16 (14.0%) 6 (16.7%) 24 (21.1%) 12 18 (33.3%) (15.8%) 3 (8.3%) 9 (7.9%) 6 (16.7%) 17 (14.9%) 0.091 pvalor G5 2 (33.3%) 3 (50.0%) 0 G6 19 (22.9%) 10 (12.0%) 16 (19.3%) 1 12 (16.7%) (14.5%) 0 7 (8.4%) 0 14 (16.9%) 0.263 G7 0 1 (33.3%) 2 (66.7%) 0 0 0 Tabla 38e. Comparación por grupos de las proporciones de pacientes según la localización de los nódulos pulmonares. No se detectaron diferencias significativas en cuanto a la localización de los nódulos pulmonares entre los grupos estudiados. 40 60 todos los lóbulos lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 Porcentaje 80 100 Localización G1 G3 G4 G5 G6 G7 Figura 8. Gráfico de barras apiladas con los porcentajes de pacientes con calcificación en los micronódulos calcificados en diferentes localizaciones por grupo. 106 o Nódulo pulmonar calcificado: G1 G2 G3 G4 G5 G6 G7 Si No 0 0 0 4 (6.2%) 1 (20%) 2 (4.9%) 0 1 (100%) 0 24 (100%) 61 (93.8%) 4 (80%) 39 (95.1%) 2 (100%) p-valor comparación proporciones (*) 1 0.5706 0.2978 Total Tabla 38f. Comparación por grupos de las proporciones de pacientes con nódulos pulmonares calcificados. No se detectaron diferencias estadísticamente significativas en cuanto a las proporciones de pacientes con calcificaciones en los nódulos pulmonares. NÓDULO CALCIFICADO G1+G3+G5 G7 p-valor Si 1 (3.3%) 0 NO 29 (96.7%) 2 (100%) 1 Tabla 38g. Comparación de las proporciones de pacientes con nódulos pulmonares calcificados entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. Al comparar los grupos de pacientes con cultivo positivo para tuberculosis con el grupo con cultivo positivo para micobacterias atípicas, no se observaron diferencias significativas en relación a la proporción de pacientes con calcificación en sus nódulos pulmonares. TBC Calcificación Nódulo Si No Si (G1+G3+G5) 1 (3.3%) 29 (96.7%) No (G2+G4+G6) 6 (5.7%) 100 (94.3%) p-valor=0.9671 RR de tener TBC debido Calcificación de Nódulo=0.635 I.C. 95%: [0.0.100 – 4.011] Odds ratio: 0.64 (I.C. 95%) (0.02 – 4.11) p-val=0.685 Tabla 38h. Comparación de las proporciones de pacientes con nódulos pulmonares calcificados entre los grupos con y sin cultivo positivo para tuberculosis. No se objetivaron diferencias significativas al comparar las proporciones de pacientes con nódulos pulmonares calcificados entre los grupos con cultivo positivo para tuberculosis y aquellos que presentaron un cultivo negativo. 107 LOCALIZACIÓN DE LOS NÓDULOS PULMONARES CALCIFICADOS G1 0 0 0 0 0 1 2 3 14 34 G2 0 0 0 0 0 G3 0 0 0 0 0 G4 1 0 2 1 0 G5 0 1 0 0 0 G6 1 0 0 0 1 G7 0 0 0 0 0 Tabla 38i. Clasificación por grupos de las diferentes localizaciones de nódulos calcificados observadas en este estudio. Tras esto, se procedió a estudiar la presencia de nódulos pulmonares calcificados en cada lóbulo por separado, obteniéndose la siguiente tabla: 1 2 3 4 5 6 pvalor G1 0 0 0 0 0 0 G2 0 0 0 0 0 0 G3 0 0 0 0 0 0 G4 2 0 2 1 0 0 G5 0 1 0 0 0 0 G6 1 0 1 1 0 0 G7 0 0 0 0 0 0 0.333 Tabla 38j. Comparación por grupos de las proporciones de pacientes según la localización de los nódulos pulmonares. No se detectaron diferencias significativas en cuanto a la localización de los nódulos pulmonares calcificados entre los grupos estudiados. o Nódulo pulmonar cavitado: G1 G2 G3 G4 G5 G6 G7 Total Si No 1 0 19 (79.2%) 17 (26.2%) 2 (40.0%) 6 (14.6%) 0 45 (32.61%) 0 0 5 (20.8%) 48 (73.8%) 3 (60.0%) 35 (85.4%) 2 93 (67.39%) p-valor comparación proporciones (*) 1 <0.0001 0.203 Tabla 38k. Comparación por grupos de las proporciones de pacientes con nódulos pulmonares cavitados. Se observó una diferencia estadísticamente significativa en cuanto a la proporción de cavitación de los nódulos al comparar el grupo G3 con el G4, en favor del primero. 108 CAVITACION NÓDULO G1+G3+G5 G7 p-valor Si 22 (73.3%) 0 NO 8 (26.7%) 2 0.168 Tabla 38l. Comparación de las proporciones de pacientes con nódulos pulmonares cavitados entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas. No fueron detectadas diferencias significativas en la proporción de cavitación de los nódulos, al comparar los grupos con cultivo positivo para M. tuberculosis con el grupo con cultivo positivo para micobacterias atípicas. TBC Cavitación Nódulo Si (G1+G3+G5) 22 (73.3%) 8 (26.7%) Si No No (G2+G4+G6) 23 (21.7%) 83 (78.3%) p-valor<0.0001 RR de tener TBC debido Cavitación nódulo=5.56 I.C. 95%: [2.69 – 11.49] Odds ratio: 9.61 (I.C. 95%) (3.89 – 26.01) p-val<0.0001 Tabla 38m. Comparación de las proporciones de pacientes con nódulos pulmonares cavitados entre los grupos con y sin cultivo positivo para tuberculosis. Al comparar el conjunto de grupos con cultivo positivo para M. tuberculosis con los grupos que presentaron un cultivo negativo para dicho microorganismo, se objetivó una mayor proporción de cavitación en los nódulos pulmonares de los primeros. LOCALIZACIÓN DE LOS NÓDULOS PULMONARES CAVITADOS 1 123456 1346 14 146 23 2356 25 3 35 4 456 6 G1 1 0 0 0 0 0 0 0 0 0 0 0 0 G2 0 0 0 0 0 0 0 0 0 0 0 0 0 G3 3 0 1 3 0 0 0 0 3 1 4 1 3 G4 3 2 1 1 1 0 1 0 2 4 2 G5 1 0 0 0 0 0 0 0 0 0 1 0 0 G6 1 0 0 0 0 1 1 1 1 0 0 0 1 G7 0 0 0 0 0 0 0 0 0 0 0 0 0 Tabla 38n. Clasificación por grupos de las diferentes localizaciones de nódulos pulmonares cavitados observadas en este estudio. A continuación, se analizó la presencia de nódulos pulmonares cavitados en cada lóbulo por separado, obteniéndose la siguiente tabla: 109 1 2 3 4 5 6 pvalor G1 1 0 0 0 0 0 G2 0 0 0 0 0 0 G3 7 0 5 9 2 5 G4 8 3 6 9 3 7 G5 1 0 0 1 0 0 0.741 G6 1 3 3 0 2 2 G7 0 0 0 0 0 0 0.1154 Tabla 38ñ. Comparación por grupos de las proporciones de pacientes según la localización de los nódulos pulmonares cavitados. No se detectaron diferencias significativas en cuanto a la localización de los nódulos pulmonares cavitados entre los grupos estudiados. 40 60 todos los lóbulos lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 Porcentaje 80 100 Cavitación G1 G3 G4 G5 G6 Figura 9. Gráfico de barras apiladas con los porcentajes de pacientes con nódulos pulmonares cavitados en diferentes localizaciones por grupo. o Nódulo pulmonar con halo: G1 G2 G3 G4 G5 G6 G7 Si No 0 0 0 4 (6.2%) 0 4 (9.8%) 0 8 (5.8%) 1 (100%) 0 24 (100%) 61 (93.8%) 5 (100%) 37 (90.2%) 2 (100%) 130 (94.2%) p-valor comparación proporciones (*) 1 0.576 1 Total Tabla 38o. Comparación por grupos de las proporciones de pacientes con nódulos pulmonares con halo. 110 Con una confianza del 95%, no se detectaron diferencias estadísticamente significativas en cuanto a la proporción de pacientes con nódulos pulmonares con halo. HALO NÓDULO G1+G3+G5 G7 p-valor Si 0 0 NO 30 (100%) 2 (100%) 1 Tabla 38p. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con nódulos pulmonares con halo. TBC Halo Nódulo Si (G1+G3+G5) 0 30 (100%) Si No No (G2+G4+G6) 8 (7.5%) 98 (92.5%) p-valor= 0.2663 Tabla 38q. Comparación de las proporciones de pacientes con nódulos pulmonares con halo entre los grupos con y sin cultivo positivo para tuberculosis. No se detectaron diferencias significativas en cuanto a la proporción de pacientes con nódulos pulmonares con halo entre los grupos con cultivo positivo para tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. Tampoco al comparar los grupos de pacientes con cultivo positivo para M. tuberculosis y aquellos con cultivo negativo para dicho microorganismo.  Masa pulmonar: G1 G2 G3 G4 G5 G6 G7 Si No 1 (50%) 2 (7.1%) 23 (27.4%) 71 (8.2%) 5 (33.3%) 60 (11.0%) 0 162 (10.46%) 1 (50%) 26 (92.9%) 61 (72.6%) 793 (91.8%) 10 (66.7%) 487 (89.0%) 9 (100%) 1387 (89.54%) p-valor comparación proporciones (*) 0.1931 <0.0001 0.02124 Total Tabla 39a. Comparación por grupos de las proporciones de pacientes con masas pulmonares. Con una confianza del 95%, se observó una mayor proporción de masas pulmonares en los grupos con cultivo positivo para tuberculosis para los intervalos de edad de 18 a 65 años (G3-G4) y en mayores de 65 (G5-G6). 111 MASA G1+G3+G5 G7 p-valor Si 29 (28.7%) 0 NO 72 (71.3%) 9 (100%) 0.1393 Tabla 39b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con masa pulmonares. No se detectaron diferencias significativas en cuanto a la proporción de pacientes con masas pulmonares al comparar los grupos con cultivo positivo para tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. TBC MASA Si (G1+G3+G5) 29 (28.7%) 72 (71.3%) Si No No (G2+G4+G6) 133 (9.2%) 1306 (90.8%) p-valor<0.0001 RR de tener TBC debido a si MASA=3.426 I.C. 95%: [2.29 – 5.11] Odds ratio: 3.96 (I.C. 95%) (2.45 – 6.27) p-val<0.0001 Tabla 39c. Comparación de las proporciones de pacientes con masas pulmonares entre los grupos con y sin cultivo positivo para tuberculosis. Al comparar los grupos con cultivo positivo para M. tuberculosis y los que presentaron un cultivo negativo, se obtuvo una diferencia estadísticamente significativa en favor de los primeros en cuanto a la presencia de masas pulmonares. LOCALIZACION DE LA MASA PULMONAR 1 12 123 13 14 145 146 16 2 23 234 236 25 3 34 345 36 4 45 46 5 6 G1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G2 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G3 7 0 1 0 4 0 1 0 0 0 0 0 0 1 0 0 0 5 0 0 1 3 G4 23 0 0 1 2 1 0 2 6 1 1 0 0 7 0 0 0 15 1 0 3 8 G5 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 G6 23 1 0 0 2 0 0 0 4 1 0 1 1 6 1 1 0 11 0 0 3 5 G7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Tabla 39d. Clasificación por grupos de las diferentes localizaciones de masas pulmonares observadas en este estudio. 112 A continuación, se procedió a estudiar la presencia de masas en cada lóbulo por separado, obteniéndose la siguiente tabla: G1 1 0 0 0 0 0 1 2 3 4 5 6 pvalor G2 2 0 0 0 0 0 G3 13 1 2 10 1 4 1 G4 29 8 10 20 5 10 G5 3 0 2 2 0 2 0.78 G6 26 8 10 15 5 6 G7 0 0 0 0 0 0 0.70 Tabla 39e. Comparación por grupos de las proporciones de pacientes según la localización de las masas pulmonares. No se detectaron diferencias significativas en cuanto a la localización de las masas pulmonares. 40 60 lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 Porcentaje 80 100 Localización Masa G1 G2 G3 G4 G5 G6 Figura 10. Gráfico de barras apiladas con los porcentajes de pacientes con masas pulmonares en diferentes localizaciones por grupo. o Masa pulmonar calcificada: G1 G2 G3 G4 G5 G6 G7 Si No 0 0 0 1 (1.4%) 0 2 (3.3%) 0 3 (1.85%) 1 (100%) 2 (100%) 23 (100%) 70 (98.6%) 5 (100%) 58 (96.7%) 0 159 (98.15%) p-valor comparación proporciones (*) 1 1 1 Total Tabla 39f. Comparación por grupos de las proporciones de pacientes con masas pulmonares calcificadas. No se encontraron diferencias estadísticamente significativas en la proporción de masas pulmonares calcificadas entre los grupos estudiados. 113 MASA CALCIFICADA G1+G3+G5 G7 p-valor Si 0 0 NO 29 0 Tabla 39g. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con masa pulmonares calcificadas. TBC MASA CALCIFICADA Si (G1+G3+G5) 0 29 Si No No (G2+G4+G6) 3 (2.3%) 130 (97.7%) p-valor=0.9551 Tabla 39h. Comparación de las proporciones de pacientes con masas pulmonares calcificadas entre los grupos con y sin cultivo positivo para tuberculosis. Tampoco se detectaron diferencias significativas en cuanto a la proporción de masas pulmonares calcificadas, al comparar a los pacientes con cultivo positivo para tuberculosis y los pacientes con cultivo negativo, ni al comparar a los que tienen el cultivo positivo para M. tuberculosis con los que presentaron un cultivo positivo para micobacterias atípicas. LOCALIZACIÓN DE LA MASA PULMONAR CALCIFICADA 1 6 14 G1 0 0 0 G2 0 0 0 G3 0 0 0 G4 0 1 0 G5 0 0 0 G6 1 0 1 G7 0 0 0 Tabla 39i. Clasificación por grupos de las diferentes localizaciones de las masas pulmonares calcificadas observadas en este estudio. Si consideramos cada lóbulo pulmonar de forma independiente, obtenemos la siguiente tabla: 1 4 6 pvalor G1 0 0 0 G2 0 0 0 G3 0 0 0 G4 0 0 1 G5 0 0 0 G6 2 1 0 G7 0 0 0 Tabla 39j. Comparación por grupos de la presencia masas pulmonares calcificadas en cada lóbulo. No se objetivaron diferencias significativas en cuanto a la localización de las masas pulmonares calcificadas. 114 o Masa pulmonar cavitada: G1 G2 G3 G4 G5 G6 G7 Si No 1 (100%) 0 20 (87%) 34 (47.9%) 4 (80%) 23 (38.3%) 0 82 (50.62%) 0 2 (100%) 3 (13%) 37 (52.1%) 1 (20%) 37 (61.7%) 0 80 (49.38%) p-valor comparación proporciones (*) 0.33 0.0012 0.1513 Total Tabla 39k. Comparación por grupos de las proporciones de pacientes con masas pulmonares cavitadas. Se detectó una mayor proporción de pacientes con masas pulmonares cavitadas en el grupo G3 con respecto al grupo G4, siendo esta diferencia estadísticamente significativa. No fueron encontradas diferencias significativas al comparar los otros grupos. MASA CAVITADA G1+G3+G5 G7 p-valor Si 25 (86.2%) 0 NO 4 (13.8%) 0 Tabla 39l. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con masa pulmonares cavitadas. No fueron objetivadas diferencias significativas al comparar los grupos con tuberculosis con el grupo con cultivo positivo para micobacterias atípicas. TBC MASA CAVITADA Si No Si (G1+G3+G5) 25 (86.2%) 4 (13.8%) No (G2+G4+G6) 57 (42.9%) 76 (57.1%) p-valor<0.0001 RR de tener TBC debido a MASA CAVITADA = 6.09 I.C. 95%: [2.22 – 16.7] Odds ratio: 7.99 (I.C. 95%) (2.88 – 29.05) p-val<0.0001 Tabla 39m. Comparación de las proporciones de pacientes con masas pulmonares cavitadas entre los grupos con y sin cultivo positivo para tuberculosis. Se observó una diferencia estadísticamente significativa en cuanto a la presencia de masas pulmonares cavitadas al comparar los grupos con cultivo positivo para tuberculosis con los grupos con cultivo negativo para este microorganismo. 115 LOCALIZACIÓN DE LA MASA PULMONAR CAVITADA G1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 2 3 4 5 6 13 14 16 34 36 45 46 123 145 234 G2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G3 7 0 0 3 1 3 0 5 0 0 0 0 0 1 0 0 G4 10 2 3 9 0 3 1 1 2 0 0 1 0 0 1 1 G5 1 0 0 0 0 0 0 1 0 0 1 0 1 0 0 0 G6 11 1 5 1 3 1 0 0 0 1 0 0 0 0 0 0 G7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Tabla 39n. Clasificación por grupos de las diferentes localizaciones de masas pulmonares cavitadas observadas en este estudio. A continuación, se analizó la presencia de masas pulmonares cavitadas en cada lóbulo por separado, obteniéndose la siguiente tabla: G1 1 0 0 0 0 0 1 2 3 4 5 6 pvalor G2 0 0 0 0 0 0 G3 13 1 1 8 1 3 1 G4 15 3 5 13 2 5 G5 2 0 1 2 0 2 0.8596 G6 11 1 6 2 3 1 0.2469 G7 0 0 0 0 0 0 Tabla 39ñ. Comparación por grupos de la presencia masas pulmonares cavitadas en cada lóbulo. 40 60 lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 Porcentaje 80 100 No se observaron diferencias significativas en cuanto a la localización de las masas pulmonares cavitadas alLocalización comparar los grupos. Masa Cavitación G1 G3 G4 G5 G6 Figura 11. Gráfico de barras apiladas con los porcentajes de pacientes con masas pulmonares cavitadas en diferentes localizaciones por grupo. 116 o Micetoma: G1 G2 G3 G4 G5 G6 G7 Si No 0 0 0 1 (0.1%) 0 6 (1.1%) 0 7 (0.45%) 2 28 84 863 (99.9%) 15 541 (98.9%) 9 1542 (99.54%) p-valor comparación proporciones 1 1 1 Total Tabla 39o. Comparación por grupos de las proporciones de pacientes con micetoma. Con una confianza del 95%, no se detectaron diferencias estadísticamente significativas entre los grupos estudiados en cuanto a la presencia de micetomas. MICETOMA G1+G3+G5 G7 p-valor Si 0 0 NO 101 9 Tabla 39p. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con micetoma. TBC MICETOMA Si No Si (G1+G3+G5) 0 101 No (G2+G4+G6) 7 (0.5%) 1432 (99.5%) p-valor=1 Tabla 39q. Comparación de las proporciones de pacientes con micetoma entre los grupos con y sin cultivo positivo para tuberculosis. No fueron objetivadas diferencias significativas al comparar los grupos con cultivo positivos para tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. Tampoco fueron detectadas diferencias significativas al comparar los grupos con cultivo positivo para M. tuberculosis y aquellos que presentaron un cultivo negativo para dicho microorganismos. 117 LOCALIZACIÓN DE LOS MICETOMAS 1 4 5 pvalor G1 0 0 0 G2 0 0 0 G3 0 0 0 G4 0 1 0 G5 0 0 0 G6 4 1 1 G7 0 0 0 Tabla 39r. Clasificación por grupos de las diferentes localizaciones de micetoma observadas en este estudio. o Sobreinfección de cavidad pulmonar: G1 G2 G3 G4 G5 G6 G7 Si No 0 0 1 (1.2%) 4 (0.5%) 0 0 0 5 (0.32%) 2 28 83 (98.8%) 860 (99.5%) 15 547 9 1544 (99.68%) p-valor comparación proporciones 1 0.378 1 Total Tabla 39s. Comparación por grupos de las proporciones de pacientes con sobreinfección de una cavidad pulmonar. Con una confianza del 95%, no se detectaron diferencias estadísticamente significativas entre los grupos estudiados en cuanto a la presencia de cavidades pulmonares sobreinfectadas. SOBREINFECCION G1+G3+G5 G7 p-valor Si 1 0 NO 100 9 1 Tabla 39t. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con sobreinfección de una cavidad pulmonar. TBC SOBREINFECCION Si No Si (G1+G3+G5) 1 (1%) 100 (99%) RR de tener TBC debido a Sobreinfección=3.079 Odds ratio: 3.94 (I.C. 95%) (0.14 – 28.8) No (G2+G4+G6) 4 (0.3%) 1435 (99.7%) p-valor=0.75 I.C. 95%: [0.53 – 17.9] p-val=0.325 Tabla 39u. Comparación de las proporciones de pacientes con cavidades sobreinfectadas entre los grupos con y sin cultivo positivo para tuberculosis. 118 No se observaron diferencias significativas al comparar los grupos con cultivo positivos para tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. Tampoco fueron detectadas diferencias significativas al comparar los grupos con cultivo positivo para M. tuberculosis y aquellos que presentaron un cultivo negativo para dicho microorganismos. LOCALIZACIÓN DE LAS CAVIDADES PULMONARES SOBREINFECTADAS 1 3 6 13 G1 0 0 0 0 G2 0 0 0 0 G3 0 0 1 0 G4 2 1 0 1 G5 0 0 0 0 G6 0 0 0 0 Tabla 39v. Clasificación por grupos de las diferentes localizaciones de sobreinfectadas observadas en este estudio. G7 0 0 0 0 las cavidades Analizando cada lóbulo por separado, obtenemos la siguiente tabla: 1 3 6 pvalor G1 0 0 0 G2 0 0 0 G3 0 0 1 G4 3 2 0 0.1667 G5 0 0 0 G6 0 0 0 G7 0 0 0 Tabla 39w. Comparación por grupos de la presencia cavidades pulmonares sobreinfectadas en cada lóbulo. No hay diferencias significativas en cuanto a la sobreinfección de cavidades entre los distintos grupos de estudio. o Masas pulmonares con halo: G1 G2 G3 G4 G5 G6 G7 Si No 0 0 0 6 (8.5%) 0 5 (8.3%) 0 11 (6.79%) 1 2 23 (100%) 65 (91.5%) 5 (100%) 55 (91.7%) 0 151 (93.21%) p-valor comparación proporciones (*) 1 0.33 1 Total Tabla 39x. Comparación por grupos de las proporciones de pacientes con masas pulmonares con halo. Con una confianza del 95%, no se detectaron diferencias estadísticamente significativas entre los grupos estudiados en cuanto a la presencia de masas pulmonares con halo. 119 HALO. MASA G1+G3+G5 G7 p-valor Si 0 0 NO 29 (100%) 0 Tabla 39y. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con masas pulmonares con halo. TBC HALO. MASA Si (G1+G3+G5) 0 29 Si No No (G2+G4+G6) 11 (8.3%) 122 (91.7%) p-valor=0.2314 Tabla 39z. Comparación de las proporciones de pacientes con masas pulmonares con halo entre los grupos con y sin cultivo positivo para tuberculosis. No fueron objetivadas diferencias significativas al comparar los grupos con cultivo positivos para tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. Tampoco fueron detectadas diferencias significativas al comparar los grupos con cultivo positivo para M. tuberculosis y aquellos que presentaron un cultivo negativo para dicho microorganismos. LOCALIZACIÓN DE LAS MASAS PULMONARES CON HALO 1 2 4 5 pvalor G1 0 0 0 0 G2 0 0 0 0 G3 0 0 0 0 G4 3 (50%) 2 (33.3%) 1 (16.7%) 0 G5 0 0 0 0 G6 2 (40%) 1 (20%) 1 (20%) 1 (20%) G7 0 0 0 0 Tabla 39aa. Clasificación por grupos de las diferentes localizaciones de las masas pulmonares con halo observadas en este estudio.  Consolidación pulmonar: G1 G2 G3 G4 G5 G6 G7 Si No 2 (100%) 7 (25%) 44 (52.4%) 237 (27.4%) 4 (26.7%) 173 (31.6%) 2 (22.2%) 469 (30.28%) 0 21 (75%) 40 (47.6%) 627 (72.6%) 11 (73.3%) 374 (68.4%) 7 (77.8%) 1080 (69.72%) p-valor comparación proporciones 0.08 <0.0001 0.7853 Total Tabla 40a. Comparación por grupos de las proporciones de pacientes con consolidaciones pulmonares. 120 Se observó una mayor proporción de pacientes con consolidación pulmonar en el grupo G3 en comparación con el G4. CONSOLIDACION G1+G3+G5 G7 p-valor Si 50 (49.5%) 2 (22.2%) NO 51 (50.5%) 7 (77.8%) 0.2215 Tabla 40b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con consolidaciones pulmonares. No se detectaron diferencias estadísticamente significativas al comparar los grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. TBC No (G2+G4+G6) 417 (29%) 1022 (71%) p-valor<0.0001 RR de tener TBC debido a Consolidación=2.25 I.C. 95%: [1.55 – 3.28] Odds ratio: 2.40 (I.C. 95%) (1.59 – 3.61) p-val<0.0001 CONSOLIDACION Si (G1+G3+G5) 50 (49.5%) 51 (50.5%) Si No Tabla 40c. Comparación de las proporciones de pacientes con consolidaciones pulmonares entre los grupos con y sin cultivo positivo para tuberculosis. Fue detectada una mayor proporción de pacientes con consolidación pulmonar en el conjunto de grupos con cultivo positivo para tuberculosis en comparación con los grupos que tuvieron un cultivo negativo. LOCALIZACIÓN DE LAS CONSOLIDACIONES PULMONARES 5 1 2 3 4 5 6 12 13 14 15 16 23 24 25 26 36 45 5 G1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G2 0 0 2 0 0 1 0 1 1 0 0 0 0 0 0 1 0 G3 9 0 3 8 0 2 2 1 4 0 1 1 0 0 2 1 0 G4 21 20 38 18 6 32 3 7 6 2 0 11 1 1 3 16 3 Continúa en la siguiente página. 121 G5 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 1 G6 24 9 36 11 3 30 3 5 2 0 1 6 0 1 3 13 1 G7 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 46 56 123 125 134 135 136 146 235 236 245 256 346 356 456 1245 1246 1346 1356 1456 2356 12346 12456 23456 123456 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 1 0 2 0 1 0 0 1 0 0 0 0 0 0 1 2 0 2 0 1 0 0 0 6 9 2 2 1 0 2 2 0 4 1 1 1 4 1 1 0 4 2 0 2 2 2 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 1 0 1 0 1 3 1 2 5 0 0 0 3 0 1 0 1 1 0 3 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Tabla 40d. Clasificación por grupos de las diferentes localizaciones de las consolidaciones pulmonares observadas en este estudio. Analizando la localización de consolidaciones en cada lóbulo por separado, obtenemos la siguiente tabla: 1 G1 1(33.3%) 2 0 3 0 4 5 6 pvalor G2 3 (23.1%) 0 4 (30.8%) 1 2 (33.3%) (15.4%) 0 1 (7.7%) 1 3 (33.3%) (23.1%) 0.8857 G3 27 (32.1%) 7 (8.3%) G4 63 (15.1%) 58 (13.9%) 14 101 (16.7%) (24.6%) 21 54 (25.0%) (12.9%) 2 (2.4%) 42 (9.9%) 13 95 (15.5%) (23.1%) <0.0001 G5 2 (33.3%) G6 48 (17.0%) G7 0 0 35 (12.2%) 0 1 (16.7%) 83 (29.9%) 2 (33.3%) 24 (8.1%) 2 (66.7%) 0 1 (16.7%) 0 22 (7.4%) 69 (24.7%) 0 1 (33.3%) 0.1086 Tabla 40e. Comparación por grupos de la proporción de pacientes con consolidaciones pulmonares en cada lóbulo. Se observaron diferencias significativas en la distribución de las consolidaciones pulmonares entre los grupos G3 y G4, detectándose una mayor frecuencia de consolidaciones en los dos lóbulos superiores del grupo G3 respecto al G4. 122 40 60 todos los lóbulos lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 Porcentaje 80 100 Consolidación G1 G2 G3 G4 G5 G6 G7 Figura 12. Gráfico de barras apiladas con los porcentajes de pacientes con consolidaciones pulmonares cavitadas en diferentes localizaciones por grupo. o Consolidación pulmonar cavitada: G1 G2 G3 G4 G5 G6 G7 Si No 2 0 17 (38.6%) 31 (13.1%) 2 (50.0%) 8 (4.6%) 0 60 (12.79%) 0 7 27 (61.4%) 206 (86.9%) 2 (50%) 165 (95.4%) 2 409 (87.21%) p-valor comparación proporciones 0.028 0.0001 0.016 Total Tabla 40f. Comparación por grupos de las proporciones de pacientes con consolidaciones pulmonares cavitadas. Con una confianza del 95 %, se observó una diferencia significativa en cuanto a la presencia de consolidaciones cavitadas al comparar el grupo G1 con el G2, el G3 con el G4 y el G5 con el G6, estando más presentes dichas lesiones en los grupos con cultivo positivo para tuberculosis. CONSOLIDACION CAVITADA G1+G3+G5 G7 p-valor Si NO 21 (42%) 0 29 (58%) 2 0.651 Tabla 40g. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con consolidaciones pulmonares cavitadas. No se detectaron diferencias estadísticamente significativas en cuanto a la proporción de consolidaciones pulmonares cavitadas al comparar los grupos con tuberculosis con el grupo con cultivo positivo para micobacterias atípicas. 123 TBC No (G2+G4+G6) 39 (9.4%) 378 (90.6%) p-valor<0.0001 RR de tener TBC debido a Consolidación cavitada=4.91 I.C. 95%: [3.00 – 8.03] Odds ratio: 6.97 (I.C. 95%) (3.60 – 13.42) p-val<0.0001 CONSOLIDACION CAVITADA Si (G1+G3+G5) 21 (42%) 29 (58%) Si No Tabla 40h. Comparación de las proporciones de pacientes con consolidaciones pulmonares cavitadas entre los grupos con y sin cultivo positivo para tuberculosis. De forma significativa, se objetivó una mayor proporción de consolidaciones pulmonares cavitadas en el conjunto de grupos con cultivo positivo para M. tuberculosis y con el conjunto de grupos con cultivo negativo para dicho microorganismo. LOCALIZACIÓN DE LAS CONSOLIDACIONES PULMONARES CAVITADAS 1 2 3 4 6 12 14 23 36 45 46 134 236 1346 1356 1456 G1 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 G2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G3 3 0 2 7 0 1 0 0 0 0 0 1 1 1 0 1 G4 5 1 3 5 9 1 2 1 1 1 0 0 1 1 0 0 G5 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 G6 2 0 3 0 1 0 0 0 1 0 0 0 0 0 1 0 G7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Tabla 40i. Clasificación por grupos de las diferentes localizaciones de consolidaciones cavitadas observadas en este estudio. Si consideramos cada lóbulo pulmonar de forma independiente, obtenemos la siguiente tabla: 1 2 3 4 5 6 pvalor G1 1 0 0 1 0 1 G2 0 0 0 0 0 0 G3 7 2 5 10 1 3 G4 9 4 7 9 1 12 0.5025 G5 1 0 0 1 1 0 G6 3 0 5 0 1 3 G7 0 0 0 0 0 0 0.1033 Tabla 40j. Comparación por grupos de la presencia consolidaciones pulmonares cavitadas en cada lóbulo. 124 No se detectaron diferencias en cuanto a la localización de las consolidaciones cavitadas entre los grupos G3 y G4 ni entre los grupos G5 y G6. 40 60 lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 Porcentaje 80 100 Cavitación Consolidación G1 G3 G4 G5 G6 Figura 13. Gráfico de barras apiladas con los porcentajes de pacientes con consolidaciones pulmonares cavitadas en diferentes localizaciones por grupo.  Tractos fibróticos cicatriciales: G1 G2 G3 G4 G5 G6 G7 Si No 0 2 (7.1%) 26 (31%) 165 (19.1%) 5 (33.3%) 112 (20.5%) 1 (11.1%) 311 (20.08%) 2 26 (92.9%) 58 (69%) 699 (80.9%) 10 (66.7%) 435 (79.5%) 8 (88.9%) 1238 (79.92%) p-valor comparación proporciones 1 0.015 0.212 Total Tabla 41a. Comparación por grupos de las proporciones de pacientes con tractos fibróticos. De forma significativa, se objetivó una mayor proporción de pacientes con tractos fibróticos en el grupo G3 en comparación con el grupo G4. Tractos Fibróticos G1+G3+G5 G7 p-valor Si 31 (30.7%) 1 (11.1%) NO 70 (69.3%) 8 (88.9%) 0.3918 Tabla 41b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con tractos fibróticos. No se observó diferencias estadísticamente significativas, en cuanto a la presencia de tractos fibróticos, al comparar los grupos con cultivo positivo para tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. 125 TBC No (G2+G4+G6) 279 (19.4%) 1160 (80.6%) p-valor=0.009 RR de tener TBC debido a Tractos Fib.=1.75 I.C. 95%: [1.17 – 2.63] Odds ratio: 1.84 (I.C. 95%) (1.17 – 2.85) p-val=0.009 Tractos Fibróticos Si (G1+G3+G5) 31 (30.7%) 70 (69.3%) Si No Tabla 41c. Comparación de las proporciones de pacientes con tractos fibróticos entre los grupos con y sin cultivo positivo para tuberculosis. Se detectó una mayor proporción de tractos fibróticos en el conjunto de grupos con tuberculosis en comparación con el conjunto de grupos con cultivo negativo para dicho microorganismo. LOCALIZACIÓN DE TRACTOS FIBRÓTICOS 1 2 3 4 5 6 13 14 24 25 36 134 146 346 1245 1346 1356 1456 12346 123456 G1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G2 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 G3 6 0 0 4 0 1 0 13 1 0 0 0 1 0 0 0 0 0 0 0 G4 34 2 2 25 1 1 0 88 0 2 1 1 2 1 1 0 1 1 1 1 G5 1 0 1 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 G6 27 4 1 12 1 2 1 59 0 1 0 0 0 1 1 2 0 0 0 0 G7 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 Tabla 41d. Clasificación por grupos de las diferentes localizaciones de tractos fibróticos en este estudio. Si consideramos cada lóbulo por separado, obtenemos la siguiente tabla: 126 G1 0 0 0 0 0 0 1 2 3 4 5 6 pvalor G2 2 0 0 2 0 0 G3 20 1 0 19 0 2 G4 131 8 9 122 8 10 0.9116 G5 4 0 1 3 0 0 G6 90 6 5 75 3 5 G7 1 0 0 1 0 0 0.6507 Tabla 41e. Comparación por grupos de la presencia de tractos fibróticos en cada lóbulo. No se detectaron diferencias en cuanto a la localización de los tractos fibróticos entre los grupos analizados. 40 60 todos los lóbulos lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 Porcentaje 80 100 Tractos Fib. G2 G3 G4 G5 G6 G7 Figura 14. Gráfico de barras apiladas con los porcentajes de pacientes con tractos fibróticos en diferentes localizaciones por grupo.  Atelectasias: G1 G2 G3 G4 G5 G6 G7 Si No 0 5 (17.9%) 10 (11.9%) 161 (18.6%) 0 147 (26.9%) 2 (22.2%) 325 (20.98%) 2 23 (82.1%) 74 (88.1%) 703 (81.4%) 15 400 (73.1%) 7 (77.8%) 1224 (79.01%) p-valor comparación proporciones 1 0.1387 0.0151 Total Tabla 42a. Comparación por grupos de las proporciones de pacientes con atelectasias pulmonares. De forma significativa, se observó una mayor proporción de atelectasias pulmonares en el grupo G6 en comparación con el G5. 127 Atelectasias G1+G3+G5 G7 p-valor Si 10 (9.9%) 2 (22.2%) NO 91 (90.1%) 7 (77.8%) 0.5631 Tabla 42b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con atelectasias pulmonares. No se detectó diferencias estadísticamente significativas, en cuanto a la proporción de atelectasias pulmonares, al comparar los grupos con cultivo positivo para tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. TBC Si (G1+G3+G5) 10 (9.9%) 91 (90.1%) No (G2+G4+G6) Atelectasias Si 313 (21.8%) No 1126 (78.2%) p-valor=0.006 RR de tener TBC debido a Atelectasias=0.41 I.C. 95%: [0.22 – 0.79] Odds ratio: 0.40 (I.C. 95%) (0.19 – 0.74) p-val=0.002 Tabla 42c. Comparación de las proporciones de pacientes con atelectasias pulmonares entre los grupos con y sin cultivo positivo para tuberculosis. Se objetivó una mayor proporción de pacientes con atelectasia pulmonar en el conjunto de grupos con cultivo negativo para tuberculosis en comparación con el conjunto de grupos con cultivo positivo. LOCALIZACIÓN DE LAS ATELECTASIAS PULMONARES 6 1 12 123 12346 1236 12456 125 13 13456 1346 136 14 15 16 2 6 G1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G3 1 0 0 0 0 0 0 0 0 0 0 0 1 0 1 G4 6 0 1 1 0 0 1 3 0 0 1 0 0 1 21 G5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Continúa en la siguiente página. 128 G6 6 1 2 0 1 1 1 0 1 1 2 2 1 0 19 G7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 23 235 2356 236 24 245 2456 25 256 26 3 34 35 356 36 4 45 456 46 5 56 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 1 1 0 0 1 0 2 5 0 1 1 1 1 1 11 2 4 25 0 1 4 17 5 0 0 1 19 7 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 1 5 0 0 0 1 1 3 18 3 5 1 14 9 0 2 0 13 2 27 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 Tabla 42d. Clasificación por grupos de las diferentes localizaciones de las atelectasias en este estudio. Si se considera la presencia de atelectasias en cada lóbulo de forma independiente, obtenemos la siguiente tabla: 1 2 3 4 5 6 pvalor G1 0 0 0 0 0 0 G2 0 1 2 1 3 1 G3 2 1 2 2 4 2 G4 14 51 60 9 48 61 0.1055 G5 0 0 0 0 0 0 G6 19 40 58 19 32 60 G7 0 0 1 0 0 2 Tabla 42e. Comparación por grupos de la presencia de atelectasias en cada lóbulo. No se observaron diferencias significativas en cuanto a la localización de las atelectasias entre los grupos analizados. 129 40 60 lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 Porcentaje 80 100 Atelectasias G2 G3 G4 G6 G7 Figura 15. Gráfico de barras apiladas con los porcentajes de pacientes con atelectasias pulmonares en diferentes localizaciones por grupo.  Engrosamiento septal: G1 G2 G3 G4 G5 G6 G7 Si No 0 0 1 (1.2%) 26 (3%) 1 (6.7%) 23 (4.2%) 1 (11.1%) 52 (3.36%) 2 28 83 (98.8%) 838 (97%) 14 (93.3%) 524 (95.8%) 8 (88.9%) 1497 (96.64%) p-valor comparación proporciones 1 0.5031 0.4848 Total Tabla 43a. Comparación por grupos de las proporciones de pacientes con engrosamiento septal. Con una confianza del 95 %, no se identificaron diferencias significativas entre grupos al comparar las proporciones de pacientes con engrosamiento septal. Engrosamiento septal G1+G3+G5 G7 p-valor Si 2 (2%) 1 (11.1%) NO 99 (98%) 8 (88.9%) 0.5867 Tabla 43b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con engrosamiento septal. TBC Si (G1+G3+G5) 2 (2%) 99 (98%) No (G2+G4+G6) Engrosamiento Si 49 (3.4%) septal No 1390 (96.6%) p-valor=0.627 RR de tener TBC debido a engrosamiento septal =0.59 I.C. 95%: [0.15 – 2.32] Odds ratio: 0.61 (I.C. 95%) (0.09 – 2.02) p-val=0.48 Tabla 43c. Comparación de las proporciones de pacientes engrosamiento septal entre los grupos con y sin cultivo positivo para tuberculosis. 130 No se detectó diferencias estadísticamente significativas, en cuanto a la presencia de engrosamiento septal, al comparar los grupos con cultivo positivo para tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. Tampoco al comparar el conjunto de grupos con cultivo positivo para M. tuberculosis y los grupos con cultivo negativo para dicho microorganismo. LOCALIZACIÓN DE ENGROSAMIENTO SEPTAL G1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 2 3 4 6 13 14 25 36 56 123 134 136 236 1246 2356 123456 G2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G3 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 G4 3 0 0 5 1 1 1 0 8 0 1 1 0 0 0 0 5 G5 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 G6 2 2 2 0 2 0 3 1 5 0 1 0 1 2 1 1 0 G7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 Tabla 43d. Clasificación por grupos de las diferentes localizaciones de engrosamiento septal en este estudio. Si analizamos la presencia de engrosamiento septal en cada lóbulo por separado, obtenemos la siguiente tabla. 1 2 3 4 5 6 pvalor G1 0 0 0 0 0 0 G2 0 0 0 0 0 0 G3 0 0 0 1 0 0 G4 17 11 21 17 10 14 0.7576 G5 0 0 0 0 1 1 G6 8 8 12 4 2 12 G7 1 1 1 1 1 0 0.234 Tabla 43e. Comparación por grupos de la presencia de engrosamiento septal en cada lóbulo. No se objetivaron diferencias significativas en cuanto a la localización del engrosamiento septal entre los grupos analizados. 131  Pérdida de volumen pulmonar: G1 G2 G3 G4 G5 G6 G7 Si No 1 (50%) 1 (3.6%) 3 (3.6%) 7 (0.8%) 0 9 (1.6%) 0 21 (1.35%) 1 (50%) 27 (96.4%) 81 (96.4%) 857 (99.2%) 15 538 (98.4%) 9 1528 (98.65%) p-valor comparación proporciones 0.131 0.051 1 Total Tabla 44a. Comparación por grupos de las proporciones de pacientes con pérdida de volumen pulmonar. Con una confianza del 95 %, no se identificaron diferencias significativas entre grupos al comparar las proporciones de pacientes con pérdida de volumen pulmonar. Pérdida volumen G1+G3+G5 G7 p-valor Si 4 (4%) 0 NO 97 (96%) 9 1 Tabla 44b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con pérdida de volumen. TBC Pérdida de volumen Si No Si (G1+G3+G5) 4 (4%) 97 (96%) RR de tener TBC debido a Pérdida volumen.=2.98 Odds ratio: 3.54 (I.C. 95%) (0.98 – 9.89) No (G2+G4+G6) 17 (1.2%) 1422 (98.8%) p-valor=0.059 I.C. 95%: [1.21 – 7.35] p-val=0.0536 Tabla 44c. Comparación de las proporciones de pacientes con pérdida de volumen pulmonar entre los grupos con y sin cultivo positivo para tuberculosis. No se detectó diferencias estadísticamente significativas, en cuanto a la pérdida de volumen pulmonar, al comparar los grupos con cultivo positivo para tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. Tampoco al comparar el conjunto de grupos con cultivo positivo para M. tuberculosis y los grupos con cultivo negativo para dicho microorganismo. 132 LOCALIZACIÓN DE LA PÉRDIDA DE VOLUMEN PULMONAR 1 2 3 4 5 6 pvalor G1 0 0 0 1 1 1 G2 0 0 0 1 1 1 G3 1 0 1 1 1 1 1 G4 2 0 0 5 3 3 0.6937 G5 0 0 0 0 0 0 G6 4 4 3 4 3 4 G7 0 0 0 0 0 0 Tabla 44d. Clasificación por grupos de las diferentes localizaciones de la pérdida de volumen en este estudio. Al considerar la presencia de pérdida de volumen en cada lóbulo por separado, obtenemos la siguiente tabla: 1 2 3 4 5 6 pvalor G1 0 0 0 1 1 1 G2 0 0 0 1 1 1 G3 1 0 1 1 1 1 1 G4 2 0 0 5 3 3 0.6937 G5 0 0 0 0 0 0 G6 4 4 3 4 3 4 G7 0 0 0 0 0 0 Tabla 44e. Comparación por grupos de la presencia de pérdida de volumen en cada lóbulo. No hay diferencias significativas en cuanto a la localización de la pérdida de volumen entre los grupos G3 y G4. 40 60 lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 Porcentaje 80 100 Pérdida volumen G1 G2 G3 G4 G6 Figura 16. Gráfico de barras apiladas con los porcentajes de pacientes con pérdida de volumen pulmonar en diferentes localizaciones por grupo . 133  Enfisema: G1 G2 G3 G4 G5 G6 G7 Si No 0 3 (10.7%) 15 (17.9%) 220 (25.5%) 4 (26.7%) 145 (26.5%) 3 (33.3%) 390 (25.18%) 2 25 (89.3%) 69 (82.1%) 644 (74.5%) 11 (73.3%) 402 (73.5%) 6 (66.7%) 1159 (74.82%) p-valor comparación proporciones 1 0.1589 1 Total Tabla 45a. Comparación por grupos de las proporciones de pacientes con enfisema. Con una confianza del 95%, no se observaron diferencias estadísticamente significativas en cuanto a la distribución de enfisema en los diferentes grupos estudiados. Enfisema G1+G3+G5 G7 p-valor Si 19 (18.8%) 3 (33.3%) NO 82 (81.2%) 6 (66.7%) 0.5427 Tabla 45b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con enfisema. TBC No (G2+G4+G6) 368 (25.6%) 1071 (74.4%) p-valor=0.1628 RR de tener TBC debido a enfisema=0.69 I.C. 95%: [0.42 – 1.12] Odds ratio: 0.67 (I.C. 95%) (0.39 – 1.11) p-val=0.126 Enfisema Si (G1+G3+G5) 19 (18.8%) 82 (81.2%) Si No Tabla 45c. Comparación de las proporciones de pacientes con enfisema entre los grupos con y sin cultivo positivo para tuberculosis. Tampoco se observaron diferencias significativas en cuanto a la presencia de enfisema al comparar los grupos con cultivo positivo para M. tuberculosis con el conjunto de grupos con cultivo negativo para dicho microorganismo o con el grupo de micobacterias atípicas. LOCALIZACIÓN DEL ENFISEMA 1 2 3 4 12 13 14 23 24 123 124 G1 0 0 0 0 0 0 0 0 0 0 0 G2 1 2 0 0 0 0 0 0 0 0 0 G3 1 4 1 0 6 2 0 0 1 0 0 G4 91 52 11 15 42 1 1 2 0 4 1 G5 1 0 0 0 3 0 0 0 0 0 0 G6 50 29 5 10 45 5 1 0 0 0 0 Tabla 45d. Clasificación por grupos de las diferentes localizaciones de enfisema en este estudio. 134 G7 2 0 0 1 0 0 0 0 0 0 0 Al analizar la distribución del enfisema en lóbulos independientes, obtenemos la siguiente tabla: G1 G2 1 2 0 0 1 2 3 4 pvalor G3 9 11 3 1 G4 140 101 18 17 0.3732 G5 4 3 0 0 G6 101 74 10 11 G7 2 0 0 1 1 Tabla 45 e. Comparación por grupos de la presencia enfisema en cada lóbulo. No se hallaron diferencias significativas en cuanto a las localizaciones del enfisema en los diferentes grupos estudiados.  Patrón de “árbol en brote”: G1 G2 G3 G4 G5 G6 G7 Si No 1 (50%) 2 (7.1%) 15 (17.9%) 20 (2.3%) 4 (26.7%) 8 (1.5%) 0 50 (3.23%) 1 (50%) 26 (92.9%) 67 (82.1%) 844 (97.7%) 11 (73.3%) 539 (98.5%) 9 1499 (96.77%) p-valor comparación proporciones 0.19 <0.0001 <0.0001 Total Tabla 46a. Comparación por grupos de las proporciones de pacientes con patrón de “árbol en brote”. De forma significativa, se observó una mayor proporción de pacientes con patrón de “árbol en brote” en el grupo G3 en comparación con el G4 y en el G5 con respecto al G6. Árbol en brote G1+G3+G5 G7 p-valor Si 20 (19.8%) 0 NO 81 (80.2%) 9 (100%) 0.3054 Tabla 46b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con patrón de “árbol en brote”. No fueron detectadas diferencias significativas al comparar la proporción de pacientes con patrón de “árbol en brote” de los grupos con cultivo positivo para M. tuberculosis con el grupo con cultivo positivo para micobacterias atípicas. 135 TBC No (G2+G4+G6) 30 (2.1%) 1409 (97.9%) p-valor<0.0001 RR de tener TBC debido a Árbol en brote=7.35 I.C. 95%: [4.93 – 10.98] Odds ratio: 11.57 (I.C. 95%) (6.20 – 21.22) p-val<0.0001 Árbol en brote Si (G1+G3+G5) 20 (19.8%) 81 (80.2%) Si No Tabla 46c. Comparación de las proporciones de pacientes con patrón de “árbol en brote” entre los grupos con y sin cultivo positivo para tuberculosis. Se objetivó una mayor proporción de pacientes con patrón de “árbol en brote” en el conjunto de grupos con cultivo positivo para tuberculosis en comparación con el conjunto de grupos con cultivo negativo para tuberculosis. LOCALIZACIÓN DEL PATRÓN “DE ÁRBOL EN BROTE” 1 2 3 4 6 12 13 14 23 25 36 45 56 123 124 136 146 235 356 456 2356 12356 123456 G1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 G3 2 1 0 1 1 1 0 1 0 1 1 1 0 0 0 0 2 0 0 1 0 0 2 G4 4 0 0 1 2 0 1 0 1 1 0 0 1 2 0 0 0 1 1 0 1 0 4 G5 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 2 G6 2 0 1 0 1 0 0 0 0 0 2 1 0 0 0 1 0 0 0 0 0 0 0 G7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Tabla 46d. Clasificación por grupos de las diferentes localizaciones de patrón de “árbol en brote” observadas en este estudio. Al considerar la presencia de patrón de “árbol en brote” en cada lóbulo por separado. 136 G1 1 0 0 0 0 0 1 2 3 4 5 6 pvalor G2 3 3 3 2 3 3 G3 10 7 5 10 7 9 G4 15 14 15 9 13 13 0.5458 1 G5 5 5 4 5 5 5 G6 3 0 4 1 1 4 0.4219 G7 0 0 0 0 0 0 Tabla 46e. Comparación por grupos de la presencia de patrón de “árbol en brote” en cada lóbulo. No se observaron diferencias significativas entre los distintos grupos en cuanto a la presencia de árbol en brote. 40 60 todos los lóbulos lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 Porcentaje 80 100 Arbol en brote G1 G2 G3 G4 G5 G6 Figura 17. Gráfico de barras apiladas con los porcentajes de pacientes con patrón de “árbol en brote” en diferentes localizaciones por grupo.  Patrón en vidrio deslustrado: G1 G2 G3 G4 G5 G6 G7 Si No 0 3 (10.7%) 11 (13.1%) 181 (20.9%) 0 115 (21.0%) 2 (22.2%) 312 (20.14%) 2 25 (89.3%) 73 (86.9%) 683 (79.1%) 15 432 (79.0%) 7 (77.8%) 1237 (79.86%) p-valor comparación proporciones 1 0.089 0.049 Total Tabla 47a. Comparación por grupos de las proporciones de pacientes con patrón en vidrio deslustrado. Con una confianza del 95 %, se identificó una mayor proporción de pacientes con patrón en vidrio deslustrado en el grupo G6 en comparación con el grupo G5. 137 vidrio G1+G3+G5 G7 p-valor Si 11 (10.9%) 2 (22.2%) NO 90 (89.1%) 7 (77.8%) 0.6382 Tabla 47b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con patrón en vidrio deslustrado. No fueron detectadas diferencias significativas al comparar la proporción de pacientes con patrón en vidrio deslustrado de los grupos con cultivo positivo para M. tuberculosis con el grupo con cultivo positivo para micobacterias atípicas. TBC Vidrio deslustrado Si (G1+G3+G5) 11 (10.9%) 90 (89.1%) Si No RR de tener TBC debido a vidrio deslustrado=0.485 Odds ratio: 0.472 (I.C. 95%) (0.23 – 0.86) No (G2+G4+G6) 299 (20.8%) 1140 (79.2%) p-valor=0.023 I.C. 95%: [0.26 – 0.895] p-val=0.012 Tabla 47c. Comparación de las proporciones de pacientes con patrón en vidrio deslustrado entre los grupos con y sin cultivo positivo para tuberculosis. Se observó una mayor proporción de patrón en vidrio deslustrado en el conjunto de grupos con cultivo negativo para M. tuberculosis en comparación con el conjunto de grupos con cultivo positivo para dicho microorganismo. LOCALIZACIÓN DEL PATRÓN EN VIDRIO DESLUSTRADO7 1 2 3 4 5 6 12 13 14 15 16 23 24 25 26 34 7 G1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G2 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 G3 1 0 1 0 0 1 1 0 1 0 0 1 0 0 0 0 G4 20 13 8 10 1 7 5 3 18 1 2 5 2 3 0 3 Continúa en la siguiente página. 138 G5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G6 16 6 7 6 2 3 3 4 9 0 1 4 0 0 1 0 G7 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 35 36 45 46 56 123 124 125 134 136 145 146 156 234 236 246 346 356 456 1234 1235 1236 1245 1346 1356 2356 12345 12356 23456 123456 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 2 8 2 2 3 5 2 0 0 0 2 0 1 1 1 1 1 0 3 0 1 1 5 2 1 0 0 1 1 34 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 8 4 1 1 3 1 1 1 1 0 0 0 0 0 0 0 3 1 1 0 0 0 0 0 4 1 0 0 21 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 Tabla 47d. Clasificación por grupos de las diferentes localizaciones de patrón en vidrio deslustrado en este estudio. Al considerar la presencia de vidrio deslustrado en cada lóbulo por separado, se obtuvo la siguiente tabla: 1 2 3 4 5 6 pvalor G1 0 0 0 0 0 0 G2 2 2 3 3 2 3 G3 8 5 5 5 4 6 G4 138 115 110 123 93 103 0.9863 G5 0 0 0 0 0 0 G6 84 67 79 67 59 66 G7 3 2 2 2 2 2 Tabla 47e. Comparación por grupos de la presencia de vidrio deslustrado en cada lóbulo. No se observaron diferencias significativas en cuanto a la localización del patrón en vidrio deslustrado entre los grupos G3 y G4. 139 todos los lóbulos lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 40 60 Porcentaje 80 100 Vidrio deslustrado G2 G3 G4 G6 G7 Figura 18. Gráfico de barras apiladas con los porcentajes de pacientes con patrón en vidrio deslustrado en diferentes localizaciones por grupo.  Bronquiectasias: G1 G2 G3 G4 G5 G6 G7 Si No 0 5 (17.9%) 15 (17.9%) 187 (21.6%) 1 (6.7%) 155 (28.3%) 4 (44.4%) 367 (23.69%) 2 23 (82.1%) 69 (82.1%) 677 (78.4%) 14 (93.3%) 392 (71.7%) 5 (55.6%) 1182 (76.31%) p-valor comparación proporciones 1 0.4864 0.0796 Total Tabla 48a. Comparación por grupos de las proporciones de pacientes con bronquiectasias. No se detectaron diferencias significativas al comparar la proporción de pacientes con bronquiectasias pulmonares en los diferentes grupos. Bronquiectasias G1+G3+G5 G7 p-valor Si 16 (15.8%) 4 (44.4%) NO 85 (84.2%) 5 (55.6%) 0.0677 Tabla 48b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con bronquiectasias. TBC No (G2+G4+G6) 347 (24.1%) 1092 (75.9%) p-valor=0.076 RR de tener TBC debido a bronquiectasias=0.61 I.C. 95%: [0.36 – 1.03] Odds ratio: 0.59 (I.C. 95%) (0.33 – 1.00) p-val=0.053 Bronquiectasias Si No Si (G1+G3+G5) 16 (15.8%) 85 (84.2%) Tabla 48c. Comparación de las proporciones de pacientes con bronquiectasias entre los grupos con y sin cultivo positivo para tuberculosis. 140 No se observaron diferencias significativas al comparar los grupos con tuberculosis con el grupo con cultivo positivo para micobacterias atípicas en lo que respecta a la proporción de pacientes con bronquiectasias. Tampoco fueron identificadas diferencias estadísticamente significativas al comparar los grupos con cultivo positivo para M. tuberculosis con los grupos con cultivo negativo para dicho microorganismo. LOCALIZACIÓN DE LAS BRONQUIECTASIAS8 1 2 3 4 5 6 12 13 14 16 23 24 25 26 34 35 36 46 56 123 124 134 135 136 146 234 235 236 245 256 356 456 1235 1236 1245 1346 1456 2345 2356 8 G1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G2 0 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G3 2 0 0 3 2 1 0 1 1 0 0 0 0 0 0 0 0 1 1 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 G4 15 18 6 12 6 19 1 2 7 0 3 0 14 2 2 2 15 0 3 1 1 1 0 1 0 0 2 4 1 5 5 4 0 2 0 0 0 0 21 Continúa en la siguiente página. 141 G5 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G6 11 9 12 5 4 18 1 1 4 0 4 2 4 0 1 1 24 0 5 3 0 0 0 4 0 2 1 10 0 3 2 1 1 0 2 3 0 1 10 G7 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 12345 12346 12356 23456 123456 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 1 2 0 8 0 0 0 0 0 0 0 1 3 2 0 0 0 0 0 Tabla 48d. Clasificación por grupos de las diferentes localizaciones de bronquiectasias en este estudio. Al considerar la presencia de bronquiectasias en cada lóbulo por separado, se obtuvo la siguiente tabla: 1 G1 0 2 3 0 0 4 0 5 0 6 0 G2 2 (8.3%) 4 (25%) 3 (16.7%) 2 (8.3%) 3 (16.7%) 3 (16.7%) G3 7 (26.9%) 0 2 (7.7%) 7 (26.9%) 5 (19.2%) 5 (19.2%) pvalor G4 51 (10.2%) G6 35 (9.9%) G7 1 (10%) 95 (20.7%) 87 (18.8%) G5 1 (50%) 0 0 61 (17.7%) 88 (25.8%) 2 (20%) 2 (20%) 46 (9%) 0 28 (7.8%) 0 82 (17.6%) 0 43 (12.3%) 1 (10%) 100 (21.9%) 1 (50%) 88 (25.8%) 4 (40%) 0.0014 0.4248 Tabla 48e. Comparación por grupos de la presencia de bronquiectasias en cada lóbulo. 40 60 todos los lóbulos lób. sup. derecho lób. medio lób inferior derecho lób. sup. izquierdo língula lób. inferior izquierdo 0 20 Porcentaje 80 100 Se detectaron diferencias estadísticamente significativas en la localización de las bronquiectasias entre los grupos G3 y G4, observándose una mayor proporción de bronquiectasias en los lóbulos superior derecho e izquierdo en el grupo G3, y una mayor proporción de bronquiectasias en lóbulo inferior izquierdo en el grupo G4. No hay diferencias significativas entre el grupo G5 y G6. Bronquiectasias G2 G3 G4 G5 G6 G7 Figura 19. Gráfico de barras apiladas con los porcentajes de pacientes con bronquiectasias en diferentes localizaciones por grupo. 142 o Bronquiectasias sobreinfectadas: G1 G2 G3 G4 G5 G6 G7 Si No 0 1 (20%) 4 (26.7%) 56 (29.9%) 0 50 (32.3%) 2 (50%) 113 (30.79%) 0 4 (80%) 11 (73.3%) 131 (70.1%) 1 105 (67.7%) 2 (50%) 254 (69.21%) p-valor comparación proporciones 1 1 1 Total Tabla 49a. Comparación por grupos de las proporciones de pacientes con bronquiectasias sobreinfectadas Con una confianza del 95 %, no se detectaron diferencias significativas al comparar la proporción de pacientes con bronquiectasias pulmonares en los diferentes grupos. Bronquiectasias sobreinf. G1+G3+G5 G7 p-valor Si 4 (25%) 2 (50%) NO 12 (75%) 2 (50%) 0.7144 Tabla 49b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con bronquiectasias sobreinfectadas. Bronquiectasias sobreinf.= Bronquiectasias sobreinfectadas. TBC Si (G1+G3+G5) 4 (25%) 12 (75%) No (G2+G4+G6) Bronquiectasias Si 107 (30.8%) sobreinfectadas No 240 (69.2%) p-valor=0.8275 RR de tener TBC debido a Bronquiectasias sobreinf.=0.75 I.C. 95%: [0.25 – 2.29] Odds ratio: 0.77 (I.C. 95%) (0.20 – 2.29) p-val=0.651 Tabla 49c. Comparación de las proporciones de pacientes con bronquiectasias sobreinfectadas entre los grupos con y sin cultivo positivo para tuberculosis. Bronquiectasias sobreinf.= Bronquiectasias sobreinfectadas. No se observaron diferencias significativas al comparar los grupos con tuberculosis con el grupo con cultivo positivo para micobacterias atípicas en lo que respecta a la proporción de pacientes con bronquiectasias. Tampoco fueron identificadas diferencias estadísticamente significativas al comparar los grupos con cultivo positivo para M. tuberculosis con los grupos con cultivo negativo para dicho microorganismo. 143 LOCALIZACIÓN DE LAS BRONQUIECTASIAS SOBREINFECTADAS G1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 2 3 4 5 6 14 23 24 25 26 36 56 134 146 235 236 245 356 1236 2356 G2 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G3 3 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G4 7 4 3 3 2 9 1 1 0 3 2 9 3 1 1 1 0 1 2 2 2 G5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G6 1 3 8 6 1 8 1 1 1 2 2 11 2 0 0 0 1 0 0 0 2 G7 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 Tabla 49d. Clasificación por grupos de las diferentes localizaciones de bronquiectasias sobreinfectadas en este estudio. Al considerar la presencia de bronquiectasias sobreinfectadas en cada lóbulo pulmonar por separado, se obtuvo la siguiente tabla: 1 G1 0 G2 0 2 3 4 0 0 0 1 0 0 5 6 pvalor 0 0 0 0 G3 G4 4 11 (12.5%) (66.7%) 0 16 (18.2%) 0 19 (21.6%) 2 6 (6.8%) (33.3%) 0 9 (10.2%) 0 27 (30.7%) 0.00246 G5 0 G6 2 G7 0 0 0 0 12 21 8 1 2 0 0 0 3 24 0 1 Tabla 49e. Comparación por grupos de la presencia bronquiectasias sobreinfectadas en cada lóbulo. Se observaron diferencias significativas en la localización de bronquiectasias infectadas entre los grupos G3 y G4, objetivándose una mayor proporción de bronquiectasias sobreinfectadas en el lóbulo superior derecho, en el grupo G3, y en el lóbulo inferior izquierdo, en el grupo G4. 144 4.1.4.2. Hallazgos mediastínicos:  Adenopatías: G1 G2 G3 G4 G5 G6 G7 Si No 0 7 (25%) 27 (32.1%) 158 (18.3%) 7 (46.7%) 100 (18.3%) 3 (33.3%) 302 (19.5%) 2 21 (75%) 57 (67.9%) 706 (81.7%) 8 (53.3%) 447 (81.7%) 6 (66.7%) 1247 (80.5%) p-valor comparación proporciones 1 0.003 0.013 Total Tabla 50a. Comparación por grupos de las proporciones de pacientes con adenopatías. De forma significativa, se objetivó una mayor proporción de adenopatías en el grupo G3 en comparación con el G4 y en el G5 en comparación con el G6. ADENOPATIAS G1+G3+G5 G7 p-valor Si 34 (33.7%) 3 (33.3%) NO 67 (66.3%) 6 (66.7%) 1 Tabla 50b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con adenopatías. No fueron identificadas diferencias estadísticamente significativas al comparar los grupos con cultivo positivo para tuberculosis con el grupo con cultivo positivo para micobacterias atípicas. TBC No (G2+G4+G6) 265 (18.4%) 1174 (81.6%) p-valor<0.0001 RR de tener TBC debido a Adenopatías=2.11 I.C. 95%: [1.42 – 3.12] Odds ratio: 2.25 (I.C. 95%) (1.44 – 3.45) p-val=0.0004 ADENOPATIAS Si No Si (G1+G3+G5) 34 (33.7%) 67 (66.3%) Tabla 50c. Comparación de las proporciones de pacientes con adenopatías entre los grupos con y sin cultivo positivo para tuberculosis. Se observó una mayor proporción de pacientes con adenopatías en el conjunto de grupos con cultivo positivo para tuberculosis en comparación con aquellos en los que fue negativo. Localización: Para la recogida de datos en nuestro estudio, se codificaron las estaciones ganglionares más representativas con las letras del alfabeto, de la forma que se expone a continuación: 145 A: mediastino anterosuperior (ganglios mediastínicos alto). B: paratraqueales C: prevasculares y retrotraqueales D: subaórtica E: paraaórticas F: subcarinales G: paraesofágicas H: hiliares I: interlobares y lobulares. A9 AB ABC ABCDF ABD ABDE ABDEF ABDEFI ABDF ABDFHI ABDFI ABDH ABE ABEH ABF ADE AI B BC BCD BCDE BCE BCEF BCEH BCF BCFGH BCFH BD BDE BDEF BDEFI BDEI 9 G1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G2 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 G3 0 1 0 0 0 0 0 0 1 0 0 0 1 0 1 0 0 3 0 0 0 1 1 0 0 0 0 2 0 1 1 0 G4 3 1 1 1 1 2 1 1 2 1 0 1 0 1 1 0 1 34 1 1 0 0 0 0 1 0 0 12 3 8 2 0 Continúa en la siguiente página 146 G5 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G6 1 1 0 0 0 2 0 0 3 0 0 0 2 0 1 0 0 23 2 0 2 0 0 1 0 1 1 12 1 3 0 1 G7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 BDF BDFG BDFH BDFHI BDH BE BEF BEFH BEI BF10 BFH BFI BH BI C CD CDFH CF D DE DEF DEFH DF DFH DFHI DH DI E EF EH EI F FH FHI FI H HI I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 1 0 0 0 1 0 0 0 0 1 1 0 0 1 1 0 0 1 0 0 0 0 1 0 0 0 0 0 1 0 1 0 2 4 1 2 1 4 4 2 1 1 8 0 2 1 1 1 0 1 1 19 1 0 1 4 0 1 0 1 2 2 1 1 3 2 0 1 3 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 1 0 0 4 1 1 0 0 1 1 0 0 8 2 1 0 4 1 0 1 0 3 0 0 0 5 0 0 0 3 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 Tabla 50d. Clasificación por grupos de las diferentes localizaciones de adenopatías en este estudio. Al considerar la presencia de adenopatías en cada cadena ganglionar por separado, se obtuvo la siguiente tabla: A B C S 10 G1 0 0 0 0 G2 1 3 0 6 G3 4 17 4 11 G4 18 108 8 76 Continúa en la siguiente página. 147 G5 2 3 0 5 G6 10 67 9 46 G7 0 0 0 2 E F G H I pvalor 0 0 0 0 0 4 2 0 0 0 8 9 0 3 4 34 55 1 21 15 2 3 0 1 1 0.666 19 31 1 10 4 0 2 0 0 0 0.5219 Tabla 50e. Comparación por grupos de la presencia de adenopatías en cada lóbulo. No hay diferencias significativas en la localización de las adenopatías en los distintos grupos. 40 60 mediastino anterosuperior paratraqueales prevasculares y retrotraqueales subaórtica paraaórticas subcarinales paraesofágicas hiliares interlobares y lobulares 0 20 Porcentaje 80 100 Adenopatías G2 G3 G4 G5 G6 G7 Figura 20. Gráfico de barras apiladas con los porcentajes de pacientes con adenopatías en diferentes localizaciones por grupo. 4.1.4.3. Hallazgos pleuropericárdicos:  Derrame pleural: G1 G2 G3 G4 G5 G6 G7 Si No 0 4 (14.3%) 9 (10.7%) 140 (16.2%) 2 (13.3%) 122 (22.3%) 2 (22.2%) 279 (18.01%) 2 24 (85.7%) 75 (89.3%) 724 (83.8%) 13 (86.7%) 425 (77.7%) 7 (77.8%) 1270 (81.99%) p-valor comparación proporciones 1 0.2116 0.5404 Total Tabla 51a. Comparación por grupos de las proporciones de pacientes con derrame pleural. Con una confianza del 95 %, no se detectaron diferencias estadísticamente significativas entre los diferentes grupos al comparar la proporción de pacientes con derrame pleural. 148 Derrame pleural G1+G3+G5 G7 p-valor Si 11 (10.9%) 2 (22.2%) NO 90 (89.1%) 7 (77.8%) 0.6382 Tabla 51b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con derrame pleural. TBC No (G2+G4+G6) 266 (18.5%) 1173 (81.5%) p-valor=0.074 RR de tener TBC debido a derrame pleural=0.56 I.C. 95%: [0.30 – 1.03] Odds ratio: 0.54 (I.C. 95%) (0.27 – 0.99) p-val=0.047 Derrame Pleural Si (G1+G3+G5) 11 (10.9%) 90 (89.1%) Si No Tabla 51c. Comparación de las proporciones de pacientes con derrame pleural entre los grupos con y sin cultivo positivo para tuberculosis. No se objetivaron diferencias significativas en cuanto a la proporción de pacientes con derrame pleural al comparar los grupos con cultivo positivo para M. tuberculosis con el grupo con cultivo positivo para micobacterias atípicas. Tampoco al comparar los grupos con cultivo positivo para tuberculosis con los que presentaron un cultivo negativo para dicho microorganismo. LOCALIZACIÓN DEL DERRAME PLEURAL Bilateral G1 0 Der. 0 Izq. 0 p-valor G2 1 (25%) 2 (50%) 1 (25%) G3 G4 2 47 (33.6%) (22.2%) 2 53 (37.9%) (22.2%) 5 40 (28.6%) (55.6%) 0.2618 G5 1 (50%) 1 (50%) 0 G6 63 (51.6%) G7 1 (50%) 29 (23.8%) 0 30 (24.6%) 1 (50%) 0.7356 Tabla 51d. Clasificación por grupos de las diferentes localizaciones del derrame pleural en este estudio. No hay diferencias significativas en cuanto a la localización de derrame pleural entre los distintos grupos.  Empiema: G1 G2 G3 G4 G5 G6 G7 Si No 0 0 3 (3.6%) 27 (3.1%) 0 15 (2.7%) 0 45 (2.90%) 2 28 81 (96.4%) 837 (96.9%) 15 532 (97.3%) 9 1504 (97.1%) p-valor comparación proporciones 1 0.7434 1 Total Tabla 52a. Comparación por grupos de las proporciones de pacientes con empiema. 149 No se observaron diferencias estadísticamente significativas en cuanto al porcentaje de pacientes con empiema, al comparar los diferentes grupos. Empiema G1+G3+G5 G7 p-valor Si 3 (3%) 0 NO 98 (97%) 9 1 Tabla 52b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con empiema. TBC Empiema Si (G1+G3+G5) 3 (3%) 98 (97%) Si No No (G2+G4+G6) 42 (2.9%) 11397 (97.1%) p-valor=1 RR de tener TBC debido a empiema =1.01 I.C. 95%: [0.33 – 3.08] Odds ratio: 1.06 (I.C. 95%) (0.25 – 3.01) p-val=0.92 Tabla 52c. Comparación de las proporciones de pacientes con empiema entre los grupos con y sin cultivo positivo para tuberculosis. No se detectaron diferencias significativas en cuanto a la proporción de pacientes con empiema al comparar los grupos con tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. Tampoco se objetivaron diferencias significativas al comparar los grupos con cultivo positivo para M. tuberculosis y los grupos con cultivo negativo para dicho microorganismo. Localización: Der. G1 0 G2 0 Izq. 0 0 G3 2 (66.7%) 1 (33.3%) p-valor G4 15 (55.6%) G5 0 G6 9 (60%) G7 0 12 (44.4%) 0 6 (40%) 0 1 Tabla 52d. Clasificación por grupos de las diferentes localizaciones del empiema en este estudio. No hay diferencias significativas en cuanto a la localización del empiema entre los distintos grupos.  Engrosamiento pleural: G1 G2 G3 G4 G5 G6 G7 Si No 0 0 3 (3.6%) 34 (3.9%) 0 34 (6.2%) 0 71 (4.58%) 2 28 81 (96.4%) 830 (96.1%) 15 513 (93.8%) 9 1478 (95.42%) p-valor comparación proporciones 1 1 1 Total Tabla 53a. Comparación por grupos de las proporciones de pacientes con engrosamiento pleural. 150 Con una confianza del 95 %, no se detectaron diferencias estadísticamente significativas entre los diferentes grupos al comparar la proporción de pacientes con engrosamiento pleural. Engrosamiento pleural G1+G3+G5 G7 p-valor Si 3 (3%) 0 NO 98 (97%) 9 1 Tabla 53b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con engrosamiento pleural. TBC No (G2+G4+G6) 68 (4.7%) 1371 (95.3%) p-valor=0.5703 RR de tener TBC debido a engrosamiento pleural =0.63 I.C. 95%: [0.20 – 1.95] Odds ratio: 0.65 (I.C. 95%) (0.15 – 1.78) p-val=0.44 Engrosamiento Pleural Si (G1+G3+G5) 3 (3%) 98 (97%) Si No Tabla 53c. Comparación de las proporciones de pacientes con engrosamiento pleural entre los grupos con y sin cultivo positivo para tuberculosis. No se detectaron diferencias significativas en cuanto a la proporción de pacientes con engrosamiento pleural al comparar los grupos con tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. Tampoco se objetivaron diferencias significativas al comparar los grupos con cultivo positivo para M. tuberculosis y los grupos con cultivo negativo para dicho microorganismo. Localización: 1 2 3 4 5 6 pvalor G1 0 0 0 0 0 0 G2 0 0 0 0 0 0 G3 1 1 2 0 0 1 G4 21 3 12 15 3 10 0.3183 G5 0 0 0 0 0 0 G6 17 3 13 18 2 12 G7 0 0 0 0 0 0 Tabla 53d. Clasificación por grupos de las diferentes localizaciones de engrosamiento pleural en este estudio. Al considerar la presencia del engrosamiento pleural en cada lóbulo por separado, se obtuvo la siguiente tabla: 151 G1 0 0 0 0 0 0 1 2 3 4 5 6 G2 0 0 0 0 0 0 G3 1 1 2 0 0 1 G4 20 2 11 14 2 9 G5 0 0 0 0 0 0 G6 16 2 12 17 1 11 G7 0 0 0 0 0 0 Tabla 53e. Comparación por grupos de la presencia de engrosamiento pleural en cada lóbulo.  Paquipleuritis cálcica: G1 G2 G3 G4 G5 G6 G7 Si No 0 1 (3.6%) 0 16 (1.9%) 1 (6.7%) 19 (3.5%) 0 37 (2.39%) 2 27 (96.4%) 84 848 (98.1%) 14 (93.3%) 528 (96.5%) 9 1512 (97.61%) p-valor comparación proporciones 1 0.3862 0.4234 Total Tabla 54a. Comparación por grupos de las proporciones de pacientes con paquipleuritis cálcica. Con una confianza del 95 %, no se detectaron diferencias estadísticamente significativas entre los diferentes grupos al comparar la proporción de pacientes con paquipleuritis cálcica. Paquipleuritis cálcica G1+G3+G5 G7 p-valor Si 1 (1%) 0 NO 100 (99%) 9 1 Tabla 54b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con paquipleuritis cálcica. TBC No (G2+G4+G6) 36 (2.5%) 1403 (97.5%) p-valor=0.5334 RR de tener TBC debido a Paquipleuritis cálcica =0.41 I.C. 95%: [0.06 – 2.83] Odds ratio: 0.44 (I.C. 95%) (0.02 – 2.06) p-val=0.36 Paquipleuritis cálcica Si No Si (G1+G3+G5) 1 (1%) 100 (99%) Tabla 54c. Comparación de las proporciones de pacientes con paquipleuritis cálcica entre los grupos con y sin cultivo positivo para tuberculosis. No se detectaron diferencias significativas en cuanto a la proporción de pacientes con paquipleuritis cálcica al comparar los grupos con tuberculosis y el grupo con cultivo positivo para micobacterias atípicas. Tampoco se 152 objetivaron diferencias significativas al comparar los grupos con cultivo positivo para M. tuberculosis y los grupos con cultivo negativo para dicho microorganismo.  Derrame pericárdico: G1 G2 G3 G4 G5 G6 G7 Si No 0 1 (3.6%) 0 8 (0.9%) 0 4 (0.7%) 0 13 (0.84%) 2 27 (96.4%) 84 856 (99.1%) 15 543 (99.3%) 9 1536 (99.16%) p-valor comparación proporciones 1 1 1 Total Tabla 55a. Comparación por grupos de las proporciones de pacientes con derrame pericárdico. Con una confianza del 95 %, no se detectaron diferencias estadísticamente significativas entre los diferentes grupos al comparar la proporción de pacientes con derrame pleural. Derrame pericárdico G1+G3+G5 G7 p-valor Si 0 0 NO 101 9 Tabla 55b. Comparación entre grupos con cultivo positivo para M. tuberculosis y el grupo con cultivo positivo para M. atípicas en cuanto a la proporción de pacientes con derrame pericárdico. TBC Derrame Pericárdico Si No Si (G1+G3+G5) 0 101 No (G2+G4+G6) 13 (0.9%) 1426 (99.1%) p-valor=0.6916 Tabla 55c. Comparación de las proporciones de pacientes con pérdida de derrame pericárdico entre los grupos con y sin cultivo positivo para tuberculosis. No se objetivaron diferencias significativas en cuanto a la proporción de pacientes con derrame pericárdico al comparar los grupos con cultivo positivo para M. tuberculosis con el grupo con cultivo positivo para micobacterias atípicas. Tampoco al comparar los grupos con cultivo positivo para tuberculosis con los que presentaron un cultivo negativo para dicho microorganismo. 4.2. Análisis multivariante: 4.2.1. Variables epidemiológicas: Exploramos el poder predictivo de las variables: sexo, edad, anemia, TB previa, VIH, trasplante, alcoholismo, esteroides, colagenopatia, EPOC, fumador. Al ajustar el modelo para predecir la presencia de tuberculosis en función de 153 todas estas variables, obtenemos un resultado que hay que depurar mediante un “proceso de eliminación hacia atrás”. Es entonces cuando conseguimos un modelo que habrá eliminado aquellas variables no influyentes a la hora de realizar la predicción. En la siguiente tabla se incluye las que han resultado significativas tras el "proceso de eliminación hacia atrás" (beta estimada (β) con error estándar (EE) y odds ratio (OR) con su valor p correspondiente). Tabla 56. Variables epidemiológicas influyentes a la hora de predecir la presencia de una tuberculosis pulmonar. EE = Error Estandar; OR = Odds Ratio; TBC. Previa = tuberculosis previa. Códigos de significación: 0-0.001 = “***”; 0.001-0.01 = “**”; 0.01-0.05 = “*”; 0.05-0.1 = “.”; 0.1-1 =” “. 4.2.2. Variables clínicas: Se explora el poder predictivo de las variables: fiebre, febrícula, pérdida de peso, tos, dolor torácico, sudoración, disnea, hemoptisis, mal estado general (MEG), síndrome constitucional. Ninguna de ellas resulta significativa en el modelo de predicción. 4.2.3. Variables en radiografía de tórax: Se explora el poder predictivo de las variables: nódulos (No; Únicos; Múltiples), masa en radiografía (No; Si; cavitada sí/no), consolidación (No; Si; cavitada sí/ no), atelectasia, derrame pleural, masa mediastínica, masa en hilio, bronquiectasia, patrón Intersticial. En la siguiente tabla se incluye las que han resultado significativas tras el “proceso de eliminación hacia atrás” (beta estimada (β) con error estándar (EE) y odds ratio (OR) con su valor de p correspondiente.). Tabla 57. Variables de radiografía de tórax influyentes a la hora de predecir la presencia de una tuberculosis pulmonar. EE = Error Estándar; OR = Odds Ratio; Rx = radiografía; (C) = cavitación; Masa. Med = masa mediastínica. Códigos de significación: 0-0.001 = “***”; 0.001-0.01 = “**”; 0.01-0.05 = “*”; 0.05-0.1 = “.”; 0.1-1 =” “. 154 4.2.4. Variables en TC:  Hallazgos pulmonares: Se explora el poder predictivo de las variables: micronódulos (No; Únicos; Múltiples), patrón miliar (Si/No), nódulo (No; Si; cavitado sí/ no), masa pulmonar (No; Si; cavitado sí/no), micetoma (Si/No), sobreinfección (Si/No), consolidación pulmonar (No; Si; cavitado sí/ no), vidrio deslustrado (Si/No), bronquiectasia (Si/No), tractos fibróticos (Si/No), atelectasia, pérdida de volumen, engrosamiento septal, enfisema, árbol en brote. Al ajustar el modelo para predecir la presencia de tuberculosis pulmonar en función de todas estas variables, obtenemos el siguiente resultado. Esta tabla no muestra los OR. Sólo muestra si la variable es significativa o no en la regresión. Null deviance: 745.56 on 1539 degrees of freedom Residual deviance: 480.93 on 1520 degrees of freedom (9 observations deleted due to missingness) AIC: 520.93 Tabla 58a. Variables de TC de tórax relacionadas con la tuberculosis pulmonar. EE = Error Estándar; (intercept) = punto de corte con el eje Y, es decir, el valor que toma Y cuando X = 0; Z = valor estadístico de contraste; Pr(>[z]) = p-valor; (C) = cavitación. Códigos de significación: 0-0.001 = “***”; 0.001-0.01 = “**”; 0.01-0.05 = “*”; 0.05-0.1 = “.”; 0.1-1 =” “. Se depura el modelo mediante un “proceso de eliminación hacia atrás”, y entonces obtenemos un modelo que eliminó aquellas variables no influyentes a la hora de realizar la predicción. De las variables resultantes, se muestra los valores beta (β) estimada con Error Estándar (EE) y la odds ratio (OR) con su correspondiente p-valor. 155 Null deviance: 745.56 on 1539 degrees of freedom Residual deviance: 486.47 on 1525 degrees of freedom (9 observations deleted due to missingness) AIC: 516.47 Tabla 58b. Variables de TC (hallazgos pulmonares) influyentes a la hora de predecir la presencia de una tuberculosis pulmonar. EE = Error Estándar; OR = Odds Ratio; (C) = cavitación. Códigos de significación: 0-0.001 = “***”; 0.001-0.01 = “**”; 0.01-0.05 = “*”; 0.05-0.1 = “.”; 0.1-1 =” “.  Hallazgos de pleura y mediastino: Exploramos el poder predictivo de los hallazgos en pleura y mediastino: adenopatías, derrame pleural, derrame pericárdico, empiema, engrosamiento pleural. En la siguiente tabla se incluye las que han resultado significativas tras el “proceso de eliminación hacia atrás” (beta estimada (β) con error estándar (ES) y odds ratio (OR) con su valor de p correspondiente.). Tabla CD. Variables de TC (hallazgos pleuromediastínicos) influyentes a la hora de predecir la presencia de una tuberculosis pulmonar. ES = Error Estándar; OR = Odds Ratio. Códigos de significación: 0-0.001 = “***”; 0.001-0.01 = “**”; 0.01-0.05 = “*”; 0.05-0.1 = “.”; 0.1-1 =” “. 156 4.2.5. Modelo global: Para construir un modelo global, inicialmente reunimos todos los tipos de variables, eligiendo sólo aquellas que fueron significativas. Null deviance: 745.42 on 1538 degrees of freedom Residual deviance: 410.50 on 1512 degrees of freedom (10 observations deleted due to missingness) AIC: 464.5 Tabla 59a. Variables epidemiológicas, de radiografía y TC de tórax relacionadas con la tuberculosis pulmonar. EE = Error Estándar; OR = Odds ratio; Pr(>[z]) = p-valor; (C) = cavitación; RX = radiografía. Códigos de significación: 0-0.001 = “***”; 0.001-0.01 = “**”; 0.01-0.05 = “*”; 0.05-0.1 = “.”; 0.1-1 =” “. Para establecer una puntuación para cada una de las variables que nos dé una puntuación global a partir de estos resultados, redondeamos los coefientes estimados () al entero más próximo, dando puntuación de 0 a todas aquellas variables que no han quedado incluidas en el modelo (ver tabla 60b). 157 Null deviance: 745.42 on 1538 degrees of freedom Residual deviance: 410.50 on 1512 degrees of freedom (10 observations deleted due to missingness) AIC: 464.5 Number of Fisher Scoring iterations: 7 Tabla 59b. Variables epidemiológicas, de radiografía y TC de tórax relacionadas con la tuberculosis pulmonar con su respectiva puntuación (score). EE = Error Estándar; OR = Odds ratio; Pr(>[z]) = p-valor; (C) = cavitación; RX = radiografía. Códigos de significación: 0-0.001 = “***”; 0.001-0.01 = “**”; 0.01-0.05 = “*”; 0.05-0.1 = “.”; 0.1-1 =” “. Tras esto, asignamos una puntuación a cada persona de la base de datos según su valor en cada una de las variables, tal como establece el modelo expuesto (anexo 3). La variante edad, al tener un valor numérico, tiene un tratamiento distinto: cada año de más contribuye en la puntuación con 0.026679, por lo que a la puntuación de cada persona, además del resto de variables, hay que contar con -0.03 x Edad (en años). La tabla 59c muestra la frecuencia de las puntuaciones obtenidas por los pacientes del estudio, relacionándolas con la presencia o no de tuberculosis confirmada (por ejemplo: un paciente obtuvo una puntuación de -6 y no presentó cultivo para tuberculosis; 13 pacientes presentaron una puntuación de -5 y no mostraron cultivo positivo para tuberculosis; y así, sucesivamente). 158 Tabla 59c. Frecuencias de las diferentes puntuaciones entre los pacientes con o sin tuberculosis. A partir de los datos de la tabla anterior se puede obtener la siguiente curva ROC: Figura 21. Curva ROC correspondiente a la distribución teórica de los resultados de la aplicación del modelo de puntuación propuesto. El área bajo la curva es 0.9163 (95% CI: 0.8858 – 0.9467) y el mejor punto de corte, según el método de Youden, es 1.845 (sensibilidad de 0.851 y especificidad de 0.836). El resto de medidas asociadas a este test obtenidas las mostramos en la siguiente tabla: RESULTADO TEST POSITIVO NEGATIVO TUBERCULOSIS SI (G1, G3, G5) NO (G2, G4, G6) 86 237 (verdadero positivo) (falso positivo) 15 1210 (falso negativo) (verdadero negativo) Tabla 59d. Tabla de contingencia para analizar la relación entre la tuberculosis y el resultado de nuestro test. 159 Así, tal como se ve en la tabla 59d, este modelo predictivo con un punto de corte en 1.845 tiene una sensibilidad del 85.1 %, especificidad del 83.6 %, VPP de 26.6 % y VPN de 98.7 % para diferenciar entre pacientes con tuberculosis pulmonar activa frente a aquellos que presentan otra infección pulmonar. 160 5. DISCUSIÓN 161 162 Los resultados de este estudio han mostrado que utilizando los modelos desarrollados sobre la base de los datos de TC, es posible predecir la presencia de tuberculosis activa en pacientes de distintos grupos de edad. El objetivo de desarrollar dichos modelos es orientar la toma de decisiones en pacientes potencialmente contagiosos, aislando fiablemente a los que presenten una tuberculosis activa y minimizando la cantidad de ellos que son aislados innecesariamente. 5.1. VARIABLES EPIDEMIOLÓGICAS  Edad: En nuestro trabajo, la edad media de los 1549 pacientes fue de 54.44 +- 19,90 años, con una mediana de 56 años. La edad media de los pacientes fue algo inferior en los grupos con cultivo positivo para tuberculosis en la población adulta. Por el contrario, dicha relación se invirtió en los grupos de menos de 18 años, siendo entonces más frecuente encontrar cultivos positivos en pacientes de más edad.  Sexo: Dos tercios de los pacientes estudiados fueron varones, observándose diferencias estadísticamente significativas (p<0.0001) en la distribución de pacientes por género en los distintos grupos.  País de procedencia: Tal y como ha sido propuesto por otros autores, el fenómeno de inmigración está modificando la dinámica de la tuberculosis en Castellón. Se está produciendo un freno en el declive de las tasas anuales de incidencia de tuberculosis a expensas del aumento de casos en ciudadanos extranjeros, especialmente en rumanos (144). En el ámbito nacional, Marruecos constituye el país más habitual de procedencia de los positivos declarados de tuberculosis en pacientes extranjeros (145), siendo el segundo en frecuencia en nuestro estudio, por detrás de Rumanía. Esta diferencia entre Castellón y el resto de España en cuanto a la procedencia de los positivos para tuberculosis en la población extranjera puede ser debido a que, con sus 40.697 habitantes, los rumanos constituyen casi la mitad de la población inmigrante en la provincia de Castellón (Padrón de 2016) (126). Por el contrario, Marruecos, aunque es uno de los países de procedencia más habituales de los inmigrantes en Castellón, con sus 15.638 habitantes, tiene una representación demográficamente menor (126). Nuestros resultados reflejan que el 30.69% de los cultivos positivos para M. tuberculosis provenían de la población extranjera (31/101), porcentaje que está alineado con el 30% referido por el Informe epidemiológico sobre la situación de la tuberculosis en España en el 2014 (145). No obstante, como dicho informe indica, el porcentaje de casos nacidos fuera de España varía mucho entre las distintas Comunidades Autónoma, siendo las comunidades de Cataluña y Madrid las que presentan un porcentaje más elevado de casos extranjeros (46% cada una), mientras que otras como Asturias tienen una menor proporción (3 %). 163 El principal factor de riesgo medible para la infección por tuberculosis en el colectivo inmigrante es el hacinamiento en contexto de privación socioeconómica, tal y como ya demostró el estudio geoestadístico de GómezBarroso y cols. (146).  Institucionalización en centros penitenciarios: En el conjunto de los centros penitenciarios de España se ha producido un descenso en el número de notificaciones de tuberculosis en los últimos años, observándose una tasa de incidencia/ 1.000 internos de 8,2 en 1998 que bajó progresivamente hasta los 2,2 en 2008 (147). La infección por tuberculosis en el medio penitenciario español tiene una alta razón de masculinidad, siendo la tasa de incidencia por nacionalidad mayor en internos españoles que en extranjeros en ambos sexos (147). Estos datos están en consonancia con lo observado en nuestro estudio en el que tres pacientes institucionalizados en centros penitenciarios presentaron cultivo positivo para M. tuberculosis, siendo todos ellos varones de entre 41 y 54 años, y de nacionalidad española.  Anemia: La anemia puede asociarse a estados carenciales, siendo la desnutrición crónica en sí misma un factor predisponente a padecer dicha anemia. Aunque existe un estudio que establece una relación clara entre la tuberculosis y la anemia, sugiriendo que ésta es más probable en un paciente con tuberculosis que en otro que no la tenga (93), aun hoy existe controversia con respecto a este tema. Dicho estudio llevado a cabo por Karyadi y cols. (93) fue realizado en un país en vías de desarrollo como Indonesia, observándose una mayor prevalencia de la asociación entre anemia y tuberculosis en las clases más deprimidas. No obstante, en dicho país existen algunas características demográficas distintas a las de la población objeto de nuestro estudio: en nuestro medio también es probablemente más común detectar tuberculosis en clases sociales marginales aunque, dentro de éstas, los grados de desnutrición no suelen ser tan severos y, por tanto, su asociación con anemia carencial no siempre estará presente. Por tanto, el bajo nivel socioeconómico y los estados carenciales consiguientes pueden ser considerados, por sí mismos, un factor predisponente tanto para la infección por M. tuberculosis como para la anemia. Es por ello que los futuros estudios sobre el tema debieran tener presente esta consideración, con el fin de arrojar más luz sobre el tema. A diferencia de lo establecido en otros estudios, en el presente trabajo se detectó una asociación inversa entre anemia y tuberculosis en el grupo de 18 a 65 años de edad, siendo esta relación también observable al separar a los pacientes en grupos con cultivo positivo o negativo para M. tuberculosis, independientemente de su edad.  Antecedente de tuberculosis pulmonar previa: Según los resultados de nuestro estudio, el antecedente de tuberculosis previa es un factor predisponente para el desarrollo de una nueva infección pulmonar por M. tuberculosis. 164 La recurrencia de la tuberculosis se convierte en un reto a veces insalvable para las técnicas convencionales de diagnóstico debido a que en muchos casos puede existir una alteración del sistema inmunitario de base. La inadecuada activación del sistema inmune puede dar lugar a la reactivación de una infección tuberculosa latente. Existe otro escenario posible, en el cual una nueva cepa de la Mycobacterium tuberculosis, distinta a la originaria de la primera infección, puede llegar del exterior y causar nuevamente la enfermedad en el paciente, independiente de su estado inmunitario. Esta modalidad de reincidencia es la más habitual en los pacientes inmunocompetentes (148). Algunas cifras indican que la tuberculosis recurre entre el 2-7% de pacientes con cepas susceptibles tratadas con la terapia DOTS (Directly Observed Therapy: Short-course) (149). Del total de 6,2 millones de casos de tuberculosis reportados en 2011, el 5% correspondía a recurrencias (150).  Antecedente de cirugía: A diferencia de otros estudios precedentes (78-80), nuestro trabajo no detectó una mayor proporción de tuberculosis pulmonar en pacientes con antecedentes de gastrectomía ni en aquellos que fueron sometidos a cualquier tipo de cirugía de las recogidas en este estudio. Si bien es cierto, sólo se registraron los antecedentes de cirugía referidos en la historia clínica electrónica del paciente, no siendo recogidos aquellos antecedentes que no constaran en la misma ni otros antecedentes de cirugía de menor relevancia como apendicectomía, amigdalectomía o colecistectomía, entre otras.  Antecedente de trasplante: Aunque otros estudios (84, 85) han establecido una mayor prevalencia de tuberculosis pulmonar activa en los receptores de trasplante de órgano sólido, nuestro estudio no ha conseguido objetivar dicha diferencia. En nuestro trabajo han sido pocos los casos de trasplante registrado y ninguno de ellos ha presentado un cultivo positivo para M. tuberculosis en el periodo de tiempo analizado.  Antecedente de diabetes: Aunque existe una evidencia robusta que apoya la asociación entre diabetes y tuberculosis pulmonar activa, tal y como argumentó Jeon y cols. en una revisión del 2008 (64), nuestro estudio no ha sido capaz de constatar dicho punto. Aunque en la población adulta se demostró una mayor proporción de pacientes con diabetes mellitus en los grupos con cultivo positivo para tuberculosis, dicha diferencia no fue estadísticamente significativa. Este estudio sí que demostró una diferencia significativa al comparar el grupo con cultivo positivo para M. tuberculosis y el grupo de las micobacterias atípicas, observándose una mayor proporción de pacientes con diabetes en el grupo de micobacterias atípicas. Según Panagiotou y cols., la incidencia de infecciones por micobacterias atípicas se está incrementando a nivel mundial debido a una también creciente presencia de enfermedades que alteran el sistema inmune, entre ellas, la diabetes mellitus (151). 165  Alcoholismo: Dentro de la población adulta, la proporción de pacientes con alcoholismo fue mayor en los grupos con cultivo positivo para tuberculosis. Esta diferencia fue estadísticamente significativa para el intervalo de edad de 18 a 65 años y al comparar el conjunto de grupos con cultivo positivo para tuberculosis con el conjunto de grupos con cultivo negativo para dicho microorganismo. Por tanto, en la línea de lo establecido por otros trabajos (53-55), nuestro estudio sugiere que el alcoholismo es un factor predisponente para la infección pulmonar por M. tuberculosis. Dicha relación entre alcoholismo y tuberculosis puede estar relacionada con el menoscabo que dicha conducta adictiva puede producir en el sistema inmunitario. Tampoco hay que olvidar la relación existente entre el abuso de alcohol y la pertenencia a clases sociales más bajas, así como los patrones de interacción social que conlleva, predisponentes a la transmisión de infecciones por vía aérea.  Tratamiento con fármacos esteroideos: Aunque el tratamiento con fármacos esteroideos es una causa reconocida de depresión inmunitaria, nuestro estudio no ha demostrado una relación significativa entre dicho tratamiento y la infección pulmonar por tuberculosis. Es probable que algunas variables no recogidas en este trabajo, como son la duración del tratamiento o la dosis utilizada, sean decisivas en relación al estado de inmunosupresión producido por los fármacos esteroideos.  Enfermedades hematológicas: La mayor proporción de tuberculosis en los pacientes con enfermedades hematológicas es probablemente debido al tratamiento con corticoides y agentes quimioterápicos que conlleva (87). En este estudio no se encontró diferencias significativas en cuanto a la proporción de pacientes con enfermedades hematológicas al comparar los grupos con cultivo positivo para tuberculosis y aquellos con cultivo negativo para dicho microorganismo.  Enfermedad renal crónica: En este estudio no se identificó una mayor proporción de enfermedad renal crónica en los pacientes con tuberculosis. La inmunodeficiencia observada en la enfermedad renal crónica se produce como resultado de las situaciones de inflamación crónica, del uso de terapias de reemplazo renal y de la insuficiencia renal crónica, así como la existencia de comorbilidades (74). Otros factores como el estado nutricional, la deficiencia de vitamina D y el hiperparatiroidismo también contribuyen al deterioro de la inmunidad en este tipo de pacientes (75).  Colagenopatías: Dado que la mayoría de las colagenopatías requieren tratamiento con esteroides e inmunosopresores en su fase activa, parece lógico pensar que 166 detrás de este grupo de enfermedades y la terapéutica que conllevan puede existir una mayor prevalencia de tuberculosis. En este sentido, el presente trabajo no ha conseguido demostrar una mayor proporción de tuberculosis en los pacientes con colagenopatías.  EPOC: La enfermedad pulmonar obstructiva crónica ha demostrado en nuestro estudio ser un factor asociado a la infección por M. tuberculosis, especialmente en la población adulta menor de 65 años. En la línea de lo que propusieron otros autores, como Byrne y cols., la asociación de tuberculosis y EPOC se suele dar más en los adultos jóvenes (<40 años) (116). Más que tratarse de un factor predisponente para la tuberculosis, la tuberculosis es un factor predisponente para la EPOC. A diferencia de la EPOC relacionada con el tabaquismo, la EPOC vinculada con la tuberculosis se produce en adultos jóvenes puesto que tienen una mayor incidencia de esta infección y el daño pulmonar asociado se produce durante el proceso de la enfermedad aguda. Incluso existen estudios que han documentado una asociación más fuerte entre la TB y la EPOC entre las personas que nunca han fumado (117). Al comparar los grupos con cultivo positivo para tuberculosis con el grupo con cultivo positivo para micobacterias atípicas, se obtuvo una diferencia significativa en favor de este último en cuanto a la proporción de pacientes con EPOC. La infección pulmonar por micobacterias atípicas afecta comúnmente a pacientes con EPOC o con alguna otra enfermedad estructural pulmonar previa, lo que incluye a la silicosis, neumoconiosis o incluso a una infección pulmonar pasada por M. tuberculosis. De forma similar a lo que sucede en la EPOC, el paciente tipo con infección pulmonar por micobacterias no tuberculosas es un varón de edad avanzada y con una enfermedad pulmonar de base (152).  VIH: La co-infección con el VIH y M. tuberculosis es el factor de riesgo más fuerte conocido, tanto para la progresión inmediata como tardía de la infección de tuberculosis activa (43, 44). En nuestro trabajo, los pacientes con VIH presentaron 3,6 veces más riesgo de tener un cultivo positivo para tuberculosis. El grupo de edad de 18 a 65 años fue en el que esta asociación se mostró más patente. Es posible que, además del aumento de la susceptibilidad individual a desarrollar tuberculosis activa tras la infección por mycobacterium tuberculosis, la asociación de tuberculosis y VIH también aumenta las tasas de transmisión de las micobacterias. En varios países, el VIH se ha asociado con brotes epidémicos de TB, y muchos de los brotes notificados involucraron cepas multirresistentes, con las dificultades desde el punto de vista epidemiológico que esto conlleva (47). Según defienden algunos autores, la inmunodepresión producida por el VIH predispone a la infección tuberculosa, mientras que, por su parte, la tuberculosis puede influenciar también la progresión de la infección por VIH, al producir de Factor de necrosis tumoral alfa e Interleukina 1 y 6 , citokinas que estimularían la replicación del virus (48). 167  Tabaquismo: El tabaquismo se relaciona con un aumento de riesgo de ser infectado por el Mycobacterium tuberculosis y de desarrollar enfermedad pulmonar activa, además de que propicia resultados pobres en el tratamiento tuberculostático (68). Se estima que hasta un 16% de todos los casos de TB se podrían evitar si la exposición al tabaco (activa y pasiva) fuera eliminada (69). En todos los estudios revisados se mostró algún efecto de la exposición al tabaquismo en el riesgo de infección por tuberculosis: en uno de ellos se halló un efecto significativo solamente en los exfumadores (153), en cinco estudios se observaron efectos uniformes en las mediciones relativas a la exposición al tabaquismo (154, 155, 156, 157, 158) y en dos estudios se halló una fuerte relación dosis-efecto (159, 160). Por otra parte, la prevalencia de tabaquismo entre los pacientes con TB es mayor que la prevalencia en la población general (70). Nuestro trabajo, al contrario de lo publicado, demuestra una menor proporción de tuberculosis en pacientes que han abandonado el hábito tabáquico. Por el contrario, no consigue demostrar una menor prevalencia de la infección en pacientes no fumadores. La proporción de abandono del hábito tabáquico es, en ambos géneros, mayor entre los casados, con un nivel socioeconómico más alto y con indicadores de estilo de vida más saludables (actividad física de ocio moderada o intensa y consumo de alcohol moderado) (161), hechos que pueden justificar una menor morbilidad para tuberculosis que los pacientes de estratos sociales inferiores. También es probable que parte de los pacientes catalogados como “nofumadores” sean realmente “fumadores” o “exfumadores” pero que dicho punto no haya sido registrado en la historia clínica. Otra circunstancia de difícil valoración es la existencia de “fumadores pasivos”, condición que no ha sido incluida a la hora de diseñar este trabajo y cuyo potencial para facilitar la infección por M. tuberculosis aún no ha sido del todo estudiada. 5.2. VARIABLES CLÍNICAS: Ninguna de las variables clínicas analizadas ha demostrado tener una clara asociación con la presencia de tuberculosis pulmonar. Las manifestaciones de la tuberculosis pulmonar son inespecíficas, e incluso puede detectarse en personas asintomáticas en el curso de estudios de contactos u otras exploraciones. Lo habitual es que el paciente presente síntomas de varias semanas de duración consistentes en tos, expectoración, en ocasiones hemoptoica, dolor torácico y sintomatología general (febrícula o fiebre, sudoración, astenia, anorexia y pérdida de peso). La exploración torácica puede ser anodina, o bien pueden auscultarse ruidos o estertores localizados o asimetrías en los ruidos respiratorios. Es fundamental que el médico sospeche esta patología en todo paciente con síntomas respiratorios y/o síntomas generales de más de 2–3 semanas de duración, así como en todo paciente con hemoptisis, independientemente de la duración de la misma. Una correcta orientación clínica y la solicitud de las pruebas diagnósticas consiguientes son de vital importancia para el control de la enfermedad. 168 5.3. VARIABLES EN RADIOGRAFÍA SIMPLE DE TÓRAX: 5.3.1. Hallazgos pulmonares:  Nódulo pulmonar: Se observó una mayor proporción de pacientes con nódulos pulmonares en radiografía simple de tórax en los grupos con cultivo positivo para M. tuberculosis, siendo más frecuente que estos fueran únicos. Estos resultados están alineados para la TC de tórax, aunque es posible que los casos catalogados como nódulo único pudieran esconder algún nódulo adicional no detectable debido a su pequeño tamaño y a la menor sensibilidad de la radiografía para su detección (162). Según Krysl y cols., los nódulos pulmonares mal definidos y las opacidades reticulares son hallazgos radiográficos detectados en el 25% de los pacientes con tuberculosis posprimaria (163).  Masa pulmonar: Ha sido observada una mayor proporción de pacientes con masas pulmonares en radiografía en los grupos con cultivo positivo para tuberculosis. También se ha detectada una asociación entre tuberculosis y la visualización de masas cavitadas en radiografía. Tal y como ha sido referido por algún autor clásico (164), aunque lo más frecuente es que las consolidaciones pulmonares en la tuberculosis se manifiesten como opacidades homogéneas, también es posible que tengan una presentación “mass-like” que las haga indistinguibles de las masas sólidas. Los márgenes bien definidos, la ausencia de broncograma aéreo y la elevada densidad pueden llevar a que esto suceda. Es por ello que algunas de las lesiones que en la radiografía han sido identificadas como masas podrían esconder realmente una consolidación.  Consolidación pulmonar: La consolidación fue un hallazgo más comúnmente observado en las radiografías de pacientes con tuberculosis pulmonar en comparación con aquellos no presentaban dicha infección. Asimismo, los grupos con cultivo positivo para M. tuberculosis demostraron una mayor proporción de consolidaciones con cavitación. Las cavitaciones, el sello radiológico de la reactivación de la tuberculosis, son evidentes radiográficamente en el 20-45% de los pacientes, según lo referido por diferentes autores (163-165). No obstante, en nuestro caso, sólo estuvieron cavitadas el 3.35 % (15/448) de las consolidaciones pero sí que presentaban cavitación el 24.19 % (15/62) de las masas pulmonares vistas en radiografía simple. 169  Patrón intersticial: Fue identificada una mayor proporción de pacientes con patrón intersticial en los grupos con tuberculosis. Este estudio no identificó diferencias entre variante focal y difusa de patrón intersticial. Aunque no es lo más habitual, la presentación radiográfica de la tuberculosis en forma de patrón intersticial también ha sido descrita (164).  Otros hallazgos pulmonares: Los otros hallazgos pulmonares detectados en radiografía simple de tórax, como son las atelectasias o las bronquiectasias, no mostraron una asociación estadísticamente significativa con la tuberculosis. 5.3.2. Hallazgos mediastínicos:  Masas mediastínicas: En nuestro estudio fue observada una mayor proporción de masas mediastínicas en los grupos con tuberculosis en comparación con los que no tenían dicha infección. Estas masas mediastínicas observadas en la radiografía de los pacientes con tuberculosis, en la mayoría de las ocasiones traducen la presencia de adenopatías mediastínicas. Estas adenopatías usualmente son unilaterales y se localizan en las cadenas paratraqueales y en hilios pulmonares en la forma primaria de la infección (166). Las adenopatías sólo afectan al 5-10% de los pacientes con tuberculosis posprimaria (163).  Agrandamiento hiliar: El ensanchamiento de los hilios pulmonares en la radiografía de tórax suele ser un indicio de la presencia de adenopatías hiliares. Aunque las adenopatías hiliares suelen ser habituales en la forma primaria de tuberculosis (166), nuestro estudio no ha conseguido demostrar diferencias significativas en cuanto al porcentaje de pacientes con agrandamiento hiliar entre los grupos cultivo positivo o negativo para M. tuberculosis. 5.3.3. Hallazgos pleurales:  Derrame pleural: En la forma primaria de tuberculosis, el derrame pleural suele ser unilateral y del mismo lado que el foco primario de la tuberculosis (165). También puede ser grande y ocurrir en pacientes sin evidencia de afectación parenquimatosa en las radiografías de tórax. En la tuberculosis posprimaria, el derrame pleural ocurre entre el 15 y el 20% de los pacientes y suele ser unilateral (165). En este estudio no ha sido demostrada una asociación clara entre tuberculosis y derrame pleural observable en radiografía simple de tórax. 170 5.4. VARIABLES EN TOMOGRAFÍA COMPUTARIZADA: 5.4.1. Hallazgos pulmonares:  Micronódulo pulmonar: En nuestro estudio se detectó una mayor proporción de pacientes con micronódulos pulmonares en los grupos con cultivo positivo para M. tuberculosis que en los grupos con cultivo negativo para dicho microorganismo. Además, al analizar por separado a los pacientes con micronódulos, se observó una mayor probabilidad de tuberculosis cuando dichos micronódulos eran múltiples. En cuanto a la distribución, se estudió la presencia de micronódulos centrolobulillares, perilinfáticos y distribuidos al azar, objetivándose una mayor presencia de micronódulos distribuidos al azar entre los pacientes tuberculosos y una mayor proporción de pacientes con patrón centrolobulillar en los grupos con cultivo negativo para M. tuberculosis. Un estudio llevado a cabo por Mo y cols. informó que los micronódulos con distribución perilinfática son comunes en la tuberculosis pulmonar, lo cual está en relación con la diseminación linfática de la enfermedad (167). Se cree que existe una participación linfática pulmonar en las fases tempranas de la infección tuberculosa pero sólo ha conseguido demostrarse en un estudio con animales (168). Según su localización, los micronódulos de distribución perilinfática pueden ser intersticiales, peribroncovasculares y subpleurales, observándose, respectivamente, en un 67, 63 y 41% de los TCAR de pacientes con tuberculosis pulmonar activa. Muchos de ellos suelen desaparecer después del tratamiento (169). Los micronódulos centrilobulares son una consecuencia de la diseminación broncogénica del M. tuberculosis (164, 170) y se correlacionan bien con los resultados de las pruebas de esputo, tal y como demostró Ko y cols. en un trabajo reciente (171). Por tanto, la distribución centrolobulillar de los nódulos, al sugerir propagación bronquial e invasión local en los pacientes con tuberculosis pulmonar activa, puede ser una herramienta útil no sólo para el diagnóstico sino también para la predicción de infectividad. No obstante, la diseminación broncogénica no es exclusiva de la tuberculosis y puede observarse en otras muchas infecciones pulmonares, motivo que puede justificar que en nuestro estudio no se haya asociado significativamente a la tuberculosis. Otra de las posibles explicaciones por las que los paciente con tuberculosis han mostrado una menor presencia del patrón centrolobulillar puede explicarse por la existencia de patologías de base como el EPOC y la fibrosis pulmonar idiopática, que asocian un patrón de destrucción pulmonar que trastoca los mecanismos de diseminación endobronquial (172). Por ello, no es raro comprobar que, en nuestro estudio, este patrón está menos presente en los grupos de edad de más de 65 años. En nuestro estudio, el patrón de distribución al azar de los micronódulos, o patrón miliar, ha mostrado una prevalencia mayor que la observada en otros trabajos (173), siendo su asociación a tuberculosis estadísticamente significativa. Según Andreu y cols., no es raro que los micronódulos 171 centrolobulillares adopten un patrón seudomiliar, imposible de diferenciar por imagen de un patrón miliar puro (174). Con ello, es posible que alguno de los casos catalogados como de distribución miliar realmente escondieran un patrón centrolobulillar de presentación atípica. Por el contrario, si dejamos atrás las series clínicas y abordamos las series de autopsias en adultos, las proporciones de patrón miliar se asemejan más a las observadas en este estudio, siendo la tuberculosis miliar documentada en un 0,3% -13,3% de todas las autopsias y en el 11,9% -40,5% de todos los casos de tuberculosis (175). En cuanto a la calcificación de los micronódulos, su presencia ha sido más manifiesta en los pacientes con cultivo negativo para tuberculosis. Dicho resultado se explica porque la calcificación del micronódulo orienta a sospechar su cronicidad. Además, los nódulos calcificados pueden tener múltiples orígenes más allá de la infección por M. tuberculosis (enfermedades inhalatorias, hamartomas, varicela, etc.).  Nódulo pulmonar: En este estudio se demostró una asociación estadísticamente significativa entre la presencia de nódulos pulmonares y la tuberculosis. La proporción de pacientes con nódulos pulmonares en los grupos con cultivo para tuberculosis ha sido del 29.7%, resultado que incluso es menor al 52.4% obtenido por Cho y cols. sobre un grupo de pacientes con tuberculosis pulmonar confirmada histológicamente (172). Según nuestro estudio, la característica diferencial más evidente de los nódulos pulmonares observados en enfermos con tuberculosis es la cavitación. Así que, aunque el diagnóstico diferencial de los nódulos pulmonares cavitados es muy amplio (nocardiosis, artritis reumatoide, histiocitosis de las células de Langerhans, etc.), un contexto clínico adecuado nos puede poner en la pista de la infección tuberculosa. Según Eerlij y cols., en el contexto de un paciente con mal control metabólico de su diabetes, que se presenta con nódulos pulmonares cavitados, se debe sospechar la presencia de infecciones como tuberculosis y fúngicas invasoras, en especial Mucormicosis y Aspergilosis (176).  Masa pulmonar: En este estudio se demostró una mayor proporción de masas pulmonares en TC en los pacientes con cultivo positivo para M. tuberculosis. Estos resultados son equiparables a los detectados en otros trabajos al respecto (172). Las masas pulmonares observadas en la TC de pacientes con tuberculosis han demostrado una mayor proporción de cavitación que las masas de otros orígenes. Al igual que en otros estudios (172), el porcentaje de masas cavitadas en este trabajo (50.62%) es mayor al porcentaje de cavitación detectado en los nódulos (32.61%), lo cual puede hacer pensar que la probabilidad de cavitación de las lesiones tuberculosis es directamente proporcional a su tamaño. 172  Consolidación pulmonar: Los grupos con cultivo positivo para tuberculosis demostraron una mayor proporción de pacientes con consolidación. La consolidación pulmonar es un hallazgo que se observa de forma muy habitual en los estudios de imagen de los individuos con tuberculosis. No obstante, nuestro porcentaje de consolidaciones en pacientes con tuberculosis (49.5%) puede ser algo mayor con respecto a lo referido en otros estudios consultados (172). En nuestro trabajo se ha observado una marcada correlación entre los resultados obtenidos en radiografía (52.5% de los pacientes con tuberculosis) y TAC (49.5% de los pacientes con tuberculosis), en lo que a consolidaciones parenquimatosas se refiere, hecho que hace hincapié en la utilidad de técnicas sencillas como la radiografía para llevar a cabo un primer acercamiento diagnóstico ante la sospecha de infección tuberculosa. La localización más habitual de las consolidaciones en los pacientes con tuberculosis fue en los lóbulos superiores, hecho esperable en la reactivación tuberculosa en individuos inmunocompetentes (174). Los individuos sometidos a una depresión inmunitaria severa presentan más comúnmente una forma primaria de tuberculosis, con localización de las consolidaciones en campos pulmonares inferiores (170, 177). Según los datos de este estudio (173), las consolidaciones pulmonares debidas a la infección por M. tuberculosis se cavitan de forma más frecuente que las consolidaciones de otros orígenes. La cavitación es considerada el sello distintivo de la tuberculosis posprimaria y se visualiza hasta en el 50 % de los casos (173), porcentaje que no queda lejos del 42% obtenido en este trabajo. No se objetiva una localización más frecuente ni diferencias significativas en cuanto a localización de las consolidaciones cavitadas al comparar los diferentes grupos.  Tractos fibróticos cicatriciales: Las cicatrices o tractos fibróticos se corresponden al intento de curación de infiltrados y cavidades mediante calcificación y retracción fibrosa del segmento o lóbulo afectado, con distorsión de su estructura. La práctica habitual en nuestro medio frente a las lesiones fibróticas es o bien tratarlas como una tuberculosis pulmonar activa y dar tratamiento estándar (138), o bien ninguna actuación (a lo sumo seguimiento clínico-radiológico). No obstante, existe cierta controversia al respecto puesto que existen pocas investigaciones al respecto y las indicaciones de tratamiento se basan en trabajos de hace tres o más décadas (178). Un 30% de los pacientes con tuberculosis presentaron cicatrices fibróticas en la TC de tórax, hallazgo que es difícil de correlacionar con los resultados de otros trabajos al respecto, basados en el seguimiento de pacientes que ya superaron la enfermedad, dejando atrás la fase aguda (179). En nuestro estudio, de forma estadísticamente significativa se detectó una mayor proporción de pacientes con cicatrices fibróticas en los grupos con cultivo positivo para M. tuberculosis, lo cual podría apoyar la necesidad de tratamiento en dichos individuos. Además, la localización predominante de este hallazgo fue en los lóbulos 173 superiores, tanto en los casos con tuberculosis como en aquellos con cultivo negativo para esta micobacteria. Es de vital importancia consensuar las indicaciones de tratamiento existentes y adaptar las experiencias publicadas a la práctica clínica actual, teniendo en cuenta los factores de riesgo de reactivación tuberculosa y el contexto clínico en el cual se identifican las cicatrices fibróticas.  Atelectasias: En este estudio se mostró una menor proporción de pacientes con atelectasia en el grupo con cultivo positivo para M. tuberculosis en comparación con los que presentaron un cultivo negativo para este microorganismo. Dicha diferencia fue especialmente evidente en los grupos de más de 65 años (G5 y G6). Por tanto, la presencia de atelectasias es un factor predictor negativo para el diagnóstico radiológico de esta infección. La atelectasia relacionada con la infección por tuberculosis suele ser más notable en los segmentos anteriores de los lóbulos superiores y en el segmento medial del lóbulo medio. Dicha atelectasia suele producirse por la compresión extrínseca del bronquio por parte de un ganglio hiliar o mediastínico, pudiendo resolverse cuando la linfadenopatía disminuye de tamaño. Una resolución repentina de atelectasia no tiene por qué significar una mejoría del paciente, puesto que puede representar la perforación de la vía aérea por un ganglio linfático infectado, mejorando la obstrucción bronquial. Parece que la capacidad de la tuberculosis para producir atelectasias no es superior a la detectada en otros tipos de infecciones pulmonares e incluso puede ser inferior, por lo observado en nuestro trabajo. Por su parte, la menor probabilidad de tener tuberculosis en pacientes con atelectasias puede venir justificada por la preexistencia de atelectasias crónicas y otros daños estructurales pulmonares que hacen más dificultosa la diseminación endobronquial de la infección. El hecho de que esta asociación sea estadísticamente significativa en los pacientes de más edad, en los que es esperable una mayor desestructuración pulmonar, refuerza este concepto. No obstante, nuevos estudios parecen necesarios para clarificar la verdadera fisiopatogenia de la infección con M. tuberculosis en pacientes con atelectasias pulmonares.  Patrón de árbol en brote: En el presente estudio se ha demostrado una asociación significativa entre la presencia de un patrón de árbol en brote en la TC de tórax y el cultivo positivo para M. tuberculosis. Dicho resultado está en consonancia con el resto de la literatura (180-182). No en balde, este patrón es considerado uno de los hallazgos más comunes en la reactivación de una tuberculosis pulmonar. El patrón de árbol en brote refleja la inflamación endobronquiolar debida, principalmente a una causa infecciosa. No obstante, ésta no es una manifestación exclusiva de infecciones (183), por lo que su asociación con otros hallazgos radiológicos y un contexto clínico adecuada será lo que nos haga sospechar una tuberculosis pulmonar. 174  Patrón en vidrio deslustrado: Según nuestros resultados, la presencia de vidrio deslustrado en TC disminuye las probabilidades de que el paciente tenga, en el momento de la exploración, una infección pulmonar por M. tuberculosis, puesto que este hallazgo se observa con más frecuencia en pacientes con cultivo negativo para dicho microorganismo. Con todo, esto no significa que no sea un hallazgo observable en la TC de tórax de un paciente afecto por tuberculosis pulmonar. Los infiltrados parenquimatosos en vidrio deslustrado, asociados comúnmente a un patrón miliar, son una de las manifestaciones radiológicas atípicas de la tuberculosis como también lo son los nódulos o masas pulmonares, los infiltrados y los quistes múltiples reversibles, entre otros. Según lo referido por Lee y cols. la tuberculosis puede tener una presentación radiológica atípica hasta en un tercio de los casos, en la población adulta (184). En un 1,7 % de los pacientes la tuberculosis pulmonar puede desencadenar una insuficiencia respiratoria aguda, la cual compromete el pronóstico del paciente, a veces de forma fatal. Hasta en un 70% de estos pacientes, con insuficiencia respiratoria aguda asociada a una tuberculosis pulmonar activa, se detecta un patrón en vidrio deslustrado en la TC de tórax. No obstante, otros hallazgos como los micronódulos y las consolidaciones son más frecuentes, siendo observados en un 94 % y en un 76 %, respectivamente (185).  Bronquiectasias: En este estudio no se ha demostrado una mayor presencia de bronquiectasias en los pacientes con tuberculosis pulmonar, resultado que contrasta con un estudio llevado a cabo en China con una población de 10.811 adultos, en el cual se demostró una probabilidad de presentar bronquiectasias en TC tres veces mayor en aquellos pacientes con antecedente de tuberculosis previa (186). En caso de aparecer las bronquiectasias, su localización preferente en adultos (18-65 años) con tuberculosis fue en los lóbulos superiores, mientras que en el resto de pacientes fueron más frecuentes las localizaciones en lóbulos medios e inferiores. Según la literatura de referencia, las bronquiectasias, observadas en un 35-86 % de las TC de pacientes que han pasado una tuberculosis posprimaria (179), tienden a localizarse en los lóbulos superiores de los pulmones afectos (170, 173).  Otros hallazgos pulmonares: Otros hallazgos como enfisema, el engrosamiento septal o la pérdida de volumen pulmonar no demostraron asociaciones estadísticas en este estudio. 5.4.2. Hallazgos mediastínicos:  Adenopatías: Los resultados de este estudio sugieren una mayor probabilidad de presentar adenopatías en los pacientes con tuberculosis en comparación con la población general, hecho que no desentona con lo establecido en la literatura actual. La 175 linfadenitis es la principal manifestación extrapulmonar de la tuberculosis, observándose en el 35% de los casos (187). Es más frecuente en niños y en mujeres, con la edad máxima de inicio en adultos entre los 20 y los 40 años. En la niñez, la linfadenitis tuberculosa es la manifestación más común de la tuberculosis primaria (96%-100% de los casos), caracterizándose por la presencia de conglomerados localizados en múltiples cadenas ganglionares (principalmente en las áreas paratraqueal derecha, hilar y subcarinal). Otras de las características típicas en este grupo de edad son su captación en anillo de contraste y su asociación con consolidaciones parenquimatosas hasta en el 90% de los casos (188). En adultos, la linfadenopatía no asociada a consolidaciones es inusual y ha sido descrita en pacientes con el síndrome de inmunodeficiencia adquirida (SIDA) (180). La presencia de una consolidación asociada a adenopatías en adultos puede ser altamente sugestiva de tuberculosis (141). 5.4.3. Hallazgos pleuropericárdicos: Las manifestaciones radiológicas de la tuberculosis pleural son inespecíficas y de utilidad dudosa para el diagnóstico de esta infección, tal y como demuestra este estudio. Aunque algunas generalidades en relación con la afectación pleural son aceptadas, como son la mayor probabilidad de aparición de derrame en forma primaria de tuberculosis o su potencial cronicidad con formación de septos en la forma posprimaria (173), el hecho es que no existe una presentación de la enfermedad pleural que se considere patognomónica de la tuberculosis. 5.5. ANÁLISIS MULTIVARIANTE: La creación de un sistema de puntuación a partir de las variables que han demostrado predecir mejor la presencia de tuberculosis pulmonar en el análisis multivariante, ha probado ser una herramienta útil para predecir la presencia de tuberculosis pulmonar activa en pacientes de diferentes grupos de edad. En condiciones ideales, una prueba diagnóstica debería tener la mayor sensibilidad posible, para evitar omitir el diagnóstico de pacientes con enfermedad activa, y un elevado valor predictivo negativo (VPN), con el fin de asegurar los resultados negativos que representen un verdadero estado libre de enfermedad. En la tuberculosis pulmonar, al tratarse de una enfermedad con una alta tasa de transmisión en su fase activa, la correcta elección de las pruebas diagnósticas es más importante, si cabe, desde el punto de vista de la salud pública. En el caso de nuestro estudio, se ha obtenido un VPN muy alto (98.78%) con una sensibilidad elevada (85.15%). Por lo tanto, el uso del sistema de puntuación de nuestro modelo global puede ser útil para el diagnóstico de la tuberculosis pulmonar, conduciendo al aislamiento respiratorio apropiado y, presumiblemente, evitar tratamientos innecesarios con tuberculostáticos. En cuanto a nuestro valor predictivo positivo (VPP) de 26.63%, es inferior al 78.5-76.5% observado en otros estudios (189, 190), lo cual puede estar justificado por la baja prevalencia de tuberculosis en nuestro medio en 176 comparación con las áreas endémicas en las que ha sido llevados a cabo dichos trabajos. Esto es debido a que si la prevalencia de una determinada enfermedad en una población es baja, el VPP tiende a ser bajo ya que, al haber un mayor número de personas sanas, se incrementa el número de falsos positivos. 5.6. LIMITACIONES DEL ESTUDIO: Hay una serie de limitaciones de este estudio que deben ser señaladas. Sólo se realizó la validación de la fiabilidad y reproducibilidad de las variables estudiadas en un solo hospital de referencia provincial y en los otros centros adscritos a la misma área de salud. Las características demográficas y clínicas de los pacientes de los diferentes grupos de edad con tuberculosis pulmonar pueden diferir en otras áreas geográficas, especialmente en aquellas que presentan una mayor prevalencia para dicha infección. Por lo tanto, el modelo debe ser validado en otros entornos antes de que pueda ser implementado en la práctica clínica. Los ancianos fueron definidos arbitrariamente con un límite de edad de 65 años, habiendo sido utlizado este punto de corte por diferentes autores en estudios anteriores (191, 192). Por último, la TC es una técnica relativamente cara para las áreas más pobres del planeta, donde la infección por M. tuberculosis es endémica, siendo su disponibilidad cuestionable. Además, no todos los centros poseen en su plantilla a radiólogos entrenados en la interpretación de los hallazgos sugestivos de tuberculosis pulmonar. Sin embargo, el método descrito es valioso para España, así como para otros países de Europa, no siendo descartable su aplicabilidad en áreas de otros países con incidencias de tuberculosis similares a las de España. 177 178 6. CONCLUSIONES 179 180 Respecto a los hallazgos radiológicos de la tuberculosis pulmonar y su correlación con la presencia de cultivo positivo para mycobacterium tuberculosis, podemos concluir que: 1.- El análisis de la curva ROC de este sistema de puntuación para predecir el resultado del cultivo para Mycobacterium tuberculosis muestra un área bajo la curva de 0.9163 (IC del 95 %: 0.8858-0.9467) y un punto de corte óptimo de 1.845. 2.- Este modelo predictivo, con un punto de corte en 1.845 tiene una sensibilidad del 85.1 %, especificidad del 83.6 %, VPP del 26.6 % y VPN del 98.7 % para diferenciar entre pacientes con tuberculosis pulmonar activa o con otra infección pulmonar. 3.- Este sistema de puntuación basado en los hallazgos epidemiológicos y radiológicos puede ayudar en el diagnóstico precoz de la tuberculosis pulmonar activa, reduciendo así la carga asistencial y financiera de los hospitales, así como minimizando el aislamiento respiratorio inmediato innecesario y el riesgo infeccioso para los pacientes y los trabajadores sanitarios. 4.- Los micronódulos pulmonares se detectan en TC en algo más de la mitad de los pacientes con cultivo positivo para Mycobacterium tuberculosis (p<0.0001), siendo habitualmente múltiples (p<0.0001) y no presentando una localización específica, aunque sí una distribución “al azar” de forma preferente (p<0.0001). El hallazgo de micronódulos calcificados va en contra del diagnóstico de tuberculosis (p=0.045). 5.- Los nódulos y masas pulmonares se presentan significativamente con mayor frecuencia en pacientes con cultivo positivo para Mycobacterium tuberculosis, tanto en radiografía simple (p<0.0001) como en TC (p<0.0001). La diferencia también es significativa a favor de la tuberculosis si los nódulos están cavitados en TC (p<0.0001) o las masas aparecen cavitadas tanto en radiografía simple (p=0.024) como en TC (p<0.0001). 6.- Las consolidaciones pulmonares cavitadas se detectan en TC en más del 20% de los pacientes con tuberculosis, siendo su localización preferente los lóbulos superiores. Se objetivó de forma significativa (p<0.0001) una mayor proporción de pacientes con consolidaciones cavitadas en los grupos con cultivo positivo frente a los grupos con cultivo negativo para Mycobacterium tuberculosis. 7.- La observación de un patrón en “árbol en brote” en TC es altamente predictiva del diagnóstico de tuberculosis (p<0.001). 8.- La presencia de un patrón en vidrio deslustrado hace menos probable el diagnóstico de tuberculosis pulmonar (p=0.023). 9.- Aunque la presencia de adenopatías se asocia al diagnóstico de tuberculosis (p<0.001), no se ha demostrado una mayor afectación de ninguna de las cadenas mediastínicas en concreto. 10.- En pacientes con cultivo positivo para micobacterias atípicas hay una presencia significativamente mayor de diabetes (p=0.014) y de EPOC 181 (p=0.0001) que en los pacientes con cultivo positivo para Mycobacterium tuberculosis. 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EPIDEMIOLÓGICOS:  SEXO: H □ M □  NACIONALIDAD  EDAD: INSTITUCIONALIZADO  GRUPO: G1  FACTORES DE RIESGO: o o o o o o o o o o o o o G2 G3 G4 G5 G6 ANEMIA ANTECEDENTE CIRUGÍA ANTECEDENTE DE TBC ANTECEDENTE DE TRASPLANTE DIABETES MELLITUS ALCOHOLISMO TRATAMIENTO ESTEROIDEO ENF HEMATOLÓGICA IRC EPOC VIH COLAGENOPATÍA TABAQUISMO B. CLÍNICOS:  FIEBRE / FEBRÍCULA ● DISNEA  SUDORACIÓN NOCTURNA ● HEMOPTISIS  SD CONSTITUCIONAL ● MEG  PÉRDIDA DE PESO  TOS CON/ SIN EXPECTORACIÓN  DOLOR TORÁCICO 199 200 ANEXO 2: FORMULARIO DE RECOGIDA DE DATOS: Id. Paciente: Año: 2005 2006 2007 2008 2009 2010 2011 2012 C. RADIOGRAFÍA DE TÓRAX:  HALLAZGOS PULMONARES: o NÓDULO PULMONAR o o o o MASA PULMONAR CONSOLIDACIÓN PULMONAR ATELECTASIA PATRÓN INTERSTICIAL o BRONQUIECTASIAS   HALLAZGOS MEDIASTÍNICOS: o MASA MEDIASTÍNICA. o AGRANDAMIENTO HILIAR. HALLAZGOS PLEURALES: o DERRAME PLEURAL. D. TC DE TÓRAX: + HALLAZGOS PULMONARES:  MICRONÓDULOS: SOLITARIOS/ MÚLTIPLES  CENTROLOBULARES  MILIARES    PERILINFÁTICOS LOCALIZACIÓN CALCIFICACIÓN NÓDULOS LOCALIZACIÓN NÓDULO CAVITADO (LOCALIZACIÓN) CALCIFICADO (LOCALIZACIÓN) NÓDULO CON HALO (LOCALIZACIÓN) MASAS LOCALIZACIÓN MASAS CAVITADAS (LOCALIZACIÓN) MICETOMA SOBREINFECCIÓN MASAS CALCIFICADAS (LOCALIZACIÓN) 201 2013 2014           MASAS CON HALO (LOCALIZACIÓN) CONSOLIDACIÓN LOBAR LOCALIZACIÓN CONSOLIDACIÓN CAVITADA (LOCALIZACIÓN) TRACTOS FIBRÓTICOS LOCALIZACIÓN ENGROSAMIENTO SEPTAL LOCALIZACIÓN ATELECTASIA LOCALIZACIÓN ENFISEMA (CENTROACINAR/PARASEPTAL/PANACINAR/CICATRICIAL) SIGNO DEL ÁRBOL EN BROTE LOCALIZACIÓN OPACIDAD EN VIDRIO DESLUSTRADO LOCALIZACIÓN BRONQUIECTASIAS + ENGROSAMIENTO LOCALIZACIÓN PÉRDIDA DE VOLUMEN LOCALIZACIÓN PATRÓN DE ÁRBOL EN BROTE LOCALIZACIÓN + HALLAZGOS MEDIASTÍNICOS:  ADENOPATÍAS LOCALIZACIÓN +HALLAZGOS PLEUROPERICÁRDICOS:  DERRAME PLEURAL UNI/BILATERAL  EMPIEMA DER IZQ  ENGROSAMIENTO PLEURAL/ PAQUIPLEURITIS  DERRAME PERICÁRDICO 202 ANEXO 3: FORMULARIO DE APLICACIÓN DEL MODELO Id. Paciente: Variables epidemiológicas Puntuación Antecedente de tuberculosis (+2) Anemia (-2) Alcoholismo (+1) EPOC (+1) Fumador (+2), no fumador (+2), exfumador (0) A= Variables en radiografía simple de tórax Puntuación Masa pulmonar (+1) Masa pulmonar cavitada (+3) Consolidación pulmonar (+1) Consolidación pulmonar cavitada (+1) Atelectasia pulmonar (+1) B= 203 Variables en TC Puntuación Nódulo pulmonar (+1) Nódulo pulmonar cavitado (+2) Masa pulmonar (-1) Masa pulmonar cavitada (+1) Consolidación cavitada (+2) Patrón en vidrio deslustrado (-1) Bronquiectasias (-1) Tractos fibróticos (+1) Pérdida de volumen (+1) Signos de enfisema (-1) Patrón de árbol en brote (+2) Patrón miliar (+4) Adenopatías (+1) C= RESULTADO (score): A + B + C - Edad x 0.03 = 204
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https://hal.science/hal-01206067v2/file/transHumUs_icra.pdf
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transHumUs: A poetic experience in mobile robotics
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To cite this version: Guilhem Saurel, Michel Taïx, Jean-Paul Laumond. transHumUs: A poetic experience in mobile robotics. IEEE International Conference on Robotics and Automation (ICRA), May 2016, Stockholm, Sweden. pp.2908-2914, ￿10.1109/ICRA.2016.7487455￿. ￿hal-01206067v2￿ I. WHEN ROBOTICS MEETS ART The Venice Biennale [1] is the most important international exhibit for contemporary art. It takes place once every two years since 1895. The exhibition is distributed in many places in Venice, including ninety pavilions located in a park, each of them being managed by a nation. For the 56th edition in 2015, the institute in charge of the French Pavilion selected the project of artist Céleste Boursier-Mougenot. The work included the so-called transHumUs project. With transHumUs, the artist intended to roll out the choreography of three mobile trees moving according to their metabolism, i.e. the varying flow of their sap when going from shadow to light. The show ran from May to November, six days a week, eight hours a day. Figure 1 shows respectively the initial sketch and the delivery of the project. Nine months separate both pictures. The purpose of this paper is to report on the research and development performed in this period, from the first meeting between the artist and the authors to the exhibition opening on the 5th of May, 2015. (a) Artist’s drawing. (a) Artist s drawing. (b) Final result. (b) Final result. Fig. 1: From the specifications to the realisation, nine months separate both pictures. 1CNRS, LAAS, Univ. de Toulouse, UPS, INSA, 7 avenue du Colonel Roche, F-31400 Toulouse, France, {saurel,taix,jpl}@laas.fr Guilhem Saurel1, Michel Taïx1 and Jean-Paul Laumond1 (a) Artist’s drawing. (b) Final result. Fig. 1: From the specifications to the realisation, nine months separate both pictures. (a) Artist’s drawing. Abstract—transHumUs is an artistic work recently exhibited at the 56th Venice Biennale. The work aims at freeing trees from their roots. How to translate this poetic ambition into technological terms? This paper reports on the setup and the implementation of the project. It shows how state of the art mobile robotics technology can contribute to contemporary art development. The challenge has been to design original mobile platforms carrying charges of three tones, while moving noiseless according to tree metabolism, in operational spaces populated by visitors. HAL Id: hal-01206067 https://hal.science/hal-01206067v2 Submitted on 21 Jul 2016 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution - ShareAlike 4.0 International License transHumUs: a poetic experience in mobile robotics Guilhem Saurel1, Michel Taïx1 and Jean-Paul Laumond1 Guilhem Saurel1, Michel Taïx1 and Jean-Paul Laumond1 II. SPECIFICATIONS AND TECHNICAL SOLUTIONS (a) Top view of the French (left), British (top), and German (right) Pavilions around an esplanade in the Giardini. (b) Geometric model of the evolution space (10m grid for scale). (b) Geometric model of the evolution space (10m grid for scale). Fig. 3: AGV in BA Systemes’ factory. (b) Geometric model of the evolution space (10m grid for scale). Fig. 2: In the birds eye view of the Giardini, the French pavilion is the building on the left side. A tree moves inside the pavilion while two other trees share the esplanade common to British and German pavilions. The geometric model of the esplanade defines the bounds of the evolution space. It is part of an user interface allowing to monitor the motions of the trees. Fig. 3: AGV in BA Systemes’ factory. • Metabolism sensor (Fig. 4): Granier probe [4] is one of the most commonly used techniques for tree metabolism measurement [5], [6]. The probe is based on a thermal dissipation principle. It is made of two needles. A heated needle is placed above a neutral needle in the sapwood of the tree. The probe measures the difference between both temperatures. When the sap flow velocity is slow, the temperature difference is high, while it decreases as soon as the sap flow velocity increases. Granier probe sensitivity is enough to capture light changes. Light changes depend on the atmospheric conditions. They also depend on the motions of the trees’ (since they are moving!). This is why we equip each tree with three probes surrounding the trunk in three different direc- tions. When passing from sunlight through shadows, the metabolism reacts on measurable way. The measures • Motion origin: Trees are living beings. Their metabolism depends on environmental conditions and weather. The motion of the trees should take its origin in the internal state variations. This is a key point of the project: the work intends to reveal the invisible internal states of the trees. Also the two trees outside the pavilion have to coordinate themselves to avoid collision. • Motion quality: The trees are intended to move very slowly. With a velocity lower than one meter per minute, the motions should be hardly perceptible. The trees should move without any fixed direction: they should be holonomic. No noise should be perceptible by the visitors: trees should give the appearance of moving by levitation. II. SPECIFICATIONS AND TECHNICAL SOLUTIONS How to translate the poetic dimension of the project into technological terms? The question addresses all components of a robotics system, ranging from the design of machines capable of moving three tons (the tree and the root ball), the sensing architecture that should combine both egocentric and allocentric sensors, and the motion generation system that should translate the motion qualitative criteria defined by the artist in terms of motion smoothness and velocity. The poetic ambition of the work is to free trees from their determinism by allowing them to explore the world by themselves. The ambition asks for original connections between natural and technological elements. The inspiration follows the work C. Boursier-Mougenot conducted for years [2]. The very first step of the project has been to open up a process of dialogue between the artist and the authors. What kind of trees? How are they distributed in the space of the Giardini? What does it mean for trees to move by themselves? What quality of motion is expected? What are the development and operating conditions? All questions should receive technological answers. The specifications that go out from the dialogue are the following ones: This paper does not address any theoretical challenge. Our contribution in this project has been to select the tech- nology providers (Section II), develop an original software architecture, including user interfaces for non expert pavilion attendants (Section III) and to manage the entire project (Section V). The second main contribution has been to propose the artist motion planning and control strategies that give the feeling that trees move in full autonomy (Section IV). • Trees: There are three pine trees whose blooming from May coincides with the opening of the Biennale. They are about five meters high. They are alive and move together with their clod for a total mass of about three tons. • Arena: Two trees share the space in front of the French pavilion. They should remain within a well-defined 300 square meter area (Fig. 2) made of limestone soil, whose softness depends on the weather. The third tree moves alone within the pavilion in a well-defined 50 square meter area equipped with a concrete floor. Both areas are permanent obstacle free. However they can be populated by visitors without any constraints imposing special safety zones. Visitors are allowed to approach and touch the trees. II. SPECIFICATIONS AND TECHNICAL SOLUTIONS months without any possible extension of the deadline fixed by the opening of the biennale (May 5, 2015), with only two weeks of on-site test. The work should be operational until November 22, 2015, 8 hours a day, 6 days a week. It should be operated by museum staff after one-day training. These specifications gave rise to the following technolog- ical solutions. • Platform design (Fig. 3): Tailor-made platforms have been devised by BA Systemes, a company acting in automated guided vehicle (AGV) design and production for logistics. Each platform is made of a conveyor supported by three electrical driving wheels [3]. A vertical rotational axis passes through the contact point between the wheel and the floor. The noise of electrical motor is inaudible. Wheels can therefore turn on the spot. Synchronous drive system allows to steer the platform in any of the three directions in R2 × S1: the platform is therefore holonomic. The control model is presented in Section IV. The tree sods are inserted within the conveyors while synthetic shells imitating soil and roots encapsulate the AGVs. The synthetic shells also contribute to totally cancel the remaining slight noise of the motors. • Platform design (Fig. 3): Tailor-made platforms have been devised by BA Systemes, a company acting in automated guided vehicle (AGV) design and production for logistics. Each platform is made of a conveyor supported by three electrical driving wheels [3]. A vertical rotational axis passes through the contact point between the wheel and the floor. The noise of electrical motor is inaudible. Wheels can therefore turn on the spot. Synchronous drive system allows to steer the platform in any of the three directions in R2 × S1: the platform is therefore holonomic. The control model is presented in Section IV. The tree sods are inserted within the conveyors while synthetic shells imitating soil and roots encapsulate the AGVs. The synthetic shells also contribute to totally cancel the remaining slight noise of the motors. (a) Top view of the French (left), British (top), and German (right) Pavilions around an esplanade in the Giardini. (b) Geometric model of the evolution space (10m grid for scale). (a) Top view of the French (left), British (top), and German (right) Pavilions around an esplanade in the Giardini. (a) Top view of the French (left), British (top), and German (right) Pavilions around an esplanade in the Giardini. II. SPECIFICATIONS AND TECHNICAL SOLUTIONS Users may also give direct orders to the Path Planner when necessary. The variables (s1,s2,s3), (x,y,α) and (vi,θi) are developed in Section IV. • Localization: The motion arenas are well defined both inside and outside the pavilion. In order to locate the AGVs we have selected a ultra wideband (UWB) technology proposed by the Ubisense company. The technology realizes a good comprise between accuracy and stealth [7], [8]. The buildings surrounding the arenas are equipped with transmitters guaranteeing a complete coverage (Fig. 5). The trees are equipped with few small receivers hidden in the branches. An accuracy of 15 centimeters is then obtained with a high level of confidence. Fig. 6: Software architecture: Each tree has three sap flow sensor that are used as inputs for the Path Planner. The Path Planner also retrieves the current position and orientation of each robot from the geolocation system, and then computes the traction speed and orientation of each wheel of each AGV. Users may also give direct orders to the Path Planner when necessary. The variables (s1,s2,s3), (x,y,α) and (vi,θi) are developed in Section IV. II. SPECIFICATIONS AND TECHNICAL SOLUTIONS The clod basement height should be less than five centimeters. • Operation: The project had to be concluded within six • Operation: The project had to be concluded within six Fig. 5: Implantation of the beacons in the Giardini. given by the three probes are used as three inputs for motion generation (Section IV). (b) Moving the tree from shadow to sunlight and vice-versa: plots of the output of one Granier Probe (in Volts) in a test tree at LAAS-CNRS lab. on the 8th and 9th of April 2015, from 12:00PM to 6:00PM, while the shifting occurred around 3:00PM. (a) Sap flow sensors: Granier probes are composed of two thermocouple needles. (b) Moving the tree from shadow to sunlight and vice-versa: plots of the output of one Granier Probe (in Volts) in a test tree at LAAS-CNRS lab. on the 8th and 9th of April 2015, from 12:00PM to 6:00PM, while the shifting occurred around 3:00PM. (a) Sap flow sensors: Granier probes are composed of two thermocouple needles. (a) Sap flow sensors: Granier probes are composed of two thermocouple needles. (b) Moving the tree from shadow to sunlight and vice-versa: plots of the output of one Granier Probe (in Volts) in a test tree at LAAS-CNRS lab. on the 8th and 9th of April 2015, from 12:00PM to 6:00PM, while the shifting occurred around 3:00PM. (a) Sap flow sensors: Granier probes are composed of two thermocouple needles. Fig. 5: Implantation of the beacons in the Giardini. Fig. 5: Implantation of the beacons in the Giardini. Fig. 4: Sap flow measurements: Those experiments allowed us to check this solution against our specifications. We found that the tree reactions to sunlight and shadows are exploitable as inputs for our systems, because when the trees would move between the sunlight and shadows, their metabolism will react in a measurable way. User Interface Geolocation Sapflow Sensors Path Planner Robotic Tree (s1, s2, s3) (x, y, α) (vi, θi) User Interface Geolocation Sapflow Sensors Path Planner Robotic Tree (s1, s2, s3) (x, y, α) (vi, θi) Fig. 6: Software architecture: Each tree has three sap flow sensor that are used as inputs for the Path Planner. The Path Planner also retrieves the current position and orientation of each robot from the geolocation system, and then computes the traction speed and orientation of each wheel of each AGV. A. Platform modeling As we want a tree to be holonomic, in the most basic form, we need to input three variables. The specification also states that a tree should behave in a similar manner in whatever direction it goes, so we chose a polar velocity system concentric with the AGV, (v,θ,ω), where: Fig. 9: Instant center of rotation. v = s1 ω = 2s2 −1 • θ ∈[0,2π[ is the direction taken by the center of the AGV; ω = 2s2 −1 • v ∈[0,1] is its linear velocity along the direction θ; • ω ∈[−1,1] is its angular velocity. The units of those velocities are then deducted to satisfy the specifications about the overall speed. In this case, the maximum speed of the trunk of the tree is one meter per minute (≈17mm/s), but the wheels of an AGV may go up to two meters per minute. Therefore we just have to multiply each vi by 17mm/s before sending it to the AGV. With this system, we can easily compute the output needed by the AGV, which consist of the orientation θi of each wheel and its traction velocity vi: vxi = vcos(θ)−ωRi sin(αi) vyi = vsin(θ)+ωRi cos(αi) vi = √ v2xi +v2yi θi = atan2(vyi,vxi) A motion generation global strategy was elaborated with the artist which implies that the tree "remembers" where one of its metabolism variables was maximum or minimum. One tree will then alternately try to seek "sunlight" and "shadow" (as functions of its sap flow), in a place where he remembers how was its sap flow or in a place where it has not yet gone. Where (Ri,αi) is the polar position of the wheel i (Fig. 8). With this construction, we ensure the uniqueness of the instant center of rotation, so that the wheels do not tear apart an AGV (Fig. 9). The main component of the motion generation is thus the θ parameter. Geolocation gives the absolute (x,y,α) position and orientation of an AGV, and once a destination (xgoal,ygoal) is determined from the last sap flow sensor, we can compute the parameter θ: III. SOFTWARE ARCHITECTURE middleware library, that can be used for both low and high frequency sensor value retrieval and commands. ZeroMQ benefits in being programming language independent and abstracting connections and disconnection. It also implements different usual communication patterns as Client/Server, Pub- lish/Subscribe and Push/Pull. In addition to choosing the different hardware providers, the authors are in charge of the main software development. Figure 6 sums up the different components that have to communicate with each other. The software has been developed to be as modular as possible. Therefore, it is easy to switch between the simulator and the production hardware. This modularity also allows to overcome the diversity in the providers’ technologies, because converters are quick and simple to program. The general idea of this architecture is to have a centralized Path Planner which is both Pulling from the inputs and Publishing to the outputs, so that any input can start and stop Pushing and any output can start and stop Subscribing at any moment, without disturbing the main process. If needed, new functionalities can then even be implemented as a Subscriber/Pusher. Indeed, the sap flow sensors broadcast their results every 30 seconds on a serial cable, the AGV software expects commands as ASCII strings on a TCP socket, and the geolocation software stack is built on a .NET technology that can only be extended with modules from a proprietary framework where the dispatching frequency may vary from less than 1Hz up to 100Hz. A Graphical User Interface based on web technologies has also been developed in order to help the pavilion’s staff to see the current situation and to foresee unexpected problems (Fig. 7). In complex cases, this also allows engineers to work remotely so that there is no need for skilled on-site staff. Therefore, we took advantage of a software architecture built on an abstract message protocol, the ZeroMQ [9], [10] Fig. 7: Web User Interface example, geometrically displaying the current situation: two AGVs are moving on the esplanade, and we can see their position, orientation, and wheel traction speed and direction. v ω v1 v2 v3 θ θ1 θ2 θ3 (a) From (v,θ,ω) to (vi,θi). v ω v1 v2 v3 α1 α2 α3 R1 R2 R3 v v v ωR1 ωR2 ωR3 (b) Geometrical construction. Fig. 8: (vi,θi) as a function of (v,θ,ω). ω v2 Fig. III. SOFTWARE ARCHITECTURE 7: Web User Interface example, geometrically displaying the current situation: two AGVs are moving on the esplanade, and we can see their position, orientation, and wheel traction speed and direction. v ω v1 v2 v3 θ θ1 θ2 θ3 (a) From (v,θ,ω) to (vi,θi). v ω v1 v2 v3 α1 α2 α3 R1 R2 R3 v v v ωR1 ωR2 ωR3 (b) Geometrical construction. Fig. 8: (vi,θi) as a function of (v,θ,ω). v ω v1 v2 v3 θ θ1 θ2 θ3 (a) From (v,θ,ω) to (vi,θi). v ω v1 v2 v3 α1 α2 α3 R1 R2 R3 v v v ωR1 ωR2 ωR3 (b) Geometrical construction. Fig. 8: (vi,θi) as a function of (v,θ,ω). (a) From (v,θ,ω) to (vi,θi). (b) Geometrical construction. Fig. 8: (vi,θi) as a function of (v,θ,ω). v ω v1 v2 v3 ICR Fig. 9: Instant center of rotation. v ω v1 v2 v3 ICR Fig. 9: Instant center of rotation. Fig. 7: Web User Interface example, geometrically displaying the current situation: two AGVs are moving on the esplanade, and we can see their position, orientation, and wheel traction speed and direction. Please note that in nominal operation, this user interface is useless, and the system is fully autonomous. C. Smoothing issues C. Smoothing issues Algorithm 1 Goal selection. start_loop: ▷Start label loop mapsap flow[position] ←s3 ▷Updating maps maptimestamp[position] ←timestamp if ∥goal −position∥< 1 then ▷Need new goal for try ←1,N do if state = 1 then goal ←MINI(mapsap flow) else if state = 2 then goal ←MAXI(mapsap flow) else goal ←MINI(maptimestamp) end if state ←(state+1)%3 if CLEARPATH(goal −position) then goto continue ▷new goal is good end if end for goal ←position ▷Failed to find a new goal v ←0 sleep 5s goto start_loop ▷Try again end if continue: ▷Success label v ←s1 ▷See IV-B θgoal ←atan2(y−ygoal,x−xgoal)−α sleep 100ms end loop function MINI(map) return coordinates of a random minima of map end function function MAXI(map) return coordinates of a random maxima of map end function function CLEARPATH(trajectory) return trajectory∩(borders∪trajectory ) ∅ Algorithm 1 Goal selection. V. RESULTS V. RESULTS ω does not appear in this algorithm, as a tree is allowed to turn on itself even in a deadlock. B. Motion generation According to the specifications, the motion generation has to be generated from the tree metabolism. As we previously saw that we need three input variables, we chose to use the outputs of three sap flow sensors (s1,s2,s3), normalized in [0,1]. θ = atan2(y−ygoal(s3),x−xgoal(s3))−α This idea addresses the space covering problem and is developed in subsection IV-D. Two of them will be directly mapped to the linear velocity v and the angular velocity ω of the trunk of the tree: Algorithm 1 Goal selection. A. User Interface A result of such algorithm in simulation is in Fig. 12. The user interface is composed by a map of the Pavilion and the Giardini. AGVs are displayed in position and ori- entation according to the data gathered from the geolocation system (Fig. 10). An objective of motion generation is to guarantee a reasonable coverage of the entire inside and outside areas. Multiple runs in simulation (Fig. 12) helped to tune the controls through the normalization parameters of (s1,s2,s3). In outside area motion coordination of the trees is performed in a centralized way on the basis of their respective local- izations. From a formal point of view the system is not guaranteed to be free of local minima. However completeness of the algorithm is not an issue for practical reasons. Indeed operational regulations impose the staff to be permanently present to prevent any mishap. The staff is then allowed to unblock potential deadlock by giving trees new goals. In practice staff interventions occurred scarcely (less than once a week). Notice that deadlock is not an issue for the tree inside the pavilion. Below this map are provided several numerical indicators and controls that ease the maintenance operations that have to be performed by the pavilion’s attendants. This avoids most of the time the need to control an AGV by directly plugin a joystick on it in front of the public. loop If this smoothing component detects that the difference between the curent direction of an AGV and the direction of the goal is greater than 2π/3, it can also invert the direction of the wheels to have to turn only by less than π/3. This also greatly improves the behaviour on the cusps. A similar component works at the wheel level, but is adapted to ensure that the ICR remains unique. function CLEARPATH(trajectory) return trajectory∩(borders∪trajectoryotherAGV) = ∅ end function ▷No obstacle between position and goal end if return coordinates of a random minima of map end function function MAXI(map) Then, a finite state machine will alternate the goal = (xgoal,ygoal) choosing mechanism between areas with high and low sap flows and areas where the tree has not been for a while. function MAXI(map) return coordinates of a random maxima of map end function In some circumstances, none of these possible goals are reachable in straight line without crossing the borders of the evolution area or the trajectory of an other AGV. In such case, defined as deadlock, the AGV must wait in its current position = (x,y). function CLEARPATH(trajectory) D. Goal selection The motion generation defines how an AGV would evolve alone on an infinite plane. We added some rules to ensure that the goal selection will not make the tree run straight into a wall or another AGV. These rules are detailed in Algorithm 1, but the general idea is to continuously update two maps of the area of evolution with the timestamp and s3 at the current coordinates, so that the tables mapsap flow and maptimestamp will contain a heat map of respectively the areas where the sap flow is high or low (that will probably match sunlight and shadow zones) and the areas where an AGV has not recently been, in order to update the value of s3 at these places. C. Smoothing issues start_loop: ▷Start label loop mapsap flow[position] ←s3 ▷Updating maps maptimestamp[position] ←timestamp if ∥goal −position∥< 1 then ▷Need new goal for try ←1,N do if state = 1 then goal ←MINI(mapsap flow) else if state = 2 then goal ←MAXI(mapsap flow) else goal ←MINI(maptimestamp) end if state ←(state+1)%3 if CLEARPATH(goal −position) then goto continue ▷new goal is good end if end for goal ←position ▷Failed to find a new goal v ←0 sleep 5s goto start_loop ▷Try again end if continue: ▷Success label v ←s1 ▷See IV-B θgoal ←atan2(y−ygoal,x−xgoal)−α sleep 100ms end loop function MINI(map) return coordinates of a random minima of map end function function MAXI(map) return coordinates of a random maxima of map end function function CLEARPATH(trajectory) return trajectory∩(borders∪trajectoryotherAGV) = ∅ end function ▷No obstacle between position and goal g start_loop: ▷Start label loop mapsap flow[position] ←s3 ▷Updating maps maptimestamp[position] ←timestamp if ∥goal −position∥< 1 then ▷Need new goal for try ←1,N do if state = 1 then goal ←MINI(mapsap flow) else if state = 2 then goal ←MAXI(mapsap flow) else goal ←MINI(maptimestamp) end if state ←(state+1)%3 if CLEARPATH(goal −position) then goto continue ▷new goal is good end if end for goal ←position ▷Failed to find a new goal v ←0 sleep 5s goto start_loop ▷Try again end if continue: ▷Success label v ←s1 ▷See IV-B θgoal ←atan2(y−ygoal,x−xgoal)−α sleep 100ms end loop function MINI(map) return coordinates of a random minima of map end function function MAXI(map) return coordinates of a random maxima of map end function function CLEARPATH(trajectory) return trajectory∩(borders∪trajectoryotherAGV) = ∅ start_loop: start_loop: On top of that basic motion generation idea, we imple- mented a smoothing component that controls the maximum angular speed of each wheel. Indeed, the two AGVs which evolve in the gardens can get easily stuck in the mud, by turning their wheels and therefore digging the ground. In a same way, the AGV which is inside the French Pavilion can produce loud tire squeal when its wheels turn abruptly. C. Experimental During the first days of tests, we realized that as the AGVs’ wheels can be very quickly reoriented. It was in favor of some early tests, but introduced many issues: • a step response makes the orientation engines audible; • a step response makes the orientation engines audible; • a quick turn of a wheel on itself leaves a permanent tire mark on the floor inside the French pavilion; • a slide of the rubber on the concrete also makes a loud shrill noise; • depending on the esplanade’s soil moisture, a change in orientation of a wheel with a low traction speed is likely to dig a small trench. Then, if a trench is too deep or if multiple wheels are in trenches, the AGV has an high probability of getting stuck. Fig. 10: User Interface screenshot, in production (9th of September, 10:30 to 10:45), where we can see the path recently taken by the AGVs, and a table with status and controls for each AGV. This is why we implemented the smoothing component (subsection IV-C) different situations, that would take weeks to perform in real time. Moreover, the software configuration of the geolocation system was not perfect from the beginning (Fig. 13). In most cases it worked exactly as expected, but unusual signal disturbance could induce inadmissible behaviours. The software blocks are the same as those described on the Fig. 6, except for the AGV and the geolocation, that are replaced by a single simulator module. Sap sensors data simulation has been done from several days real data recorded in Toulouse on a pine-tree of the same family as the Biennale trees. Fig. 13: AGV trying to follow a square. Success rate can vary considerably even in similar conditions. On Fig. 11, we show an example the paths followed by the trees together with the tracks that would be let on the ground by the wheels in such a simulation. (a) Center of the AGVs. (b) Wheels of the AGVs. Fig. 11: Example of a simulation with the tracks of the tree’s trunk and the wheels of the AGVs. In real life, this would approximately correspond to a 45 minutes journey. A trunk must not go out of the black polygon, but a wheel can. (b) Wheels of the AGVs. (a) Center of the AGVs. Fig. 13: AGV trying to follow a square. B. Simulator The short period of development on site (only one week) together with the very low AGV velocity imposed to tune the system imposed the use of a simulator. It allows to quickly check how the motion planning system would react in many Fig. 10: User Interface screenshot, in production (9th of September, 10:30 to 10:45), where we can see the path recently taken by the AGVs, and a table with status and controls for each AGV. VI. CONCLUSION This paper contributes to disseminate the domain of art as an application field for robotics. Art history is rich in openings towards science and technology. Mobile robotics offers artists the unique possibility of making things moving and exploring the world, far beyond the possibilities of classical robot mechanisms that remain at fixed positions. Planned and controlled movements appear as new available components of artistic expression. Artist Robert Breer made floating sculptures moving at the end of the sixties [11]. Making trees moving six days a week during seven months, while revealing some autonomy in the strong operational conditions of Venice Biennale, has been a challenge. The challenge would not have been addressable thirty years ago. Robotics technology is today spreading quickly in arts field [12]. Fig. 14: The pictures show the trees inside and outside the pavilion respectively. The AGVs are moving so slowly and silently that it takes few seconds for visitors to notice the motions. The solutions we have developed for transHumUs project did not ask for any new robot algorithm. The entire sys- tem has been built from existing know-how. However the devil was in the details. Translating artistic intentions into technological terms has required a lot of discussions. Motion qualities (silence, smoothness, variability, coverage) required a lot of control law tuning. While classical application domains in robotics (e.g. manufacturing, field robotics) ask for performance in completing an action (e.g. manipulation, exploration), art imposes constraints that are better defined in terms of style than in terms of actions to perform. These constraints open new issues in robot motion generation. By its very nature, the topic is necessarily multidisciplinary (e.g. [13]) and it enlarges the scope of robotics research. Fig. 14: The pictures show the trees inside and outside the pavilion respectively. The AGVs are moving so slowly and silently that it takes few seconds for visitors to notice the motions. ACKNOWLEDGMENT The authors are supported by European Research Council (ERC) grant 340050 Actanthrope. They thank C. Boursier- Mougenot and exhibition curator E. Lavigne for attracting them in such thrilling adventure. The project has been sup- ported by French Institut from the French Ministry of Foreign Affairs. It benefited from the active participation of producers E. Albaran and T. Patrix, technical manager P. Zimmerman and his staff, G. Caverot and BA Systemes team, H. Peyre from Ubisense. The authors have been trained to Granier probe technology by biologists J. Chave (EDB-CNRS lab) and V. Le Dantec (CESBIO lab). Picture in Fig. 1 and 14 have been taken by photographer L. Lecat. [6] P. Z. P. Lu, L. Urban, “Granier’s thermal dissipation probe (tdp) method for measuring sap flow in trees: Theory and practice,” Acta Botanica Sinica, vol. 46, no. 6, pp. 631–646, 2004. [7] D. Kocur, P. Kazimir, J. Fortes, D. Novak, M. Drutarovsky, P. Galajda, and R. Zetik, “Short-range uwb radar: Surveillance robot equipment of the future,” in Systems, Man and Cybernetics (SMC), 2014 IEEE International Conference on. IEEE, 2014, pp. 3767–3772. [8] A. Bahr, A. Feldman, J. Colli-Vignarellii, S. Robert, C. Dehollaini, and A. Martinoli, “Modeling and benchmarking ultra-wideband local- ization for mobile robots,” in Ultra-Wideband (ICUWB), 2012 IEEE International Conference on, 2012, pp. 443–447. [9] B. Dias, B. Rodrigues, J. Claro, J. P. Pimentão, P. Sousa, and S. Onofre, “Architecture and message protocol proposal for robots integration in multi-agent surveillance system,” in Rough Sets and Current Trends in Computing. Springer, 2014, pp. 366–373. C. Experimental Success rate can vary considerably even in similar conditions. (b) Wheels of the AGVs. (a) Center of the AGVs. (b) Wheels of the AGVs. (a) Center of the AGVs. Fig. 11: Example of a simulation with the tracks of the tree’s trunk and the wheels of the AGVs. In real life, this would approximately correspond to a 45 minutes journey. A trunk must not go out of the black polygon, but a wheel can. Those issues were solved by tuning the configuration of the Ubisense’s software stack. Considering the speed of our robots, the need for safety has not been a major issue. The pavilion attendants may use the user interface to softly avoid casual heavy obstacles or a remote kill switch in case of emergency. At the time of the writing of this article, the AGVs have been running for four months. The nominal mode has been increasingly used, from a few hours a day before the opening to entire weeks this summer. The running shows that the museum staff interventions rarely occur (less than once a week). Fig. 12: Example of space covering simulation. On site, such coverage would have taken several days between the end of the hardware installation and the opening of the Biennale, which we had not. The main problem is the weather, as it can make the outside area impracticable for our robots. Still, the public can visit the inside of the French Pavilion in case of heavy rain. Fig. 12: Example of space covering simulation. On site, such coverage would have taken several days between the end of the hardware installation and the opening of the Biennale, which we had not. Noiseless specification was respected, as the engines are perfectly inaudible, as long as there is no sudden turn in any wheel. And the smoothing component worked for that purpose. VI. CONCLUSION [6] P. Z. P. Lu, L. Urban, “Granier’s thermal dissipation probe (tdp) method for measuring sap flow in trees: Theory and practice,” Acta Botanica Sinica, vol. 46, no. 6, pp. 631–646, 2004. pp [7] D. Kocur, P. Kazimir, J. Fortes, D. Novak, M. Drutarovsky, P. Galajda, and R. Zetik, “Short-range uwb radar: Surveillance robot equipment of the future,” in Systems, Man and Cybernetics (SMC), 2014 IEEE International Conference on. IEEE, 2014, pp. 3767–3772. [6] P. Z. P. Lu, L. Urban, “Granier’s thermal dissipation probe (tdp) method for measuring sap flow in trees: Theory and practice,” Acta Botanica Sinica, vol. 46, no. 6, pp. 631–646, 2004. REFERENCES [10] A. Dworak, M. Sobczak, F. Ehm, W. Sliwinski, and P. Charrue, “Middleware trends and market leaders 2011,” 13th International Conference on Accelerator and Large Experimental Physics Control Systems, pp. 1334–1337, Oct 2011. [Online]. Available: https://cds.cern.ch/record/1391410 [1] E. Di Martino, The history of the Venice Biennale. Papiro Arte, 2005. [2] B. A. MacAdam, “French pavilion artist Céleste Boursier-Mougenot teaches the world to sing,” ARTnews, april 2015. [3] G. Campion, G. Bastin, and B. Dandrea-Novel, “Structural properties and classification of kinematic and dynamic models of wheeled mobile robots,” Robotics and Automation, IEEE Transactions on, vol. 12, no. 1, pp. 47–62, 1996. [11] A. Pardey, “Robert Breer - Floats. On Sculpture without a Place,” in Robert Breer. Bielefeld Kerber, 2011, pp. 100–104. pp [12] C. K. Damith Herath and S. (Editors), Robots and Art: Exploring an Unlikely Symbiosis. Springer, 2015 (to appear). [4] A. Granier, “Evaluation of transpiration in a douglas-fir stand by means of sap flow measurements,” Tree Physiology, vol. 3, no. 4, pp. 309–320, 1987. [13] J. Laumond and N. A. (Editors), Dance Notations and Robot Motion, ser. Star Series. Springer, 2016 (to appear). [5] S. A. James, M. J. Clearwater, F. C. Meinzer, and G. Goldstein, “Heat dissipation sensors of variable length for the measurement of sap flow in trees with deep sapwood,” Tree Physiology, vol. 22, no. 4, pp. 277– 283, 2002.
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Association of 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphisms with type 2 diabetes in obese women candidates for bariatric surgery
Archives of Endocrinology and Metabolism
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original article original article original article Keywords Keywords Obesity; type 2 diabetes; hypertension; gene polymorphism Obesity; type 2 diabetes; hypertension; gene polymorphism Obesity; type 2 diabetes; hypertension; gene polymorphism DOI: 10.1590/2359-3997000000260 Association of 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphisms with type 2 diabetes in obese women candidates for bariatric surgery Noa Pereira Prada Schnor1, Rozangela Verlengia2, Patrícia Fátima Sousa Novais1, Alex Harley Crisp1, Celso Vieira de Souza Leite3, Irineu Rasera-Júnior4, Maria Rita Marques de Oliveira1 1 Universidade Estadual Paulista Júlio de Mesquita Filho (Unesp), Programa de Pós-Graduação em Alimentos e Nutrição, Araraquara, SP, Brasil 2 Universidade Metodista de Piracicaba (Unimep), Programa de Pós-Graduação em Ciências do Movimento Humano, Piracicaba, SP, Brasil 3 Unesp, Faculdade de Medicina, Botucatu, SP, Brasil 4 Clínica Bariátrica, Piracicaba, SP, Brasil ABSTRACT Objective: Obesity can cause systemic arterial hypertension (SAH) and type 2 diabetes mellitus (DM2) factor that is also influenced by genetic variability. The present study aims to investigate the association between gene polymorphisms related with obesity on the prevalence of SAH and DM2 in the preoperative period and 1 year after Roux-en-Y gastric bypass surgery. Subjects and methods: In total, 351 obese women in a Brazilian cohort completed the study. The clinical diagno­ sis of SAH and DM2 was monitored from medical records. Twelve gene polymorphisms (rs26802; rs572169; rs7799039; rs1137101; rs3813929; rs659366; rs660339; rs1800849; rs7498665; rs35874116; rs9701796; and rs9939609) were determined using real-time polymerase chain reaction and TaqMan assay. Results: In the preoperative period, prevalence of SAH and DM2 was 57% and 22%, respec­ tively. One year postoperatively, 86.8% subjects had remission of DM2 and 99.5% had control of SAH. Subjects with T allele from the serotonin receptor gene (5-HT2C, rs3813929) had five times greater chance of DM2, and the CC genotype from uncoupling protein 3 gene (UCP3, rs1800849) had three times greater chance in the preoperative period. Conclusion: These findings indicate that polymor­ phisms rs3813929 and rs1800849 from 5-HT2C and UCP3 genes were related to DM2 prevalence among the Brazilian obese women candidates for bariatric surgery. Arch Endocrinol Metab. 2017;61(3):326-31. Correspondence to: Maria Rita Marques de Oliveira Universidade Estadual Paulista (Unesp), Instituto de Biociências. Distrito de Rubião Junior, s/n, caixa postal 510 18618-000 – Botucatu, SP, Brasil mrmolive@ibb.unesp.br Received on April/17/2016 Accepted on Nov/25/2016 DOI: 10.1590/2359-3997000000260 Correspondence to: Maria Rita Marques de Oliveira Universidade Estadual Paulista (Unesp), Instituto de Biociências. Distrito de Rubião Junior, s/n, caixa postal 510 18618-000 – Botucatu, SP, Brasil mrmolive@ibb.unesp.br INTRODUCTION It is recognized that obesity and its associated comorbidities have multifactorial causes. Therefore, it is important to analyze several genes and the interaction between them to better understand the obesity genesis and the contribution of genetic variability. Review studies indicate a large number of gene candidates that may be related to obesity and chronic diseases (7-9). O besity prevalence is increasing at a worrying rate; with significant influence on morbidity and mor­ tality rate worldwide (1). Excessive food intake and a sedentary lifestyle contribute to the accumulation of body fat and increase the risk of chronic diseases such as systemic arterial hypertension (SAH) and type 2 dia­ betes mellitus (DM2) (2). Moreover, genetic factors are recognized as an important influence in increasing sus­ ceptibility to obesity and its associated comorbidities (3). Copy g & a g s ese ed O besity prevalence is increasing at a worrying rate; with significant influence on morbidity and mor­ tality rate worldwide (1). Excessive food intake and a sedentary lifestyle contribute to the accumulation of body fat and increase the risk of chronic diseases such as systemic arterial hypertension (SAH) and type 2 dia­ betes mellitus (DM2) (2). Moreover, genetic factors are recognized as an important influence in increasing sus­ ceptibility to obesity and its associated comorbidities (3). A genetic factor of considerable interest is the variation of a gene (DNA sequence bases) between subjects, described as gene polymorphism (4). In this context, some studies have related polymorphism of specifics genes that control appetite, energy metabolism, susceptibility to obesity (5), and chronic diseases such as DM2 and SAH (6). O Regarding the treatment of obesity, bariatric surgery is the most long-term effective method of inducing body weight loss and beneficial effects on metabolic disorders (10). However, there is a significant variability in body weight loss and control of comorbidities between subjects undergoing bariatric surgery (11). This suggests that some of these variable responses to bariatric surgery can be explained by genetic factors. In this sense, investigating the relationship between uncoupling protein 3 (UCP3) gene polymorphism 326 Arch Endocrinol Metab. 2017;61/4 Gene polymorphism and bariatric surgery (rs1800849) and results after biliopancreatic diversion surgery (BDS), Luis and cols. (12) observed no significant association with the control of SAH and DM2 1 year postoperatively. However, another study from Luis and cols. INTRODUCTION (13) indicated that fat mass and obesity- associated (FTO, rs9939609) gene polymorphism were associated with greater body weight loss 3 months after BDS, but no difference was evident after 9 and 12 months postoperatively. In addition, Luis and cols. (13) observed an association between FTO gene polymorphism (rs9939609) with a decrease in the body mass index (BMI), and improvements of glycated hemoglobin (HbA1c) levels 6 months after mini gastric bypass laparoscopic surgery. Therefore, other studies are warranted to investigate a larger number of gene polymorphisms to expand the evidence regarding the effect of genetic variability as a determinant of SAH and DM2 on obesity and bariatric surgery response. i (c) and registered on the waiting line for bariatric surgery. The exclusion criteria were as follows: (a) alcoholism; (b) genetic syndromes associated with obesity; (c) Cushing’s syndrome; (d) hypothyroidism; (e) renal or liver failure; (f) neoplasia; (g) infection with human immunodeficiency virus (HIV); (h) use of corticosteroids; (i) and postmenopausal women using estrogen replacement. In total, 441 women in a Brazilian cohort were eligible for study inclusion. However, subjects who did not attend 1 year postoperatively were not included in data analysis. This study included 351 subjects who completed the study. The number of subjects was determined using the data of the genotype with lower prevalence and a minimum 300 subjects was shown to be necessary. All subjects signed a free-and- informed consent form after being informed about the procedures involved in the research. This study was approved by the local Research Ethics Committee (protocol number: 3303/2009). The main benefit of surgical treatment of obesity is the control of comorbidities. Therefore, the current study selected 12 gene polymorphisms related to obesity in order to assess an association with SAH and DM2 prevalence, and the possible influence on bariatric surgery results. There is some evidence that suggests effects on the prevalence of DM2 with polymorphisms of ghrelin (GHRL, rs26802), uncoupling protein 2 (UCP2, rs659366), uncoupling protein 3 (UCP3, rs1800849), FTO (rs9939609), leptin (LEP, rs7799039), leptin receptor (LEPR, rs1137101), and serotonin receptor (5-HT2C, rs3813929) genes. Body weight was measured using a digital balance (Fizola, SP, Brazil) with a capacity of 200 kg, precise to within 100 g. Height measurements were obtained using a stadiometer (Seca, SP, Brazil) precise to within 0.1 cm. The procedures followed the standard recommendations (14). EW = body weight preoperatively – ideal body weight; WL = body weight preoperatively – current body weight; %EWL = percentage of body weight lost in relation to excess weight. The SAH remission was considered when the subject stopped with anti-hypertensive medications and presented: systolic blood pressure < 140 mmHg and diastolic blood pressure < 90 mmHg (16). The DM2 remission was considered when the subject stopped with anti-diabetic medications and presented: fasting glucose concentration < 126 mg/dL; postprandial glucose < 200 mg/dL after 75g oral glucose load, and hemogloblin A1c level < 6.5% (17). Arch Endocrinol Metab. 2017;61/4 INTRODUCTION Excess weight (EW, in kg); weight loss (WL, in kg), and percentage of excess weight loss (%EWL), were calculated using the followings formulas (15): Thus, the current study aimed to investigate the association of 12 gene polymorphisms on the prevalence of SAH and DM2 in the preoperative period and 1 year after Roux-en-Y gastric bypass (RYGB) surgery in obese women. EW = body weight preoperatively – ideal body weight; WL = body weight preoperatively – current body weight; %EWL = percentage of body weight lost in relation to excess weight. EW = body weight preoperatively – ideal body weight; WL = body weight preoperatively – current body weight; %EWL = percentage of body weight lost in relation to excess weight. SUBJECTS AND METHODS In the 5HT2C g carriers of the T allele had a five times g of DM2 compared to CC genotype In (rs659366); UCP2 (rs660339); UCP3 (rs1800849); SH2B1 (rs7498665); TAS1R2 (rs35874116); TAS1R2 (rs9701796); and FTO (rs9939609), was determined by real-time polymerase chain reaction (RT-PCR) and TaqMan assay (Applied Biosystems®, Branchburg, New Jersey, USA). The RT-PCR was processed in ABI 7500 fast equipment (Applied Biosystems®, Branchburg, NJ, USA) according to the manufacturer’s instructions. A random selection of 10% of the samples was again genotyped to evaluate the reproducibility of genotyping. The agreement of genotype frequencies with Hardy–Weinberg equilibrium expectations was tested by chi-square test. Multiple logistic regressions were performed to evaluate the effects of gene polymorphism (with adjustments for age, skin color, preoperative BMI, previous pregnancy, and age at onset of obesity) taking SAH and DM2 as dependent variables. When the genotype of lower frequency was < 10%, the genotype was combined with a heterozygous genotype. p < 0.05 was considered to be statistically significant. (rs659366); UCP2 (rs660339); UCP3 (rs1800849); SH2B1 (rs7498665); TAS1R2 (rs35874116); TAS1R2 (rs9701796); and FTO (rs9939609), was determined by real-time polymerase chain reaction (RT-PCR) and TaqMan assay (Applied Biosystems®, Branchburg, New Jersey, USA). The RT-PCR was processed in ABI 7500 fast equipment (Applied Biosystems®, Branchburg, NJ, USA) according to the manufacturer’s instructions. A random selection of 10% of the samples was again genotyped to evaluate the reproducibility of genotyping. (rs659366); UCP2 (rs660339); UCP3 (rs1800849); SH2B1 (rs7498665); TAS1R2 (rs35874116); TAS1R2 (rs9701796); and FTO (rs9939609), was determined by real-time polymerase chain reaction (RT-PCR) and TaqMan assay (Applied Biosystems®, Branchburg, New Jersey, USA). The RT-PCR was processed in ABI 7500 fast equipment (Applied Biosystems®, Branchburg, NJ, USA) according to the manufacturer’s instructions. A random selection of 10% of the samples was again genotyped to evaluate the reproducibility of genotyping. The agreement of genotype frequencies with Hardy–Weinberg equilibrium expectations was tested by chi-square test. Multiple logistic regressions were performed to evaluate the effects of gene polymorphism (with adjustments for age, skin color, preoperative BMI, previous pregnancy, and age at onset of obesity) taking SAH and DM2 as dependent variables. When the genotype of lower frequency was < 10%, the genotype was combined with a heterozygous genotype. p < 0.05 was considered to be statistically significant. diabetic subjects who obtained or did not obtain the control of DM2 postoperatively (Table 3). DISCUSSION This study investigated the association of 12 gene polymorphisms related to obesity on the prevalence of SAH and DM2 during the preoperative period and 1 year after RYGB surgery in obese women. Our main finding indicated that 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphisms were associated with the prevalence of DM2 in obese women candidates for bariatric surgery. The agreement of genotype frequencies with Hardy–Weinberg equilibrium expectations was tested by chi-square test. Multiple logistic regressions were performed to evaluate the effects of gene polymorphism (with adjustments for age, skin color, preoperative BMI, previous pregnancy, and age at onset of obesity) taking SAH and DM2 as dependent variables. When the genotype of lower frequency was < 10%, the genotype was combined with a heterozygous genotype. p < 0.05 was considered to be statistically significant. Table 1. Characteristics of subject study Variables Median (min–max) Age (years) 34 (20–50) Age at onset of obesity (years) 18 (0–40) Preoperative BMI (kg/m2) 46 (33–73) BMI postoperatively (kg/m2) 30 (20–55) %EWL 1 year postoperatively 69 (37–119) DM2 preoperative – n (%) Yes 76 (22) No 275 (88) DM2 1 year postoperative – n (%) Yes 10 (2.8) No 341 (97.2) SAH preoperative – n (%) Yes 201 (57) No 150 (43) SAH 1 year postoperative – n (%) Yes 01 (0.3) No 350 (99.7) Family history of obesity – n (%) Yes 279 (79.5) No 72 (20.5) Skin Color – n (%) Black 52 (14.8) White 226 (64.4) Parda 73 (20.8) Education – n (%) Middle and High School (incomplete) 123 (35) High School (complete) and superior 228 (65) Previous pregnancies – n (%) Yes 263 (74.9) No 88 (25.1) n = 351. BMI: body mass index; %EWL: percentage of excess weight loss. Table 1. Characteristics of subject study SUBJECTS AND METHODS This is a prospective study with adult women undergoing RYGB surgery, performed by the same medical staff from June 2010 to May 2013. The subjects were evaluated in the preoperative period and 1 year postoperatively. Self-reported information regarding age, skin color, education, previous pregnancies, family history of obesity, and obesity at early age was recorded. Body weight, BMI, and the presence of SAH and DM2 were provided by medical records. Genomic DNA was isolated from whole blood samples (EDTA-treated), using Illusta blood genomicPrep Kit (GE Healthcare®, New York, USA). The analysis of gene polymorphisms GHRL (rs26802); GHRS (rs572169); LEP (rs7799039); LEPR (rs1137101); 5HT2C (rs3813929); UCP2 The eligible criteria for participation were: (a) being female; (b) aged between 20 and 50 years; 327 Arch Endocrinol Metab. 2017;61/4 Gene polymorphism and bariatric surgery rved. (rs659366); UCP2 (rs660339); UCP3 SH2B1 (rs7498665); TAS1R2 (rs358741 (rs9701796); and FTO (rs9939609), wa by real-time polymerase chain reaction (R TaqMan assay (Applied Biosystems®, Bran Jersey, USA). The RT-PCR was processed fast equipment (Applied Biosystems®, Bra USA) according to the manufacturer’s A random selection of 10% of the samp genotyped to evaluate the reproducibility o The agreement of genotype frequ Hardy–Weinberg equilibrium expectation by chi-square test. Multiple logistic regr performed to evaluate the effects of gene p (with adjustments for age, skin color, BMI, previous pregnancy, and age at ons taking SAH and DM2 as dependent variab genotype of lower frequency was < 10%, was combined with a heterozygous genot was considered to be statistically significan RESULTS The genotype distributions of gene po (rs26802; rs572169; rs7799039; rs3813929; rs659366; rs660339; rs7498665; rs35874116; rs9701796; was within the expectations of the Ha equilibrium (p > 0.05). Table 1 shows ch of the study subject. One year postoperatively, of 76 subject only 10 maintained the diagnosis of DM2, r control of 86.8%. Regarding SAH, 99.5% o control of this disease. The median of %EW of which 4.3% of the subjects had less than 5 and 26.7% had DM2. One subject did not of SAH postoperatively and presented 58.7 Because of a high proportion of con comorbidities postoperatively, logistic analysis was possible only with preop Among 12 gene polymorphisms analy (rs3813929) and UCP3 (rs1800849) h on DM2 prevalence. RESULTS The genotype distributions of gene polymorphisms (rs26802; rs572169; rs7799039; rs1137101; rs3813929; rs659366; rs660339; rs1800849; rs7498665; rs35874116; rs9701796; rs9939609) was within the expectations of the Hardy-Weinberg equilibrium (p > 0.05). Table 1 shows characterization of the study subject. One year postoperatively, of 76 subjects with DM2, only 10 maintained the diagnosis of DM2, representing a control of 86.8%. Regarding SAH, 99.5% of subjects had control of this disease. The median of %EWL was 69.2%; of which 4.3% of the subjects had less than 50% of %EWL and 26.7% had DM2. One subject did not have control of SAH postoperatively and presented 58.7% of %EWL. py g g Because of a high proportion of control of these comorbidities postoperatively, logistic regression analysis was possible only with preoperative data. Among 12 gene polymorphisms analyzed, 5HT2C (rs3813929) and UCP3 (rs1800849) had influence on DM2 prevalence. In the 5HT2C gene, subject carriers of the T allele had a five times greater chance of DM2 compared to CC genotype. In UCP3 gene, CC genotype had three times greater chance of DM2 compared with T allele carriers (Table 2). However, there was no prevalence of 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphisms among 328 Gene polymorphism and bariatric surgery Table 2. Values of odds ratio (OR) and 95% confidence interval (95% CI) for diabetes mellitus type 2 (DM2) and systemic arterial hypertension (SAH) before and after bariatric surgery, according to each gene polymorphism Table 2. Arch Endocrinol Metab. 2017;61/4 RESULTS Values of odds ratio (OR) and 95% confidence interval (95% CI) for diabetes mellitus type 2 (DM2) and systemic arterial hypertension (SAH) before and after bariatric surgery, according to each gene polymorphism Gene Polymorphism Genotype n (%) DM2 Preoperative SAH Preoperative OR (95% CI) p-value OR (95% CI) p-value GHRL rs26802 CC 35 (10) 1.00 0.921 1.00 0.563 CA + AA 316 (90) 1.045 (0.436–2.507) 1.244 (0.593–2.609) GHSR rs572169 GG 211 (60) 1.00 0.547 1.00 0.443 GA + AA 140 (40) 0.722 (0.250–2.085) 1.441 (0.566–3.667) LEP rs7799039 AA 53 (15) 1.00 0.318 1.00 0.274 AG + GG 298 (85) 0.652 (0.282–1.508) 0.712 (0.388–1.307) LEPR rs1137101 GG 73 (21) 1.00 0.974 1.00 0.945 AG + AA 278 (79) 0.989 (0.514–1.902) 1.019 (0.596–1.742) 5–HT2C rs3813929 TT/CT 87 (25) 1.00 0.001 1.00 0.556 CC 264 (75) 0.251 (0.109–0.578) 1.164 (0.702–1.928) UCP2 rs659366 TT 49 (14) 1.00 0.505 1.00 0.884 CT + CC 302 (86) 1.330 (0.574–3.082) 1.056 (0.511–2.181) UCP2 rs660339 AA 49 (14) 1.00 0.238 1.00 0.675 AG + GG 302 (86) 0.568 (0.222–1.452) 1.167 (0.567–2.405) UCP3 rs1800849 CC 54 (15) 1.00 0.019 1.00 0.128 CT + TT 297 (85) 0.305 (0.113–0.821) 0.624 (0.341–1.144) SH2B1 rs7498665 GG 223 (64) 1.00 0.594 1.00 0.946 AG + AA 294 (84) 0.815 (0.385–1.726) 0.980 (0.544–1.765) TAS1R2 rs35874116 CC + CT 184 (52) 1.00 0.790 1.00 0.933 TT 167 (48) 1.706 (0.626–1.850) 1.019 (0.658–1.579) TAS1R2 rs9701796 GG + CG 117 (33) 1.00 0.353 1.00 0.341 CC 234 (67) 0.761 (0.428–1.354) 1.252 (0.788–1.989) FTO rs9939609 AA 71 (20) 1.00 0.911 1.00 0.058 AT + TT 280 (80) 0.963 (0.492–1.882) 0.595 (0.348–1.017) n = 351. Variables with adjustments for age, skin color, preoperative BMI, previous pregnancy and age at onset of obesity. n = 351. Variables with adjustments for age, skin color, preoperative BMI, previous pregnancy and age at onset of obesity. polymorphism for 5–HT2C (rs3813929) and UCP3 (rs1800849) between diabetic subjects who obtained or did not obtain t i Table 3. Prevalence of gene polymorphism for 5–HT2C (rs3813929) and UCP3 (rs1800849) between diabetic subjects who obtained or did not obtain the control of DM2 after bariatric surgery Response CT + CC CT + CT CT + TT CC + CC CC + CT CC + TT Yes 1/65 8/58 4/62 7/59 22/44 24/42 No 0/10 1/9 1/9 1/9 3/7 4/6 p-value* 0.868 1.000 0.985 1.000 1.000 0.999 * Fisher’s exact test. Table 3. RESULTS Prevalence of gene polymorphism for 5–HT2C (rs3813929) and UCP3 (rs1800849) between diabetic subjects wh the control of DM2 after bariatric surgery Unlike the results of other studies (18-23), the current study did not find an association of GHRL (rs26802), UCP2 (rs659366), FTO (rs9939609), LEP (rs7799039), and LEPR (rs1137101) gene polymorphisms with DM2. These controversies, far from discouraging the study of the effects of gene polymorphisms, instigate a deepening of this knowledge, which takes into account the characteristics This knowledge might be important in identifying, according to genetic variability, subjects who are at higher risk of DM2, because it is not obesity per se that is the lethal factor, but it’s associated chronic diseases. Thus, the analysis of 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphism may theoretically define individualized and preventive measures for DM2 in obese women on a waiting list for bariatric surgery. Arch Endocrinol Metab. 2017;61/4 329 Gene polymorphism and bariatric surgery of the populations and the interactions between the different gene polymorphisms. Although the molecular and genetic comprehension of DM2 has advanced rapidly, much of the knowledge remains unknown. Adding to this, the number of bariatric surgeries has risen as an effective treatment option for obesity and its associated comorbidities. Thus, more studies are necessary to investigate the effects of surgical and non-surgical intervention for weight loss and remission of DM2, while larger numbers of subjects, a wider range of gene polymorphism, and analyzing a later postoperative time, could better elucidate these questions. Regarding 5-HT2C gene polymorphism results, serotonin plays an important role in the nervous system, such as sleep regulation, body temperature, appetite and mood, among others (24). Thus, low levels of serotonin or problems in the signaling with the receptor have been linked to an increased desire to eat sweets and carbohydrates (25), which may explain in part the association of 5-HT2C gene polymorphism (rs3813929) with the prevalence of DM2 observed in our study. Genetic association studies are relevant to determine the risk of chronic diseases on a specific population and to improve the medical and nutritional treatment. These results enable an individualization of the treatment according to genetic variability. The current study investigates 12 obesity-related gene polymorphisms in obese women candidates for bariatric surgery. Similarly, other studies reported that subjects carrying the T allele from 5-HT2C gene polymorphism (rs3813929) were associated with greater chances of DM2. Kring and cols. RESULTS (26), investigating Caucasian young men, observed an association of T allele from 5-HT2C gene polymorphism with glucose and acute insulin response. On the other hand, Iordanidou and cols. (27) observed that T allele frequency was lower in diabetic subjects compared to nondiabetic subjects. In summary, subjects with T allele from 5-HT2C gene polymorphism (rs3813929) had five times greater chance of DM2, and the CC genotype from UCP3 gene polymorphism (rs1800849) had three times greater chance of DM2, in the preoperative period. Our results indicate that 5-HT2C and UCP3 gene polymorphism was related to prevalence of DM2 among Brazilian obese women candidates for bariatric surgery. In the present study, the UCP3 gene was also associated with prevalence of DM2, specifically the polymorphism rs1800849. The UCP3 protein is involved in promoting fatty acid oxidation in skeletal muscle and it indirectly influences glucose metabolism (28). In addition, UCP3 regulates the production of reactive oxygen species in the mitochondria (28), a factor that has established an association of this protein with DM2, because oxidative stress pathways play a key role in the development of this chronic disease (29). Disclosure: no potential conflict of interest relevant to this article was reported. Disclosure: no potential conflict of interest relevant to this article was reported. Disclosure: no potential conflict of interest relevant to this article was reported. REFERENCES Gueorguiev M, Lecoeur C, Meyre D, Benzinou M, Mein CA, Hinney A, et al. Association studies on ghrelin and ghrelin receptor gene polymorphisms with obesity. Obesity (Silver Spring) 2009;17(4):745-54. 9. Tan LJ, Zhu H, He H, Wu KH, Li J, Chen XD, et al. Replication of 6 obesity genes in a meta-analysis of genome-wide association studies diverse ancestries. Plos One. 2014;9(5):e96149. 21. Salopuro T, Pulkkinen L, Lindström J, Kolehmainen M, Tolppanen AM, Eriksson JG, et al. Variation in the UCP2 and UCP3 genes associates with abdominal obesity and serum lipids: the Finnish Diabetes Prevention Study. BMC Med Genet. 2009;10:94. 10. Neff KJ, le Roux CW. Bariatric surgery: the indications in metabolic disease. Dig Surg. 2014;31(1):6-12. 11. Benoit SC, Hunter TD, Francis DM, De La Cruz-Munoz N. Use of bariatric outcomes longitudinal database (BOLD) to study variability in patient success after bariatric surgery. Obes Surg. 2014;24(6):936-43. 22. Roszkowska-Gancarz M, Kurylowicz A, Polosak J, Mossakowska M, Franek E, Puzianowska-Kuznicka M. Functional polymorphisms of leptin and leptin receptor genes are associated with longevity and with the risk of myocardial infarction and type 2 diabetes mellitus. Endokrynol Pol. 2014;65(1):11-6. 12. de Luis DA, Pacheco D, Aller R, González Sagrado M, Izaola O, Terroba MC, et al. Influence of -55CT polymorphism of UCP3 gene on surgical results of biliopancreatic diversion. Obes Surg. 2010;20(7):895-9. 23. Xi B, Takeuchi F, Meirhaeghe A, Kato N, Chambers JC, Morris AP, et al.; DIAGRAM Consortium, AGEN-T2D Consortium, SAT2D Consortium. Associations of genetic variants in-near body mass index-associated genes with type 2 diabetes: a systematic meta- analysis. Clin Endocrinol (Oxf). 2014;81(5):702-10. 13. de Luis DA, Aller R, Conde R, Izaola O, Pacheco D, Sagrado MG, et al. Effects of RS9939609 gene variant in FTO gene on weight loss and cardiovascular risk factors after biliopancreatic diversion surgery. J Gastrointest Surg. 2012;16(6):1194-8. 24. Mohammad-Zadeh LF, Moses L, Gwaltney-Brant SM. Serotonin: a review. J Ver Pharmacol Ther. 2008;31(3):187-99. 14. Gibson RS. Nutritional assessment: a laboratory manual. New York: Oxford University Press; 1993. 25. Wurtman RJ, Wurtman JJ. Brain serotonin, carbohydrate-craving, obesity and depression. Obes Res. 1995;4:477S-80S. 15. Metropolitan height and weight tables. Stat Bul Metrop Live Found. 1983;64(1):3-9. 26. Kring SI, Werge T, Holst C, Toubro S, Astrup A, Hansen T, et al. Polymorphisms of serotonin receptor 2A and 2C genes and COMT in relation to obesity and type 2 diabetes. PLoS One. 2009;4(8):e6696. 16. REFERENCES In this study, women carrying the CC genotype on UCP3 gene polymorphism (rs1800849) showed three times more chance of DM2 compared to allele T carriers. Corroborating our data, Meirhaeghe and cols. (30) showed that UCP3 gene polymorphism (rs1800849) was related to DM2 on a French cohort, and T allele carriers presented lower risk. 1. Global Burden of Disease Study 2013 Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015;386(9995):743-800. 2. Malik VS, Willett WC, Hu FB. Global obesity: trends, risk factors and policy implications. Nat Rev Endocrinol. 2013;9(1):13-27. 3. Lu Y, Loos RJF. Obesity genomics: assessing the transferability of susceptibility loc across diverse populations. Genome Medicine. 2013;5(55):1-15. Regarding data 1 year postoperatively, the 12 gene polymorphisms investigated in our study showed no effect on the control/remission of SAH and DM2. Among the diabetic women in this study, 13% did not obtain the control of this chronic disease; thus, it is plausible to hypothesize that there are a possible genetic factors involved in the lack of this response postoperatively. On the other hand, considering that 1 year postoperatively is a relatively short time period, it is important to do a long-term investigation (> 2 years) to determine these responses. 4. Attia J, Ioannidis JP, Thakkinstian A, McEvoy M, Scott RJ, Minelli C, et al. How to use an article about genetic association: A: Background concepts. JAMA. 2009;301(1):74-81. 5. Bauer F, Elbers CC, Adan RA, Loos RJ, Onland-Moret NC, Grobbee DE, et al. Obesity genes identified in genome-wide association studies are associated with adiposity measures and potentially with nutriente-specific food. Am J Clin Nutr. 2009;90(4):951-9. 6. Rankinen T, Sarzynski MA, Ghosh S, Bouchard C. Are there genetic paths common to obesity, cardiovascular disease outcomes, and cardiovascular risk factors?. Circ Res. 2015;116(5):909-22. 7. Rankinen T, Zuberi, A, Chagnon YC, Weisnagel SJ, Argyropoulos G, Walts B, et al. The Human obesity gene map: the 2005 update. Obesity (Silver Spring). 2006;14(4):529-644. 330 Arch Endocrinol Metab. 2017;61/4 Gene polymorphism and bariatric surgery 2 diabetes and body weight: The Finnish Diabetes Prevention Study. Int J Obes (Lond). 2005;29(10):1245-51. 8. Xu X, Zeng H, Xiao D, Zhou H, Liu Z. Genome wide association study of obesity. Zhong Nan Da Xue Bao Yi Xue Ban. 2013;38(1):95- 100. 20. Arch Endocrinol Metab. 2017;61/4 REFERENCES Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JLJr, et al.; Joint National Committe on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. National Heart, Lung, and Blood Institute; National High Blood Pressure Education Program Coordinating Committe. Seventh report of Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension. 2003;42(6):1206-52. 27. Iordanidou M, Tavridou A, Vasiliadis MV, Arvanitidis KI, Petridis J, Christakidis D, et al. The -759C-T polymorphism of 5-HT2C receptor is associated with type 2 diabetes in male and female Caucasians. Pharmacogenet Genomics. 2008;18(2):153-9. 17. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care. 2013;36:S67-74. 28. Giralt M, Villarroya F. Mitochondrial Uncoupling and the Regulation of Glucose Homeostasis. Curr Diabets Rev. 2016 Feb 17. [Epub ahead of print]. 18. Gaukrodger N, Mayosi BM, Imrie H, Avery P, Baker M, Connell JM, et al. A rare variant of the leptin gene has large effects on blood pressure and carotid intima-medial thickness: a study of 1428 individuals in 248 families. J Med Gent. 2005;42(6):474-8. 29. Liu J, Li J, Wang CM. The role of uncoupling proteins in diabetes mellitus. J Diabetes Res. 2013;2013:585897. 30. Meirhaeghe A, Amouyel P, Helbecque N, Cottel D, Otabe S, Froguel P, et al. An uncoupling protein 3 gene polymorphism associated with lower risk of developing type II diabetes and with athetogenic lipid profile in a French cohort. Diabetologia. 2000;43(11):1424-8. 19. Salopuro T, Pulkkinen L, Lindström J, Eriksson JG, Valle TT, Hämäläinen H, et al.; Finish Diabetes Prevention Study Group. Genetic variation in leptin receptor gene is associated with type Arch Endocrinol Metab. 2017;61/4 331
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Relational Dynamics of MSM Population
International journal of Indian psychology
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© 2015 I A Shukla, S Srivastava; licensee IJIP. This is an Open Access Research distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any Medium, provided the original work is properly cited. 1Assistant Professor, Department of Psychology, University of Lucknow. 2Research Scholar, Department of Psychology, University of Lucknow. Relational Dynamics of MSM Population Dr. Archana Shukla1, Saurabh Srivastava2 ABSTRACT The aim of present study was to cast a glance on the dynamics of interpersonal relationships of Men who have sex with Men (MSM). Sample of 50 MSM were selected through purposive sampling technique age ranged from 16 to 40 years from Bharosa (Trust on MSM). Thematic Apperception Test (Indian adaption by Dr. Uma Roy Chowdhary) was administered on the participants. Result revealed that conflicting interpersonal relationships of MSM with their significant others specially father. The International Journal of Indian Psychology ISSN 2348-5396 (e) | ISSN: 2349-3429 (p) Volume 2, Issue 4, DIP: B00315V2I42015 http://www.ijip.in | July – September, 2015 Keywords: Dynamics, MSM Population There are lots of men who are practicing same sex activities due to various reasons like their orientation or just for pleasure and any other reason. While there is die hard rules and laws which construe same sex activities as a criminalizing and punishable act in all over the world and in India too. Same sex behavior is not a socially accepted norm in India. It’s a taboo identity and behavior both at global level. But male to male sexual behavior is major concern because it has been denied in most of the part of the world including India, and that is the major reason that MSM population is invisible and hard to trace while according to WHO SEARO, 2010 estimated population of MSM in India is 23, 52, 133 and it is growing day by day. Talking and any kind of discussion about any kind of sexual behavior are prohibited in public domain by Indian civil society and the government, and same sex behavior has been declared criminalize by section 377, Indian penal code, 1860. In spite of having sexual identities MSM share behavior identity. The term “Men who have Sex with Men” (MSM) addresses to those who engage in sexual relationships exclusively with other men (homosexuality) or who engage in sex relations with either men or women (bisexuality). Men who have sex with men have been used in HIV literature since at least 1990 by epidemiologists when many new HIV infections were identified among those who were behaviorally homosexual in Western & Asian countries (Young and Meyer, 2005). Relational Dynamics of MSM Population The acronym MSM had been in use in public health discussions the coining of the initialize by Glick et al. in 1994 "signaled the crystallization” of a new concept. MSM is not a behavior which is developed by any mental disorder nor is it any mental or physical depravity. MSM is a choice of sexual behavior it’s not all about sexual orientation. But some researchers found relational worldview aspect behind MSM behavior. As this miniscule population is considered as deviant population, people living drifted away from social norms. Crucial issues of their life which they witness are their interpersonal relationship. Hence for them relationship is a highly sensitive issue. Interpersonal relationships have been identified as more important than anything else in making our lives meaningful (Adler, Rosenfeld, & Proctor, 2001). Humans are born to relate with one very significant person from their primary caregiver to their partners and their grand children respectively at different stage of life. Interpersonal relationships are dynamic system that changes during continuously during their existence. Each and every relationship has a beginning, a lifespan and an end. From beginning to the end relationship grow improve as people know each other and become closer emotionally or it deteriorates as people set aside, not able to manage interpersonal conflicts and frictions, hence interpersonal relationships are a dynamic system and it can be called relational dynamics. Relational dynamics does not refer only to by birth relationships like father, mother, siblings, spouse, and maternal or paternal relatives but it also emerges friends, helpers, colleagues, and other whom one’s consider a significant in his circle of life. There is paucity of research concerning the interpersonal relationship of the MSM. Further the prevalence of the MSM and their increasing member day by day. As being stigmatized, sexually minor and hidden population they were studied just for stipulated issues like HIV/AIDS and STI. Facing the different kind of social, personal and interpersonal issues in their day to day life, here an effort was done to explore the dynamics of their interpersonal relationship in their relational world. The present study focuses on to explore the dynamics of interpersonal relationship of MSM population. An attempt was made to understand the relational world of MSM population. © The International Journal of Indian Psychology | 1 Participants The participants of present study compromised of MSM from Lucknow. Method of sample selection was based on purposive sampling technique age ranged from 16 to 40 years. I t t After administered TAT, analyses of the stories were done with the help of Bellak TAT Blank and Analysis Sheet. After administered TAT, analyses of the stories were done with the help of Bellak TAT Blank and Analysis Sheet. Instrument The Indian adaptation of the TAT by Dr. Uma Roy Chowdhary was used. Only 5 cards were administered which were two neutral cards- Card no. T(2) and T(6), two cards depicting interpersonal conflicts Card no T (3MB) and T(4MB) and lastly a blank card. © The International Journal of Indian Psychology | 160 160 Research Design The present study was an ex post facto research with an exploratory orientation. Procedure The researchers initially approached to Bharosa Trust in Lucknow for facilitating the study. Bharosa is basically involved in providing social and medical support to the MSM community. Bharosa assisted in establishing appropriate rapport with the participants, after that participants were introduced with the TAT with the instructions “I am going to show you some pictures, one at a time, and your task will be to make up a story for each card. In your story, be sure to tell what has led up to the event shown in the picture, describe what is happening at the moment, what the characters are feeling and thinking, and then give the outcome. Tell a complete story with a beginning, middle, and end. Do you understand? I will write your stories verbatim as you tell them. Here’s the first card.” RESULT AND DISCUSSION The perception of these participants on administered TAT cards was sought in terms of interpersonal conflicts as well as their relational dynamics. TAT responses were analyzed on some basic points as manifest contents and latent contents. Manifest content has descriptive features where latent content consists of interpretative value. One main thing to consider in the interpretation of the TAT is that the pictures are best seen psychologically as a series of social situations and interpersonal relations. Another way to consider them is that all characters in the stories are projected aspects of the self, keeping in mind that they may represent the ideal self, the real self, the feared self, etc. Bellak (1947) says "A repetitive pattern is the best assurance that one does not deal with an artifact". The data obtained was exhaustive and qualitative in nature. Keeping this in mind, data were analyzed on the major point Hero, Need, Press, Thema and Figures and objects Introduced/omitted according to Bellak TAT Blank and Analysis Sheet. © The International Journal of Indian Psychology | 161 Hero As in the interpretation of TAT; Bellak considered that hero is the figure with whom the subject identified with himself. As shown in table 1.1, it was found that participants identified themselves with same sex as hero except in the card T(4)MB. In the response story of T(4)MB participant did not identified themselves with same sex clearly. To the extent this showed that participants had little conflict with their self. Manifested content of the stories showed the demographic picture of the present MSM sample according to which it can be easily said that MSM population belong to low socio-economic status with little or no educational background. It showed that participants demographically projected themselves in their stories. In some cases heroes had maladapted behavior by getting in wrong company from their younger age or school days. Need Murray (1938) described needs as a, "potentiality or readiness to respond in a certain way under certain given circumstances”. Focusing on table 1.1; in present study it was found that participants were projected two major need and that are need of achievement and need of affiliation and they also projected achievement motivation which was depicted in stories as they quoted, “Mehnat se padhai ki aur …naukari mil gayi (study hard and got a job), “Apna sara dhyan padhai likhai pe lagaya … zile ke collector ka pad hasil kia.”(Put focus on education and became the district collector). Participants projected hero in highly need of affiliation, which was manifested in their stories as hero’s need to relate with others and it can be interpreted that this need emerged from the conflicting relationship and deprivation of love and affection from their significant others like parents. © The International Journal of Indian Psychology | 161 Relational Dynamics of MSM Population Table 1.1: TAT analysis table in terms of Hero, Need, Press, Thema and figure introduce/omitted Card no. Hero Need Press Theme Figure Introduced Figure omitted T2 Male Achievement Affiliation Lack of harmonious interpersonal relationship, Lack of education and money Down trodden family , poverty, educational & economical deprivation and interpersonal relationship conflict (with father), helplessness School, House, village Man, girl, old lady , small child T6 Male Achievement Affiliation Contamination, educational deprivation, interpersonal relationship conflicts. Down trodden family poverty, educational & economical deprivation and interpersonal relationship conflict (with father), helplessness Fisherman, people, villagers, town, pollution in the river, Boat, T(3)MB female Achievement Affiliation Lack of harmonious interpersonal relationship, capricious discipline Down trodden family, poverty, deprivation and interpersonal- relationship conflicts (father), helplessness Father, Friends, alcohol Boy T(4)MB Male Affiliation Lack of harmonious interpersonal relationship, Down trodden family Poverty, deprivation and interpersonal- relationship conflicts, helplessness Mother _ Relational Dynamics of MSM Population © The International Journal of Indian Psychology | 162 Relational Dynamics of MSM Population Blank Card Male Achievement Affiliation Lack of harmonious interpersonal relationship, education and money Down trodden family, Poverty and deprivation of education. Family and its member, education, friends, chronic illness (cancer), doctor _ Relational Dynamics of MSM Population Down trodden family, Poverty and deprivation of education. © The International Journal of Indian Psychology Thema Thema includes the instigating situation (press) and the need that is operating. Thema is a dynamical structure of need and press interaction (Murray, 1938). Table 1.1 summarizes all the themes which emerges from the plot of the stories is broadly a down trodden family which have economic deprived and conflicting interpersonal relationships between hero and his parent (Father). And another theme on the basis of latent affect in the plot is helplessness which depicted in each and every story. Press Press is effective and significant determinants one’s behavior in the environment. “The press of an object is what it can do to the subject for the subject- the power that it has to affect the well being of the subject in one way or another.”, Murray(1938). In simple words press can be any environmental attribute which facilitates the efforts of individual to drive towards need satisfaction. On analyzing manifested and latent both content of the stories presses were hero’s © The International Journal of Indian Psychology 163 Relational Dynamics of MSM Population conflicts to the extent on both interpersonal and intrapersonal level. As they manifested in their stories that hero doesn’t have cordial and harmonious relation with his significant others most importantly with his father. Other presses are poverty, and lack of affiliation and capricious discipline as mentioned in table 1.1. conflicts to the extent on both interpersonal and intrapersonal level. As they manifested in their stories that hero doesn’t have cordial and harmonious relation with his significant others most importantly with his father. Other presses are poverty, and lack of affiliation and capricious discipline as mentioned in table 1.1. Figures and objects Introduced/omitted As analyzing the story it was found that participants introduced/omitted characters and objects in their stories e.g.: father (dead), boat man, old man, doctor, fishes, children, school, village, harvest etc (table 1.1). And in TAT figures or objects were not only considered as figures but they have their own meaning as significant relations or person of life and inclusion/exclusion of figures and objects both shows dynamics of interpersonal relationship as well as high need for aggression, nurturance and money and denial, repression or anxiety respectively (Groth-Marnat, 2003) of the subject which he projected in the story through the hero. On the basis of the manifest content of the stories and introduction and omission of the figures it can be fairly said that subjects have conflicts regarding close and significant relationship like parent-child relation and subject has need for aggression, nurturance, and money for example many of the subjects quoted, “wo pita se apni baatein nahi kah pata tha…” (He did not say things to his father). This line reflected the need for nurturance and affiliation. An overview of overall analysis and interpretation we found that MSM population have friction in their family and they have entangled relational dynamics with full of conflicts in their close and significant relationship like parent-child and friendship. CONCLUSION Findings emerged from the present study: MSM population has little conflict with their physical self which was depicted on analyzing the stories in terms of Hero. MSM population has high need of affiliation and need of achievement. Deprivation in terms of nurturance, belongingness, harmonious relation with father, education and money are the major presses or environmental forces in the life of MSM population. Overall present study was an effort to explore the dynamics of interpersonal relationship among MSM population. The study of the MSM sample highlighted the fact that the relationship © The International Journal of Indian Psychology 164 Relational Dynamics of MSM Population of MSM population with their parents has been conflicting, moreover its can be said that father- son relationship was more conflicting and full of frictions. IMPLICATION After analyze of the present study brought to the fore the fact that interpersonal relationship one can understand MSM population better and further intervention can be planned which would facilitate them towards acceptance, adequate social support, healthy optimism, life satisfaction, in other words enriched quality of life. This would not only make their life more meaningful by including them in main stream, it will also send a message to our policy makers, activists and social reformers to visualize effective plans for such sample. Young, R.M. and Meyer, I.H. (2005). The Trouble with “MSM” and “WSW”: Erasure of the Sexual-Minority Person in Public Health Discourse. Am J Public Health. REFERENCES Adler, R.B., Rosenfeld, L.B. and Proctor, R.F., (2001). Interplay: The Process of Interpersonal Communication, 8th ed. Oxford University Press, USA. Bellak, L., (1997). The Thematic Apperception Test, The Children's Apperception Test, and the Senior Apperception Technique in Clinical Use, Volume 383, 6th ed. (R). Groth-Marnat,G.,(2003). Handbook of Psychological Assessment 4th ed. P.510 John Wiley & Sons, Inc, Hoboken, New Jersey. Murray, H.A., (1938). Exploration in Personality, Oxford University Press, New Young, R.M. and Meyer, I.H. (2005). The Trouble with “MSM” and “WSW”: Erasure of the Sexual-Minority Person in Public Health Discourse. Am J Public Health. © The International Journal of Indian Psychology | 165 A., (1938). Exploration in Personality, Oxford University Press, New York. Groth-Marnat,G.,(2003). Handbook of Psychological Assessment 4th ed. P.510 John Wiley & Sons, Inc, Hoboken, New Jersey. © The International Journal of Indian Psychology | 165
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Successfully treating 90 patients with obsessive compulsive disorder in eight days: the Bergen 4-day treatment
BMC psychiatry
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© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Successfully treating 90 patients with obsessive compulsive disorder in eight days: the Bergen 4-day treatment Gerd Kvale1,2, Bjarne Hansen1,2, Thröstur Björgvinsson3,4, Tore Børtveit1, Kristen Hagen1,5, Svein Haseth6, Unn Beate Kristensen7, Gunvor Launes8, Kerry J. Ressler3,4, Stian Solem1,9*, Arne Strand10, Odile A. van den Heuvel1,11 and Lars-Göran Öst1,12 Abstract Background: Oslo University Hospital, Norway, had by autumn 2016, accumulated a waiting list of 101 patients with obsessive-compulsive disorder (OCD) who had a legal right to receive treatment by a specialized OCD team. In this challenging situation, the Bergen OCD-team suggested to solve the problem by offering all patients an option for the rapid Bergen 4-day treatment (B4DT). The B4DT is an individual treatment delivered during four consecutive days in a group of six patients with the same number of therapists. The approach has previously shown a post-treatment response rate of 90% and a 3-month remission rate of 70%. Methods: Ninety-seven of the wait-list patients were available for the scheduled time slots, and 90 received the 4-day format during 8 days (45 patients each week). The therapists were recruited from 22 different specialized OCD-teams from all over Norway, and 44 (68%) had not previously delivered the 4-day format. Results: Post-treatment; 91.1% of the patients were classified as responders, and 72.2% were in remission. At 3- month follow-up; 84.4 were classified as responders and the remission rate was 67.7%. Oslo University Hospital now offers the 4-day treatment as standard treatment for OCD. Conclusions: We conclude that the B4DT is an acceptable and potentially effective OCD Conclusions: We conclude that the B4DT is an acceptable and potentially effective OCD-treatme Keywords: Obsessive-compulsive disorder, OCD, 4-day treatment, Exposure exposure and response prevention (ERP) [2] where pa- tients typically are treated over 12–16 weeks, the OCD- team at Haukeland University Hospital, Bergen, has devel- oped a novel treatment format where ERP is delivered during just four consecutive days. Despite the relatively brief treatment, the Bergen 4-day treatment (B4DT) has been shown to yield a good outcome [3–5]. At post- treatment assessment the proportion of responders varied from 83% [4] to 93.8% [5] with a weighted mean of 89.4%. At 6- or 12-month follow-up the mean response rate was 82.4%. The post-treatment remission rate varied between 73.8% [4] and 77.1% [3] with a weighted mean of 76.0%, and at follow-up the average rate was 69.7%. Also, the 4-day format has shown to be acceptable for the patients and has a 'low drop-out rate; 0.7% (only 1 out of 142 patients; [3–5]. However, these studies have RESEARCH ARTICLE Open Access Successfully treating 90 patients with obsessive compulsive disorder in eight days: the Bergen 4-day treatment Gerd Kvale1,2, Bjarne Hansen1,2, Thröstur Björgvinsson3,4, Tore Børtveit1, Kristen Hagen1,5, Svein Haseth6, Unn Beate Kristensen7, Gunvor Launes8, Kerry J. Ressler3,4, Stian Solem1,9*, Arne Strand10, Odile A. van den Heuvel1,11 and Lars-Göran Öst1,12 https://doi.org/10.1186/s12888-018-1887-4 Open Access Kvale et al. BMC Psychiatry (2018) 18:323 https://doi.org/10.1186/s12888-018-1887-4 Kvale et al. BMC Psychiatry (2018) 18:323 https://doi.org/10.1186/s12888-018-1887-4 Background In Norway, all patients with obsessive-compulsive disorder (OCD) are granted empirically supported treatment by a specialized OCD-team [1], and 30 teams have been estab- lished since 2012. At Oslo University Hospital (OUH) the consequence of this new rule of granted treatment was a tripling of patients from 2014 to 2016, and more than 100 patients were waiting for treatment by the end of 2016. The most probable reason is that many more OCD- patients than previously applied for treatment when they realized they might be granted empirically supported treatment. While OUH employed a protocol for individual * Correspondence: stian.solem@ntnu.no 1Haukeland University Hospital, OCD-team, 5021 Bergen, Norway 9Department of Psychology, Norwegian University of Science and Technology, Trondheim, Norway Full list of author information is available at the end of the article Kvale et al. BMC Psychiatry (2018) 18:323 Page 2 of 9 Page 2 of 9 had small sample sizes (N = 35, 42, and 65) so larger tri- als are warranted. the disorder and treatment were the same. Also, because the three previous studies [3–5] on the B4DT were all carried out with, to a large extent new therapists, we ex- pected that the B4DT would be effective also with a sub- stantial number of new therapists. Thus, the present study can be seen as an example of systematic replica- tion [13] having the purpose to test the outcome when a large number of OCD-patients are treated at a new clinic by new therapists. There are a few published studies on concentrated ERP for OCD (using Yale-Brown Obsessive Compulsive Scale as primary outcome measure) but no randomized trial comparing it to weekly sessions of ERP. Franklin et al. [6] and Abramowitz et al. [7] published uncon- trolled effectiveness studies on concentrated ERP using 15 2 hour sessions over 4 weeks and 12 sessions of 90 min over 4 weeks, respectively. Hiss et al. [8] and Lindsey et al. [9] published efficacy studies using 19 ses- sions of 90 min over 4 weeks and 15 2 hour sessions over 3 weeks, respectively. Finally, there are two non-randomized comparison trials involving concen- trated ERP. Abramowitz et al. [10] compared 15 2 hour sessions over 3 weeks with 15 sessions over 8 weeks (twice weekly) and found that the concentrated treat- ment yielded a significantly higher proportion of recov- ered patients post-treatment but not at 3-month follow- up. Storch et al. [11] compared 14 sessions of 90 min over 3 weeks with 14 weekly sessions and found no sig- nificant differences at post- or at 3-month follow-up as- sessment. Combining the Y-BOCS data for these six studies (N = 266) yielded a weighted mean at pre- treatment of 26.2 (SD 4.9) and at post-treatment of 11.9 (6.9), with a within-group effect size (Cohen’s d) of 2.42. This can be compared to 2.06 for standard ERP in the meta-analysis by Öst et al. [12]. Thus, previous versions of concentrated ERP have used daily sessions of 90 or 120 min over 3–4 weeks, which is quite different to the B4DT, consisting of four sessions of 3–8 h across four consecutive days. Participants and procedure In Norway, OCD-patients with a principal DSM-5 diag- nosis of OCD [14] are entitled to empirically supported treatment from an outpatient OCD-team. In February 2017, the 101 patients on the OUH waitlist were offered the 4-day treatment. Inclusion criteria were a diagnosis of OCD and patients had to be fluent in Norwegian. Ex- clusion criteria included suicidality, psychosis, and active substance abuse. Patients declining would receive stand- ard care at OUH. Four were unable to attend due to school obligations, work, and a prescheduled vacation and 97 patients were scheduled (see flow chart in Fig. 1). p ( g ) Patients referred to OUH had all been diagnosed with OCD (by a psychiatrist or clinical psychologist at the OUH OCD-team) and severity of OCD had been assessed with a Y-BOCS interview before being placed on the waiting list. Diagnoses were assessed using the MINI International Neuropsychiatric Interview [15]. For the present study, patients on the waiting list were for- mally referred to the OCD-team in Bergen, and they were re-assessed before starting treatment by using the OCD entry questions from DSM-5 as well as another Y-BOCS interview. This re-assessment was necessary as some patients had been waiting for treatment for many months. When formally referred to Bergen, patients were asked to complete a number of questionnaires ad- ministrated online (see measures) prior to the treatment, and to complete these questionnaires post-treatment as well as at 3-month follow-up. The leader of the Bergen OCD-team (GK) was made aware of the treatment delay problem at OUH and sug- gested offering the 4-day treatment to patients on the waiting list, which OUH accepted. The 4-day treatment is best described as “individual treatment in a group setting” since the ratio between therapist and patients is 1:1 in groups which usually range from three to six patients. In order to treat 100 patients, eight parallel groups during two separate weeks would be needed. It was decided to offer OCD-therapists from the other specialized OCD-teams in Norway the opportunity to participate, and by doing so to start their training in the 4-day format. This logistically demanding project of treating 100 OCD-patients in 8 days was initiated dur- ing the spring of 2017. The present paper reports on the results. Since longer versions of concentrated ERP seem to yield somewhat better effects than standard ERP it is possible that our highly concentrated version yields even better effects. Pharmacological treatment Mean age was 32.7 (SD = 9.7), 57% were women, and 58% single. A total of 78% were either working or study- ing, whereas 22% received different social benefits. Mean years of education was 12.6 (SD = 3.5). Thirty-two pa- tients reported to have family members with OCD (see Table 1 for details). Forty patients used psychotropic medications: 35 used antidepressants, one antiepileptic medication, eight antipsychotics, four stimulants, five anxiolytics, three hypnotics, and one received drug assisted treatment for substance abuse. Patients with and without psycho- tropic medication did not differ on Y-BOCS scores, t(88) = .014, p = .99, GAD-7 scores t(88) = .41 p = .69, or PHQ-9 scores, t(88) = 1.10, p = .27, at pre-treatment. Participants and procedure Y-BOCS at post-treatment, as well as at 3-month follow-up, were conducted over the phone by specially trained clinical psychologists who worked at other clinics and were not involved in the treatment. At 3-month follow-up, 20% of the sample was randomly se- lected to be re-interviewed within 1 week by another in- dependent psychologist. The inter-rater reliability, Intra Class Coefficient (3.1) = 0.93, was excellent. The treatment was conducted in Oslo by bringing in therapists from all over Norway. Three of the 97 patients became ill (two with flu, one with a minor bleeding during pregnancy). One patient turned out to have se- vere language problems, and three were misdiagnosed (one had OCD-like symptoms which turned out to be Based on the outcomes of three previous effectiveness studies from our team [3–5] we predicted that the out- come of the present study would be equally good since Kvale et al. BMC Psychiatry (2018) 18:323 Page 3 of 9 Fig. 1 Flow chart of study participants Fig. 1 Flow chart of study participants a moderate OCD (Y-BOCS score of 18–23). A total of 86.7% had received previous treatment trials, but none of the patients had received CBT for OCD with includ- ing ERP procedures. Patients with previous therapy trials (M = 26.25, SD = 3.47) did not differ significantly from treatment naïve (M = 25.45, SD = 2.62), t(88) = 0.73, p = .47. A total of 56 patients (62%) had one or more co- morbid disorders (see Table 1 for details). due to a neurological disorder; one was preparing for a transsexual operation and the OCD-like symptoms were related to this, and one patient had a primary diagnosis of generalized anxiety disorder). Thus, the number of OCD patients who received the 4-day treatment was 90. All of these patients completed treatment so the attrition rate was 0%. Staffing the groups In order to be able to treat 100 patients, two time slots were selected with eight groups of six patients in each. Therapists from 22 different OCD-teams in Norway wanted to participate, as well as a Scandinavian-speaking American colleague and three Icelandic psychologists. Therapists with the most extensive OCD-experience were given priority, as were therapists from teams that would be able to participate in both time-slots. When possible, the groups were staffed with 50% of qualified 4-day therapists, or therapists with at least one prior ex- perience with the 4-day treatment. In order to qualify as a 4-day therapist he or she must have participated in a minimum of two 4-day groups and demonstrated com- petency in the exposure procedure evaluated independ- ently by two 4-day experts. All group leaders had acted as therapists in minimum of six groups. In the Oslo case, group leaders did not have responsibility for a given pa- tient, but rather was responsible to ensure that all thera- pists and patients received the necessary supervision, intervention, and assistance. In each group, one of the therapists also acted as “second in command”. Further- more, each group had a person taking care of the logis- tics. In total, 66 therapists (including group leader) participated, 61 of them twice, and 44 of them without prior experience with the 4-day format. prohibited during and after the 4-day treatment. Patients were not receiving other treatment during the treat- ment period. Preparing the patients for treatment In order to ensure standardized information, patients re- ceived written information and watched a video presenting the outline of the 4-day treatment https://www.youtube.- com/watch?v=nqx8knpy3i4 as well as a 6 minute descrip- tion of the treatment made in 2014 by and broadcasted on Norwegian national TV (NRK) https://www.youtube.com/ watch?v=ZRSExyZ3GPg. After accepting the treatment offer, they watched the following video describing the 4-day treatment in more detail https://www.youtube.com/ watch?v=1Fnxt0_ljpY&t=1s. g Groups were scheduled from Tuesday to Friday, and therapists met the day before for 8 hours in prepar- ation, including introduction to the psychoeducation and a detailed outline of the 4-day treatment, given by the developers, Bjarne Hansen (BH) and Gerd Kvale (GK). Prior to this, all therapists received the same introduction to the 4-day treatment as the patients. The therapists also received all relevant information regarding their patients. GK and BH had daily meet- ings with each group leader and “second in command” to ensure that potential challenges were dealt with in accordance with the 4-day protocol. The patients’ expectations of treatment outcome and evaluation of the treatment credibility, were assessed with an adapted version of the Borkovec and Nau [16] Reaction to treatment scale (0–100%). A score below 100% on any of the four questions was taken as an opportunity to clarify possible misunderstandings. The patients were instructed to suggest exposure tasks, and as guidance they were told that “exposures that their OCD would appreciate the least” often were the most relevant. During the week before treatment, the group leader (see below) called each patient and performed a standard interview, focusing on clarifying possible misunderstandings and ensuring that the patients understood that they were required to prepare exposure tasks. Groups were located at different places at the OUH, minimizing external disturbances related to the number of parallel groups. Also, a centrally located “control center” led by GK and BH and staffed with support personnel was established. OCD severity and comorbidity Mean pre-treatment score on the Yale-Brown Obsessive Compulsive Scale (Y-BOCS) was 26.16 (SD = 3.37), 77% had severe OCD (Y-BOCS score of 24–38), and 23% had No changes were applied to medication, but the pa- tients were informed that the use of anxiolytics was Kvale et al. BMC Psychiatry (2018) 18:323 Page 4 of 9 Table 1 Demographic and diagnostic characteristics of the sample (N = 90) Demographics n (%)/ M (SD) Comorbidity Age 32.70 (9.72) GAD 27 (30.0%) Years of education 12.60 (3.46) Depression 20 (22.2%) Female gender 51 (56.7%) Bipolar 1 (1.1%) Single 52 (57.8%) Panic 14 (15.5%) Working 51 (56.7%) Tourette 2 (2.2%) Studying 19 (21.1%) Specific phobia 4 (4.4%) Social benefits 20 (22.2%) Social phobia 11 (12.2%) Previous treatment 79 (87.8%) BDD 1 (1.1%) Psychotropic meds 40 (44.4%) Health anxiety 2 (2.2%) GAF-S (0–100) 51.73 (5.50) Anorexia 1 (1.1%) GAF-F (0–100) 57.54 (9.96) Bulimia 2 (2.2%) Age at OCD onset 15.47 (6.55) ADHD 1 (1.1%) Years with OCD 16.12 (10.40) Note: GAF general assessment of functioning, GAD generalized anxiety disorder, BDD body dysmorphic disorder, ADHD attention deficit hyperactivity disorder Measures Yale-Brown Obsessive Compulsive Scale, Y-BOCS; [17, 18] a semi-structured clinical interview that con- sists of 10 items (each 0–4) covering the severity of both obsessions and compulsions, and is a standard approach to assess OCD severity. The psychometric properties of the interview are well established. Y- BOCS has been found to be sensitive to change after treatment (e.g., 8). Benchmarking As is customary in effectiveness studies we compared the mean Y-BOCS score and remission rate of the present sample and the average for published effectiveness studies on ERP. In order to do a fair comparison, we selected studies that had short-term follow-up assessment (3– 6 months post-treatment) as in the present study, and used any format of ERP. The following studies were included in the benchmarking analysis [21, 22, 23–25] comprising a total of 381 patients fulfilling DSM-IV cri- teria for OCD. The exposures were interspaced with brief group meetings where each participant reported on how they were doing, especially on how they were practicing the LET-technique. The patients had individual exposure tasks for the afternoon and evenings, and reported pro- gression back to the therapist with the last contact typically being at 9 p.m. Relatives and friends were in- vited to a psycho-educative meeting (one for each group) in the afternoon of day 3. The last day “lessons learnt” were summarized and plans for the next 3 weeks of self- administered exposures were made. The patients were informed how to contact the health care provider if an emergency situation should occur. The next 3 weeks, the patients were encouraged to report online every day on how they were practicing the LET-technique. The cli- nicians read the reports, without being in contact with the patients. Treatment The first of the 4 days (approximately 3 h) was allocated to psychoeducation and to prepare individual exposure tasks. The two middle days were dedicated to individually tailored and therapist assisted exposure training (8–10 h each day) in a wide range of OCD-relevant settings. Kvale et al. BMC Psychiatry (2018) 18:323 Kvale et al. BMC Psychiatry (2018) 18:323 Kvale et al. BMC Psychiatry (2018) 18:323 Page 5 of 9 Page 5 of 9 The main feature of the 4-day treatment is to teach the patients to actively approach whatever elicits the relevant anxiety or discomfort, and to help them sys- tematically use the anxiety and discomfort as a cue to “LEan into The anxiety” (LET-technique) instead of employing obvious or subtle avoidance. Typically, the therapist serves as a coach in the beginning, gradually leaving more responsibility to the patients. The thera- pists work as a team, which means that the patients are not necessarily working with the same therapist during the 2 days of exposure (see below). Rather, the team leaders allocate and reallocate therapists (during frequent and brief team-meetings) to ensure that the patients who are struggling are assisted by the most experienced therapists. Generalized Anxiety Disorder scale, GAD-7; [20] is a self-administered screening instrument for symptoms of generalized anxiety consisting of seven items each rated on a 0–3 scale. The psychometric properties for the in- strument are good [20]. Statistical analyses Statistical analyses were performed with SPSS version 24.0. Repeated measures ANOVA for Y-BOCS was con- ducted using pre-treatment, post-treatment, and follow- up scores. Effect sizes were calculated with Cohen’s d, defined as (Mpre – Mpost)/SDpre. Treatment response was calculated based on the inter- national consensus criteria [26] which requires a ≥35% re- duction of the individual patient’s pre-treatment Y-BOCS score in order to be classified as a clinically relevant re- sponse. A patient is classified as remitted if the post- treatment Y-BOCS score is ≤12 points. For Y-BOCS scores there were no missing data at pre-treatment, whereas data were missing for four patients at post-treatment and 12 at follow-up. For PHQ-9 one case was missing at pre- treatment and 10 at post-treatment. For GAD-7 there were 2 patients with missing data at pre-treatment and 10 at post-treatment. Missing data were replaced using the expectation-maximization method of SPSS, version 24. The method was chosen to allow for repeated measures ANOVA. All data presented are an integrated part of the 4-day standard quality control procedure. Three months after treatment, patients were invited to an individual session (30 min, conducted by the Oslo team) where their experiences in the period following treatment were discussed, and the principles of the LET-technique repeated. No exposure work was con- ducted in this session. Benchmarking T bl 6 h Table 6 shows the comparison between the present sam- ple and standard ERP from published uncontrolled ef- fectiveness studies. The Oslo patients, treated with the Bergen 4-day treatment, had a significantly higher mean Y-BOCS score at pre-treatment. However, both at post- treatment and at 3-month follow-up their means were significantly lower than the average for the published ef- fectiveness studies. The remission rate was also signifi- cantly higher for the Oslo patients than for the patients in the effectiveness studies. Table 3 shows the clinical improvement at post- treatment and 3-month follow-up for the individual pa- tients. Of the 65 patients who were in remission at post-treatment, 51 patients (78.5%) were classified as in remission at follow-up. Of the 17 patients who were classified as responders at post-treatment, 6 had become remitted at follow-up, 9 remained as a responder whereas 2 had deteriorated to the category of no change. Of the 8 patients who were classified as unchanged at post-treatment, 4 were in remission at follow-up, and 4 remained unchanged. Clinically significant change in OCD-severity At post-treatment, 91.1% of the patients had responded (≥35% improvement) and 72.2% were in remission (≥35% reduction and Y-BOCS score of ≤12). At 3-month follow-up, 84.4% had responded, and 67.8% were in remission. Primary measures Table 2 displays the results for Y-BOCS at pre-treatment, post-treatment, and 3-month follow-up. Repeated mea- sures ANOVA (Wilks’ Lambda) found a significant effect of time, F(2) = 428.94, p < .001, partial Eta squared = .91. Mauchly’s test of sphericity was not significant (p = .263). A large effect size was observed with a Cohen’s d of 4.6 both at post-treatment and at follow-up. There were no significant changes in symptoms from post-treatment to follow-up assessment (p = .82). Patient Health Questionnaire-9, PHQ-9; [19] is a frequently used self-administered screening instrument consisting of nine questions each rated on a 0–3 scale. A score of 10 or more is indicative of a depressive disorder [20]. The psychometric properties of the instrument are sound [20]. Kvale et al. BMC Psychiatry (2018) 18:323 Page 6 of 9 Page 6 of 9 Table 2 Means, standard deviations and effect sizes (Cohen’s d) for symptoms of OCD, anxiety, and depression M SD d Y-BOCS Pre 26.16 3.37 Post 10.54 4.61 4.63 3 months 10.68 6.31 4.59 GAD-7 Pre 11.88 4.88 Post 8.80 4.49 0.63 PHQ-9 Pre 11.75 5.36 Post 8.34 4.59 0.64 Note: N = 90. d = (Mpre – Mpost)/SDpre Remission rates at post-treatment were not signifi- cantly different between the two severity subgroups, χ2 (1) = 0.22, p = .64. The same was true at follow-up, χ2 (1) = 2.18, p = .14. Table 5 shows a comparison of the Y-BOCS data be- tween the present study and our previous three studies [3–5] carried out in Bergen. The present sample of OCD-patients starts at the same severity level as the pre- vious samples and has very similar post-treatment and 3-month follow-up results. There are no significant dif- ferences on Y-BOCS between the present and the previ- ous samples at any time point. Clinically significant change in OCD-severity Discussion Clinically significant change was also calculated for the two severity subgroups. For the moderate subgroup, 76.2% were classified as remitted at post-treatment and 23.8% showed no change. At follow-up 81.0% were re- mitted, 4.8% responded, and 14.3% showed no change. For the severe/extreme subgroup; 71.0% were remitted at post-treatment while 24.6% responded, and 4.3% showed no change. At follow-up 63.8% were remitted, 20.3% responded, and 15.9% showed no change. The joint effort to erase the waiting list of more than 100 OCD-patients at Oslo University Hospital during 8 days worked well. In comparison, during the entire year of 2016 OUH was able to treat 70 patients. Ninety-one percent of the 90 patients treated with the 4-day format responded at post-treatment, and 68% were in remission at three-month follow-up. Table 4 Severity of anxiety and depression at pre-treatment, post-treatment, and 3-months follow-up GAD-7 PHQ-9 Severity Pre Post Pre Post None 5 (5.6%) 13 (14.4%) 6 (6.7%) 15 (16.7%) Mild 22 (24.4%) 43 (47.8%) 25 (27.8%) 42 (46.7%) Moderate 37 (41.1%) 24 (26.7%) 32 (35.6%) 25 (27.8%) Severe 26 (28.9%) 10 (11.1%) 27 (30.0%) 8 (8.9%) Note: Pre Pre-treatment, Post Post-treatment. Cut-offs used for both GAD-7 and PHQ-9 were 0–4 (none), 5–9 (mild), 10–14 (moderate), 15 and above (severe) Table 4 Severity of anxiety and depression at pre-treatment, post-treatment, and 3-months follow-up Table 3 Comparison of clinical improvement rates at post- treatment and follow-up 3 months follow-up Remission Responded Unchanged Total Post Remission 51 6 8 65 Responded 6 9 2 17 Unchanged 4 0 4 8 Total 61 15 14 90 Table 3 Comparison of clinical improvement rates at post- treatment and follow-up Kvale et al. BMC Psychiatry (2018) 18:323 Page 7 of 9 Page 7 of 9 Table 5 Comparison the 4-day treatment studies on Y-BOCS scores (M and SD) Study Time point Havnen (2014) Havnen (2017) Hansen (2018) Present study N: 35 42 65 90 Pre 26.1 (4.3) 25.7 (4.3) 25.8 (4.7) 26.2 (3.4) Post 9.0 (4.8) 10.8 (3.9) 10.2 (5.1) 10.5 (4.6) 3 months 10.6 (7.0) ─ 10.5 (5.9) 10.7 (6.3) 6 months 10.3 (5.7) 12.2 (6.4) ─ ─ 1 year ─ ─ 10.6 (7.0) ─ 4 years 9.9 (7.4) Note: The 4-year time point pertain to the combination of the Havnen et al. Discussion 2014 and 2017 studies Table 5 Comparison the 4-day treatment studies on Y-BOCS scores (M and SD) previously not worked with the 4-day format and the clear majority of the therapists had never worked together before. Since each group is staffed with as many therapists as patients, the format serves as a good opportunity to work side-by-side with, and observe ex- perienced 4-day therapists, and to get hands-on supervi- sion. While model learning with hands-on supervision is an obvious approach for physicians who are going to learn surgery or any other medical discipline, it is rare in the mental health care. It is a unique experience to be able to observe six patients with OCD going through major changes in only 4 days. After the Oslo experience, all teams involved have asked to be fully trained in order to deliver the 4-day format. Note: The 4-year time point pertain to the combination of the Havnen et al. 2014 and 2017 studies Note: The 4-year time point pertain to the combination of the Havnen et al. 2014 and 2017 studies y There are different limitations of the current study. Evaluations of this treatment format has so far only been tested using open trial designs, which do not compare the effect of treatment with other established treatments or placebo control. A RCT is necessary to draw defini- tive conclusions about the efficacy and specificity of the 4-day treatment. Also, participants had not previously received evidence based treatment for OCD. This could limit generalizability of the findings. However, the present study was aimed at ordinary OCD-patients, not those who are treatment resistant. The study design and study population could be possible explanations for the good results. Another possible explanation is that the current concentrated format is different from that used in previous studies of concentrated treatment. They have used daily sessions of 90–120 min across 3–4 weeks, while we used 4 days and 3–8 h sessions. Longer sessions in highly concentrated ERP could yield better effects than standard ERP. Another issue concerns missing data as 13% of participants did not provide data at follow-up. The results from Oslo are basically the same as reported on the 4-day format in our previous effectiveness studies [3–5]. Furthermore, we have documented that the changes are maintained at 1-year follow-up [5] and at 4-year follow-up [27]. Discussion Compared to standard ERP treat- ment approaches, the 4-day format has a number of po- tential advantages. It has a low declining rate and attrition rate. Moreover, this treatment may have a significantly lar- ger response rate and remission rate at post-treatment compared to standard ERP in effectiveness studies. In the present study we demonstrated that we were able to upscale, and to help 90 moderately to severely af- fected OCD-patients in 8 days; 45 in each 4-day slot. This was done as a part of the public mental health care, with no selection of patients. The 4-day format is often labelled “individual treat- ment in a group setting” indicating that the ratio be- tween therapists and patients is 1:1. Basically, this means that each therapist is able to treat one patient in less than a week, which probably is highly cost-effective. All the participating therapists were experienced in treating patients with OCD using CBT, but 67% of them had The present study tentatively indicates that the 4-day treatment can yield good outcome at another site than the originators’ place of work. Whether it works as well for other research groups and in other countries remains to be tested in future studies. However, the treatment is currently being tested in Iceland and an uncontrolled pilot study [28] obtained equally good effects as the ori- ginal studies in Norway [3–5]. Table 6 Comparison between the Bergen 4-day treatment and standard ERP in effectiveness studies Bergen 4-day Effectiveness Statistic N M (SD) N M (SD) t-test (p) Y-BOCS Pre 90 26.2 (3.4) 381 24.4 (5.4) 3.02 (=.0026) Post 90 10.5 (4.6) 374 18.2 (7.0) 9.93 (<.0001) 3 months 90 10.7 (6.3) 317 16.2 (8.1) 5.95 (<.0001) Remission Fisher’s test Post 90 72.2% 110 35.5% p < .0001 3 months 90 67.8% 110 45.5% p = .0017 Note: Data for effectiveness studies were taken from uncontrolled studies of ERP having 3–6 month follow-up. N indicates the total number of participants across these studies. Remission was calculated as ≥35% reduction of pre- treatment Y-BOCS scores and ≤12 on post and follow-up Y-BOCS Table 6 Comparison between the Bergen 4-day treatment and standard ERP in effectiveness studies This approach has been developed as an integrated part of the specialist health care with severely affected patients and with virtually no bias in the selection of cases that are offered the treatment. Discussion Since clinical changes that are achieved during a very brief window of time are large, seen in a large majority of patients, and to a large extent are maintained at follow-up, we argue that the 4-day treatment might offer a unique longitu- dinal and nearly experimental approach to study basic mechanisms of brain plasticity. Because no other factors apart from treatment are of influence during the 4 days Page 8 of 9 Kvale et al. BMC Psychiatry (2018) 18:323 Page 8 of 9 of intervention, it is an ideal setting for studies using functional magnetic resonance brain imaging (fMRI) or biomarkers of e.g. epigenetic changes to elucidate func- tional and structural changes on a number of different explanatory levels [29, 30]. This is an interesting area for future research which should be pursued. medical center (VUmc), Amsterdam, The Netherlands. 12Department of Psychology, Stockholm University, Stockholm, Sweden. medical center (VUmc), Amsterdam, The Netherlands. 12Department of Psychology, Stockholm University, Stockholm, Sweden. Competing interests Competing interests The authors declare that they have no competing interests. 16. Borkovec TD, Nau SD. Credibility of analogue therapy rationales. J Behav Ther Exp Psychiatry. 1972;3(4):257–60 https://doi.org/10.1016/0005- 7916(72)90045-6. References 1. Kvale G, Hansen B. Dissemination and intensifying evidence-based treatment for OCD: Norway is in the lead. Nordic Psychiat. 2014;3(1):14–5. 2. Foa EB, Yadin E, Lichner TK. Exposure and response prevention for OCD: therapist guide. London: Oxford University Press; 2012. 1. Kvale G, Hansen B. Dissemination and intensifying evidence-based treatment for OCD: Norway is in the lead. Nordic Psychiat. 2014;3(1):14–5. 2. Foa EB, Yadin E, Lichner TK. Exposure and response prevention for OCD: therapist guide. London: Oxford University Press; 2012. 3. Havnen A, Hansen B, Öst L-G, Kvale G. Concentrated ERP delivered in a group setting: an effectiveness study. J Obsessive-Compulsive Relat Disord. 2014;3(4):319–24 https://doi.org/10.1016/j.jocrd.2014.08.002. 1. Kvale G, Hansen B. Dissemination and intensifying evidence-based treatment for OCD: Norway is in the lead. Nordic Psychiat. 2014;3(1):14–5. Acknowledgments Th h ld li The authors would like to thank all participants in the study as well as therapists and the assessment team. 7. Abramowitz JS, Tolin DF, Diefenbach GJ. Measuring change in OCD: sensitivity of the obsessive-compulsive scale-revised. J Psychopathol Behav Assess. 2005;27:317–24. https://doi.org/10.1017/s10862-005-2411-y. 7. Abramowitz JS, Tolin DF, Diefenbach GJ. Measuring change in OCD: sensitivity of the obsessive-compulsive scale-revised. J Psychopathol Behav Assess. 2005;27:317–24. https://doi.org/10.1017/s10862-005-2411-y. Conclusions 2. Foa EB, Yadin E, Lichner TK. Exposure and response prevention for OCD: therapist guide. London: Oxford University Press; 2012. In sum, the Bergen 4-day treatment for OCD showed promising results. Future studies should test this treat- ment format using a RCT design. 3. Havnen A, Hansen B, Öst L-G, Kvale G. Concentrated ERP delivered in a group setting: an effectiveness study. J Obsessive-Compulsive Relat Disord. 2014;3(4):319–24 https://doi.org/10.1016/j.jocrd.2014.08.002. 3. Havnen A, Hansen B, Öst L-G, Kvale G. Concentrated ERP delivered in a group setting: an effectiveness study. J Obsessive-Compulsive Relat Disord. 2014;3(4):319–24 https://doi.org/10.1016/j.jocrd.2014.08.002. 4. Havnen A, Hansen B, Öst LG, Kvale G. Concentrated ERP delivered in a group setting: a replication study. Behav Cogn Psychother. 2017;45(5):1–7 https://doi.org/10.1017/S1352465817000091. Received: 20 December 2017 Accepted: 17 September 2018 Received: 20 December 2017 Accepted: 17 September 2018 Abbreviations DSM: Diagnostic and statistical manual of mental disorders; ERP: Exposure and response prevention; fMRI: Functional magnetic resonance imaging; GAD-7: Generalized Anxiety Disorder-7; LET: Lean into The anxiety; OCD: Obsessive-compulsive disorder; OUH: Oslo University Hospital; PHQ-9: Patient health questionnaire-9; SCID: Structured clinical interview; Y-BOCS: Yale-Brown obsessive-compulsive scale 5. Hansen B, Hagen K, Öst L-G, Solem S, Kvale G. The Bergen 4-day OCD treatment delivered in a group setting: 12-month follow-up. Front Psychol. 2018;9:369 https://doi.org/10.3389/fpsyg.2018.00639. 6. Franklin ME, Abramowitz JS, Kozak MJ, Levitt J, Foa EB. Effectiveness of exposure and ritual prevention for obsessive-compulsive disorder: randomized compared with nonrandomized samples. J Consult Clin Psychol. 2000;68:594–602. https://doi.org/10.1037/0022-006X.68.4.594. Authors’ contributions GK and BH designed the study. KH, SS, and L-GÖ conducted statistical analyses. GK, BH, KH, ThB, TB, SH, UBK, and GL supervised the treatment. AS represented the OCD patient organization and helped with planning and organizing the study. KJR and OAH helped with interpretation and revising the manuscript. All authors participated with drafting and revising the manuscript. 10. Abramowitz JS, Foa EB, Franklin ME. Exposure and ritual prevention for obsessive-compulsive disorder: effects of intensive versus twice-weekly sessions. J Consult Clin Psychol. 2003;71(2):394–8 http://psycnet.apa.org/doi/ 10.1037/0022-006X.71.2.394. 11. Storch EA, Merlo LJ, Lehmkuhl H, Geffken GR, Jacob M, Ricketts E, Murphy TK, Goodman WK. Cognitive-behavioral therapy for obsessive-compulsive disorder: a non-randomized comparison of intensive and weekly approaches. J Anxiety Disord. 2008;22:1146–52 https://doi.org/10.1016/j. janxdis.2007.12.001. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 17. Goodman WK, Price LH, Rasmussen SA, Mazure C, Delgado P, Heninger GR, et al. The Yale-Brown obsessive compulsive scale. II. Validity. Arch Gen Psychiatry. 1989;46(11):1012–6 https://doi.org/10.1001/archpsyc.1989. 01810110054008. Availability of data and materials 8. Hiss H, Foa EB, Kozak MJ. Relapse prevention program for treatment of obsessive-compulsive disorder. J Consult Clin Psychol. 1994;62(4):801–8 https://doi.org/10.1037/0022-006X.62.4.801. 8. Hiss H, Foa EB, Kozak MJ. Relapse prevention program for treatment of obsessive-compulsive disorder. J Consult Clin Psychol. 1994;62(4):801–8 https://doi.org/10.1037/0022-006X.62.4.801. The datasets used and/or analyzed during the current study can be available from the corresponding author on reasonable request. 9. Lindsay M, Crino R, Andrews G. Controlled trial of exposure and response prevention in obsessive-compulsive disorder. Br J Psychiat. 1997;171:135–9 https://doi.org/10.1192/bjp.171.2.135. Consent for publication Not applicable. 15. Sheehan DV, Lecrubier Y, Sheehan KH, Amorim P, Janavs J, Weiller E, et al. The mini-international neuropsychiatric interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10. J Clin Psychiat. 1998;59(Suppl. 20):22–33. Ethics approval and consent to participate This paper uses data collected as part of the standard assessment procedure at the outpatient OCD-clinic in Helse Bergen, Norway, approved by the Data Protection Official on August 5, 2012. This treatment quality assurance strategy does not require full ethical review and approval according to national and institutional guidelines. This has been described by the Regional committees for medical and health research ethics in Norway (https://helseforskning.etikkom.no/ reglerogrutiner/soknadsplikt/sokerikkerek?p_dim=34999&_ikbLanguageCode=us). Furthermore, quality assurance data does not require informed consent. 12. Öst L-G, Havnen A, Hansen B, Kvale G. Cognitive behavioral treatments of obsessive-compulsive disorder. A systematic review and meta-analysis of studies published 1993-2014. Clin Psychol Rev. 2015;40:156–69 https://doi. org/10.1016/j.cpr.2015.06.003. 13. Barlow D, Nock MK, Hersen M. Single case experimental designs: strategies for studying behavior change (3rd ed.). Boston, MA: Pearson; 2009. 14. American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 5th ed. Washington, DC: American Psychiatric Publishing; 2013. 14. American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 5th ed. Washington, DC: American Psychiatric Publishing; 2013. Author details 1 k l d 1Haukeland University Hospital, OCD-team, 5021 Bergen, Norway. 2Department of Clinical Psychology, University of Bergen, Bergen, Norway. 3McLean Hospital, Belmont, MA, USA. 4Harvard Medical School, Boston, MA, USA. 5Molde Hospital, Molde, Norway. 6Nidaros DPS, Division of Psychiatry, St. Olav University Hospital, Trondheim, Norway. 7Oslo University Hospital, Oslo, Norway. 8Sørlandet Sykehus, Kristiansand, Norway. 9Department of Psychology, Norwegian University of Science and Technology, Trondheim, Norway. 10Norwegian OCD-foundation, Ananke, Oslo, Norway. 11Department of Psychiatry and Department of Anatomy & Neurosciences, VU university 18. Goodman WK, Price LH, Rasmussen SA, Mazure C, Fleischmann RL, Hill CL, et al. The Yale-Brown obsessive compulsive scale. I. Development, use, and reliability. Arch Gen Psychiatry. 1989;46(11):1006–11 https://doi.org/10.1001/ archpsyc.1989.01810110048007. 18. Goodman WK, Price LH, Rasmussen SA, Mazure C, Fleischmann RL, Hill CL, et al. The Yale-Brown obsessive compulsive scale. I. Development, use, and reliability. Arch Gen Psychiatry. 1989;46(11):1006–11 https://doi.org/10.1001/ archpsyc.1989.01810110048007. 19. Kroenke K, Spitzer RL, Williams JB, Lowe B. The patient health questionnaire somatic, anxiety, and depressive symptom scales: a systematic review. Gen Hosp Psychiatry. 2010;32(4):345–59 https://doi.org/10.1016/j.genhosppsych. 2010.03.00614. 19. Kroenke K, Spitzer RL, Williams JB, Lowe B. The patient health questionnaire somatic, anxiety, and depressive symptom scales: a systematic review. Gen Hosp Psychiatry. 2010;32(4):345–59 https://doi.org/10.1016/j.genhosppsych. 2010.03.00614. Page 9 of 9 Kvale et al. BMC Psychiatry (2018) 18:323 Kvale et al. BMC Psychiatry (2018) 18:323 Kvale et al. BMC Psychiatry (2018) 18:323 20. Spitzer RL, Kroenke K, Williams JB, Lowe B. A brief measure for assessing generalized anxiety disorder: the GAD-7. Arch Int Med. 2006;166(10):1092–7 https://doi.org/10.1001/archinte.166.10.1092. 20. Spitzer RL, Kroenke K, Williams JB, Lowe B. A brief measure for assessing generalized anxiety disorder: the GAD-7. Arch Int Med. 2006;166(10):1092–7 https://doi.org/10.1001/archinte.166.10.1092. 21. Boschen MJ, Drummond LM. Community treatment of severe, refractory obsessive-compulsive disorder. Behav Res Ther. 2012;50(3):203–9. https://doi. org/10.1016/j.brat.2012.01.002. 22. Håland ÅT, Vogel PA, Lie B, Launes G, Pripp AH, Himle JA. (2010). Behavioural group therapy for obsessive-compulsive disorder in Norway. An open community-based trial. Behav. Res. Ther. 2010;48(6):547–54. https://doi. org/10.1016/j.brat.2010.03.005. 23. Himle JA, Rassi S, Haghigthatgou H, Krone KP, Nesse RM, Abelson J. Group behavioral therapy of obsessive-compulsive disorder: seven-vs. twelve-week outcomes. Depress Anxiety. 2001;13(4):161–5 https://doi.org/10.1002/ (ISSN)1520-6394. 24. Tolin DF, Maltby N, Diefenbach GJ, Hannan SE, Worhunsky P. Cognitive–behavioral therapy for medication nonresponders with obsessive–compulsive disorder. a wait-list-controlled open trial J Clin Psychiat. 2004;65(7):922–31 https://doi.org/10.4088/JCP.v65n0708. 24. Tolin DF, Maltby N, Diefenbach GJ, Hannan SE, Worhunsky P. Cognitive–behavioral therapy for medication nonresponders with obsessive–compulsive disorder. a wait-list-controlled open trial J Clin Psychiat. Author details 1 k l d 2004;65(7):922–31 https://doi.org/10.4088/JCP.v65n0708. 25. Tolin DF, Hannan S, Maltby N, Diefenbach GJ, Worhunsky P, Brady RE. A randomized controlled trial of self-directed versus therapist-directed cognitive-behavioral therapy for obsessive-compulsive disorder patients with prior medication trials. Behav Ther. 2007;38:179–91 https://doi.org/10. 1016/j.beth.2006.07.001. 26. Mataix-Cols D, Fernandez dela Cruz L, Nordsletten AE, Lenhard F, Isomura K, Simpson HB. Towards an international expert consensus for defining treatment response, remission, recovery and relapse in obsessive- compulsive disorder. World Psychiatry. 2016;15(1):80–1 https://doi.org/10. 1002/wps.20299. 27. Hansen B, Kvale G, Hagen K, Havnen A, Öst L-G. The Bergen 4-day format for OCD: four years follow-up of concentrated ERP in a clinical mental health setting. Cogn Behav Ther. in press. https://doi.org/10.1080/16506073. 2018.1478447. 28. Davíðsdóttir SD, Sigurjónsdóttir O, Ludvigsdóttir SJ, Hansen B, Laukvik IL, Hagen K, Björgvinsson T, Kvale G. Implementation of the Bergen 4-day treatment for OCD in Iceland. Clin Neuropsychiatry. in press. http://www. clinicalneuropsychiatry.org/. 29. Thorsen AL, van den Heuvel OA, Hansen B, Kvale G. Neuroimaging of psychotherapy for obsessive-compulsive disorder: a systematic review. Psychiatry Res Neuroimaging 2015;233(3):306–313: https://doi.org/10.1016/j. comppsych.2016.11.011. 29. Thorsen AL, van den Heuvel OA, Hansen B, Kvale G. Neuroimaging of psychotherapy for obsessive-compulsive disorder: a systematic review. Psychiatry Res Neuroimaging 2015;233(3):306–313: https://doi.org/10.1016/j. comppsych.2016.11.011. 30. van den Heuvel OA, van Wingen G, Soriano-Mas C, Alonso P, Chamberlain SR, Nakamae T, et al. Brain circuitry of compulsivity. Eur Neuropsychopharmacol. 2016;26(5):810–27 https://doi.org/10.1016/j. euroneuro.2015.12.005.
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https://journal.ugm.ac.id/populasi/article/download/10685/8029
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PENINGKATAN PERANAN WANITA DALAM PEMBANGUNAN* Teori dan ImpiikasiKebijaksanaan
Populasi
2,016
cc-by-sa
4,100
* Ditulis kembali dengan perbaikan dari makalah yang sebelumnya disampaikan pada "Seminar Peranan Wanita Dalam Pembangunan: Antara Harapan dan Realita", yang diselenggarakan oleh Pusat Penelitian Kependudukan, Universitas Gadjah Mada, Yogyakarta, 21Maret 1990. ** Dosen Fakultas Ilmu Sosial dan Ilmu Politik, Staf Pusat Penelitian Pembangunan Pedesaan dan Kawasan, Direktur Pusat Antar Universitas Studi Sosial, Universitas GadjahMada, Yogyakarta. PENINGKATAN PERANANWANITA DALAM PEMRANGUNAN* Teori danImpiikasiKebijaksanaan Oleh Nasikun POPULASI, 1(1),1990 wanita? Atau, di dalam pengungkapan lain, implikasi pilihan kebijaksanaan pembangunan apa saja yang dapat diambil untuk mengangkat kedudukan kaum wanita? Kendati tidak tersedia dukungan bahan pustakadan datayang memadai, tulisan ini ingin mencoba menjawab kedua pertanyaan tersebut. Bagian pertama penyajian, suatu pemaparan sangat singkat mengenai beberapa teori tentang mengapa kesenjangan pembagian kerja seksual terjadihampirdisemuamasyarakatyang kitakenal sampai saat ini. Bagian kedua berupa suatu uraian singkat mengenai faktor-faktor yang mempengaruhi diskriminasi seksual. Bagian ketiga berupa implikasi kebijaksanaan untuk mengangkat harkat dan kedudukan kaum wanita. Yang terakhir berupa uraian mengenai perlunya penyusunan suatu agenda penelitiantentatif tentang wanita dan pembangunan pada akhirnya akan penulis kemukakan. wanita? Atau, di dalam pengungkapan lain, implikasi pilihan kebijaksanaan pembangunan apa saja yang dapat diambil untuk mengangkat kedudukan kaum wanita? Kendati tidak tersedia dukungan bahan pustakadan datayang memadai, tulisan ini ingin mencoba menjawab kedua pertanyaan tersebut. Bagian pertama penyajian, suatu pemaparan sangat singkat mengenai beberapa teori tentang mengapa kesenjangan pembagian kerja seksual terjadihampirdisemuamasyarakatyang kitakenal sampai saat ini. Bagian kedua berupa suatu uraian singkat mengenai faktor-faktor yang mempengaruhi diskriminasi seksual. Bagian ketiga berupa implikasi kebijaksanaan untuk mengangkat harkat dan kedudukan kaum wanita. Yang terakhir berupa uraian mengenai perlunya penyusunan suatu agenda penelitiantentatif tentang wanita dan pembangunan pada akhirnya akan penulis kemukakan. 25 lapangan kerja yang hanya dapat dimasuki oleh sejumlah laki-laki, sementara kaum laki-laki dapat ditemukan tersebar di dalam tidak kurang dari 300 lapangan kerja yang hanya dapat dimasuki oleh sejumlah wanita. Denganperkataanlain, didalam masyarakat-masyarakat di mana emansipasi wanita sudah sangat berkembangpun atribut pekerjaan laki-laki dan wanita ternyata tidaklah bersifat paralel atau tidak berada pada dataran yang sama, melainkan secara kualitatifberbeda: jikalau berbagaijenis pekerjaan wanita lebih banyak ditentukan oleh seks, tidak demikian halnya dengan pekerjaan laki-laki. Lebih dari itu, hampir semua pekerjaan wanita pada umumnya dilihat dan dialokasikan di dalam hubungannya dengan pekerjaan-pekerjaan di sektor domestik (rumahtangga). Jikalau oleh karena evolusi perkembangan jaman kaum wanita melangkah juga ke dalam pekerjaan-pekerjaan di sektor publik, maka jenis-jenis lapangan pekerjaan yang mereka masula pada umumnya terbatas pada jenis-jenis lapangan pekerjaanyangmerupakankepanjangan dari pekerjaan-pekerjaan rumahtangga: seperti bidan, juru-rawat, guru, sekretaris, dan pekerjaan-pekerjaanlain yanglebihbanyakmemerlukankeahlian manual. 1. Pendahuluan Terlalu banyak bukti-bukti yang dapat kita kemukakan untuk menunjukkan kebenaran pernyataan diatas,sehinggaolehketerbatasanruang yangtersedia di dalam penyajiantulisan ini cukuplah penulis membatasinya denganmenampilkansebuahbuktiyang paling sederhana akan tetapi cukup meyakinkan. Untuk menunjuk diantara bukti-buktiyangpalingmeyakinkan atas kebenaran pernyataan tersebut, cukuplah kiranya pada kesempatan ini disajikan sebuah gambaran tentang distribusi peluang kaum wanita untuk memperoleh Iapangan kerja di negara- negara maju, yang biasanya kita fahami sebagai masyarakat yang memiliki struktur okupasiyang lebihterbuka. Menurut Bennholdt-Thomsen (1984) bahwa posisi sosial wanita menyerupaiposisikastabawahdidalam suatu masyarakat dengan sistem kasta, yang pilihan-pilihan iapangan kerjanya laksana telah ditetapkan oleh "suratan tangan" melalui kelahiran Kenyataan tersebut terasagetir bukansajaditelinga kaum wanita akan tetapi barangkali bahkandi telinga banyakkaumIaki-laki. Akan tetapi, tidak perduli bagaimana kata suara hati kita, memang itulah agaknya ungkapan paling tajam yang dapat kita pakai untuk menggambarkan kenyataan dunia yang kita jumpai di mana-mana. Di mana-mana, sebagaimana halnya dengan kasta bawah di dalam suatu masyarakat dengan sistem kasta, kaum wanita agaknya memang dilahirkan untuk melakukan pekerjaan-pekerjaan yang jauh lebih terbatas jumlahnya, memiliki status yang rendah, dan oleh karenaitu memperoleh imbalan kerja dan pendapatanyang rendah. Di dalam laporannya kepada UNDP berjudulNorthernWomen,andtheNew Economic Order, Martha Loufti (sebagaimana dikutip Bennholdt- Thomsen, 1984) mengungkapkan bahwa 70persenwanitayang bekerjadi dalam Iapangankerjayang terorganisasi ternyata hanya terkonsentrasi di dalam 1 POPULASI, 1(1),1990 2. Teori-teori tentang pembagian kerja seksual Diantara berbagai upaya teorisasi tentang pembagian kerja seksual, tiga buah teori di dalam sistem klasifikasi Anker dan Hein (1986) sangat menarik untuk kita simak: bukan hanya oleh karena ketiganya mewakili perspektif inter-disipliner yang saling melengkapi satu sama lain, akan tetapi lebih-lebih oleh karena kemampuan eksplanatoris mereka yang dapat membantu menjelaskan mengapa diskriminasi seksual terhadap kaum wanita nyaris kitatemukandidalamsetiapmasyarakat. Didalamketerbatasanbahandanwaktu, keseluruhan pemikiran yang akan dituangkan di dalam tulisan singkat ini bahkan dapat dipandang sebagai derivasi eklektik ketiga teori tersebut. Ketiga teori yang dimaksud adalah Kenyataan-kenyataantersebut paling sedikit menyodorkan dua buah pertanyaan yang sangat mendasar. Pertama,mengingatdiskriminasiseksual tanpa kecuali nyaris lata temukan di dalamsetiap masyarakat,pertanyaannya adalah apakah kenyataan itu memiliki akarnya di dalam dinamika setiap masyarakat? Dan jikalau jawabnya ya, bagaimana lata dapat menjelaskannya? Kedua, adakah peluang yang masih terbuka untuk mengangkat harkat kaum 2 POPULASJ, 1(1),1990 yang sama orangtua dan anggota keluargawanita sendiri pada umumnya memiliki insentif yang rendah untuk menginvestasikansumber dayakeluarga untuk pendidikan dan latihan anggota-anggota keluarga wanita, suatu situasi yang pada gilirannya akan menghasilkan kualitas human capital pekerja wanita yang senantiasa berada di bawah kualitas human capital pekerja laki-laki. teori-teori "neo-klasik", teori-teori "segmentasi pasar tenaga kerja", dan teori-teori "gender" atau "feminist yang sama orangtua dan anggota keluargawanita sendiri pada umumnya memiliki insentif yang rendah untuk menginvestasikansumber dayakeluarga untuk pendidikan dan latihan anggota-anggota keluarga wanita, suatu situasi yang pada gilirannya akan menghasilkan kualitas human capital pekerja wanita yang senantiasa berada di bawah kualitas human capital pekerja laki-laki. Teori-teori neo-klasik tentang pembagian kerja seksual menekankan perbedaan-perbedaan seksual di dalam variabel-variabel yang mempengaruhi produktivitas tenaga kerja dan ketersediaan tenaga kerja (seperti tanggungjawab ruraahtangga, kekuatan fisik, pendidikan, latihan, jam kerja, absentisme, dan kelangsungan kerja) untuk menjelaskan mengapa wanita memperoleh penghasilan lebih rendah daripada laki-laki. Asumsi yang mendasarinya adalah bahwa di dalam kondisi persaingan, pekerja memperoleh upah sebesar marginal productyangdihasilkannya.Asumsi lain yang turun dari asumsi yang pertama, adalah bahwa keluarga mengalokasikan sumber dayamereka (berupawaktu dan uang) diantara anggota-anggota keluarga di dalam cara yang rasional, yang pada gilirannya mengakibatkan anggota keluarga wanita memperoleh investasi human capital yang lebih sedikit dibandingkan dengan yang diterima oleh anggota keluargalaki-laki. Menurut teori ini, wanita memperoleh penghasilanyang rendah dibandingkan dengan laki-laki oleh karena mereka memiliki human capital (terutama pendidikan, latihan, dan pengalaman kerja) yang rendah, dan dengan demikian tingkat produktivitas rendah pula. 2. Teori-teori tentang pembagian kerja seksual Sebagai contoh, oleh karena wanita memiliki absentisme dan pemutusan kerja yang tinggi untuk menikah, melahirkan, dan memelihara anak, maka pengusaha pada umumnya tidakbersediamenginvestasikansumber daya merekauntuk memberikan latihan kerja kepada buruh-buruh wanita. Pada tingkat rumahtangga, halyangsamajuga p j Meskipun penjelasan neo-klasik memberikan sumbangan yang sangat penting di dalam menunjukkan mengapa wanita pada umumnya lebih banyak terakomodasi di dalam bidang-bidang pekerjaan yang kurang produktif dan oleh karena itu memperoleh tingkat pendapatan yang lebih rendah daripada yang diterima oleh para pekerja laki-laki, ia temyata memiliki kelemahan-kelemahannya sendiri. Kelemahan pertama berkaitan dengan asumsi penjelasan neo-klasik tentang perbedaan fisik sebagai sumber mengapa wanita harus melakukan pekerjaan-pekerjaan tertentu. Kecuali untuk pekerjaan mengandung dan melahirkan anak, tidak terdapat alasan biologis mengapa wanita harus melakukan pekerjaan-pekerjaan, misalnya, mengasuh anak dan pekerjaan-pekerjaandomestikyanglain. Kelemahan lain penjelasan neo-klasik adalah asumsinya bahwa laki-laki dan wanita memiliki akses yang sama terhadap peluang-peluang kerja dan bersaing di atas landasan yang sama. Asumsi ini melupakan kenyataan adanya segmentasi pasar tenaga kerja, yangtidak dapat dijelaskan semata-mata berdasarkan perspektif perbedaan seksual di dalam humancapital. Karena kedua kelemahan itulah relevansi kedua teori berikut kita temukan. Apabila kelemahan yang 3 POPULASI, 1(1),1990 POPULASI, 1(1),1990 dan peluang untuk promosi yang terbatas. Pekerjaan-pekerjaan sektor primer adalahjenis-jenis pekerjaanyang menuntut keahlian pekerja yang firm-specific, dan yang oleh tuntutan pengusaha akan stabilitas di dalam segmen angkatan kerja tersebut menyebabkan pengusaha bersedia memberikan penawaran upah yang lebihtinggi dan peluanguntuk promosi yang lebih terbuka. Bagi pengusaha, stabilitas tenaga kerja di sektor primer adalah sangat penting. Ketidakstabilan tenaga kerja wanita di mata pengusaha yang menyebabkan mereka cenderung senantiasa terdesak ke pekerjaan- pekerjaan di sektor sekunder. Dengan demikian, bahkan seandainya pra- kualifikasiuntukmemasukipasar tenaga kerja diperlakukan sama untuk tenaga kerjalaki-laki dan wanita, peluanglebih besar untuk memenangkan persaingan senantiasa lebih besar dimiliki oleh tenaga kerjalaki-laki daripada wanita. pertama kemudian ditutup oleh penjelasan teori gender ataufeminist, kelemahanyangkeduadapat dilengkapi olehteorisegmentasipasar tenagakerja. Jikalau yang pertama menekankan faktor-faktor sosial dan kultural yang menempatkan wanita di dalam posisi sosial yang kurang beruntung di dalam pasar tenaga kerja sebagai penjelasan kesenjangan pembagian kerja seksual, yang kedua memberikan tekanan pada faktor-faktor yang berkaitan dengan struktur pasar tenaga kerja dan bagaimana laki-laki dan wanita tersalur ke dalam segmen-segmen pasar tenaga kerja yang terpisah satu sama lain sebagai penjelasannya. Teori-teori segmentasi pasar tenaga kerja dapat dipandang sebagai modifikasi atau pemurnian teori neo-klasik, dalam arti mereka melihat pasar tenaga kerja sebagai pasar yang berjenjang atau tersegmentasi oleh hambatan-hambatan kelembagaan tertentu. 2. Teori-teori tentang pembagian kerja seksual Meskipun demikian, di dalam menjelaskan operasi tiap segmen pasar, sampai tingkat tertentu teori segmentasi pasar tetap memegang asumsi-asumsi neo-klasik: antara lain, bahwa pasar tenaga kerja bersifat kompetitif di atas dasar kualitas human capital yang dimiliki oleh semua pesaing. g j p Sumbangan paling penting teori segmentasi pasar tenaga kerja adalah tekanannya pada adanya pasar tenaga kerjatersegmentasi dankemampuannya untuk menganalisa berbagai situasi di berbagai segmen pasar tenaga kerja beroperasi, dan dengan demikian menyajikan alternatif bagi penjelasan neo-klasik yang mengasumsikan kompetisi terbuka diantara semua tenaga kerja yang memasuki pasar. Adanya dua pasar tenaga kerja yang secara relatif terpisah satu sama lain bagi tenaga kerja laki-laki dan wanita memiliki konsekuensi sangat penting bagi rendahnya upah, dan pendapatan tenagakerjawanita. Olehkarenapilihan pekerjaan yang terbuka bagi tenaga kerja wanita sangat terbatas, -dengan akibat terjadinya penawaran tenaga kerja yang berlebih untuk pekerjaan- pekerjaan wanita--, persaingan tenaga Salah satu diantara berbagai penjelasan teori segmentasi pasar tenaga kerjayang paling dikenal adalah teori pasar tenaga kerja ganda dari Doeringer dan Piore (Anker and Hein, 1986), yang membedakan dua jenis tenaga kerja atau pekerjaan: (1) pekerjaan-pekerjaansektor primeryang secara relatif lebih baik di dalam hubungannya dengan upah, jaminan keamanan, dan peluanguntuk promosi; dan (2) pekerjaan-pekerjaan sektor sekunder yang memiliki upah yang rendah,jaminan keamananyangkurang, 4 POPULASI, 1(1), 1990 POPULASI, 1(1), 1990 kerja wanita menjadi overcrowded untuk pekerjaan-pekerjaan tersebut. Sebagai akibatnya, upah tenaga kerja cenderung lebih rendah untuk pekerjaan-pekerjaanwanita olehkarena mereka harus bersaing keras dengan sesama tenaga kerja wanita untuk lapangan kerja yang sangat terbatas. Sebaliknya, tenaga kerja wanita tidak dapat bersaing dengan tenaga kerja laki-laki untuk jumlah lapangan kerja laki-laki yang lebih banyak, suatu hal yang pada gilirannya membantu mempertahankan upah tenaga kerja yang relatif tinggi untuk jenis pekerjaan-pekerjaan laki-laki. dijumpai sangat universal baik di masyarakat-masyarakat sedang berkembang maupun lebih-lebih di dalam masyarakat-masyarakat modem- industrial. Dimasyarakat negara-negara sedang berkembang, pekerjaan- pekerjaan mengasuh anak dan mengurus rumah tangga, meski menjadi tugas utama ibu rumah tangga, pada umumnya dilakukan bersama-sama dengan wanita-wanita lain di dalam konteks keluarga sedarah (extended family). Dengan bergesemya extended family menuju ke keluarga batih (nuclear family) menyusul proses modemisasi, tanggung jawab mengasuh anak danmengurus mmahtangga justru semakin jatuh ke tangan ibu rumah tangga. Pada saat yang bersamaan, ideologi gender ternyata hanya mengakomodasi pekerjaan-pekerjaan bam di sektor publik yang dapat dilihat sebagai kepanjangan dari pekerjaan- pekerajaan wanita di sektor domestik juru-rawat, bidan, gum, sekretaris, dan sejenisnya. 3. Faktor-faktor yang mem- pengaruhi diskriminasi seksual 3. Faktor-faktor yang mem- pengaruhi diskriminasi seksual Ketiga teori yang baru saja kita paparkan dengan sangat jelasnya mengungkapkan bahwa diskriminasi di dalam pembagian kerja seksual berkaitan sangat eratnya dengan perbedaan kualitastenaga kerjalaki-laki dan wanita sebagai human capital (menurut teori neo-klasik), dengan struktur pasar tenaga kerja (menurut teorisegmentasipasar tenagakerja),dan dengannorma-normatentangstatus dan peran wanita di dalam masyarakat (menurutteorigender ataufeminist).Di dalam bagian ini, penyajian lebih dipusatkan pada identifikasi faktor- faktor yang pada tingkat operasional mempengaruhi diskriminasi seksual di dalam pasar tenaga kerja. Mengikuti Anker dan Hein, faktor-faktor yang dimaksud dapat dikelompokkan ke dalam dua kategori: (1) faktor-faktor yang mempengaruhi diskriminasi seksual didalam aksesterhadap peluang kerja dan (2) faktor-faktor yang mempengaruhi diskriminasi pembagian kerja seksual di dalam lapangankerja di sektor modern. (c). (c). Lokasi kerja sektor modern yang berada di luar rumah dan jam kerja yang panjang dan tertentu, yang sulit diakomodasiwanitayangmasih harus melakukan pekerjaan- pekerjaan rumah tangga, dan menyebabkan kebanyakan wanita terpaksa harus menerima pekerjaan-pekerjaan di sektor informal. (c). Lokasi kerja sektor modern yang berada di luar rumah dan jam kerja yang panjang dan tertentu, yang sulit diakomodasiwanitayangmasih harus melakukan pekerjaan- pekerjaan rumah tangga, dan menyebabkan kebanyakan wanita terpaksa harus menerima pekerjaan-pekerjaan di sektor informal. (d) (d). Pembatasan-pembatasan kultural bagi wanita untuk bekerja dengan laki-laki yang bukan mukhrimnya. Pembatasan-pembatasan tersebut bukanhanyamembatasipenawaran tenaga kerjawanita akan tetapi juga mengurangi permintaan tenaga kerja wanita oleh karena pembatasan-pembatasan yang demikian menuntut biaya yang mahal untuk menyediakan tempat kerjayang terpisah bagi wanita. (d). Pembatasan-pembatasan kultural bagi wanita untuk bekerja dengan laki-laki yang bukan mukhrimnya. Pembatasan-pembatasan tersebut bukanhanyamembatasipenawaran tenaga kerjawanita akan tetapi juga mengurangi permintaan tenaga kerja wanita oleh karena pembatasan-pembatasan yang demikian menuntut biaya yang mahal untuk menyediakan tempat kerjayang terpisah bagi wanita. (e) (e). Keyakinan-keyakinan pengusaha bahwa tenaga kerja wanita adalah lebih mahal dibandingkan dengan tenaga kerja laki-laki, oleh karena mempekerjakan wanita meng- haruskan mereka untuk mebayar sejumlah biaya jaminan sosial wanita (seperti, jaminan kehamilan, melahirkan, dan sejenisnya) dan persentase absentisme wanita yang tinggi. (e). Keyakinan-keyakinan pengusaha bahwa tenaga kerja wanita adalah lebih mahal dibandingkan dengan tenaga kerja laki-laki, oleh karena mempekerjakan wanita meng- haruskan mereka untuk mebayar sejumlah biaya jaminan sosial wanita (seperti, jaminan kehamilan, melahirkan, dan sejenisnya) dan persentase absentisme wanita yang tinggi. Penemuan-penemuan empiris dari berbagai masyarakat memperlihatkan bahwa faktor-faktor yang mem¬ pengaruhidiskriminasiseksualterhadap peluangkerjameliputi: (a). Prioritas untuk menduduki posisi sebagai breadwinners yang pada umumnya diberikan kepada kaum laki-laki di dalam situasi tingkat pengangguran yang sangat tinggi, sebagaimana yang terjadi di kebanyakan negara-negara sedang berkembang. 2. Teori-teori tentang pembagian kerja seksual Dengan semuanya itu, sumbangan teori gender ataufeminist yang paling penting adalah menunjukkan bagaimana kedudukan wanita didalam pasartenagakerjadapat dilihat tidak lebih sebagai bagian yang tidak terpisahkan dari keseluruhan sistem sosial di mana wanita memiliki kedudukan yang rendah. Dengan perkataan lain, ketidaksamaan seksual di dalam pasar tenaga kerja bertalian sangat erat dengan dan bersumber di dalam norma-norma budaya yang mendefinisikan secara berbeda status dan peranan laki-laki dan wanita di dalam masyarakat. Asumsi posisi kaum laki-laki sebagai breadwinners dan posisi kaum wanita sebagai housekeepers oleh hampir semua masyarakat yang pernah kita kenal, (demikiankesimpulanteorigender atau Meskipun teori segmentasi pasar tenaga kerja berhasil membantu mempertajam penjelasan tentang mengapa pembagian kerja seksual cenderung menyudutkan kedudukan wanita, teori tersebut ternyata tidak mampu menjelaskan mengapa segmentasi pasar tenaga kerja berdasarkan seks terjadi. Adalah teori gender ataufeminist yang memusatkan perhatiannya untuk menjelaskan fenomena tersebut. Teori tersebut menjelaskan bahwa kedudukan yang kurang beruntung dari kaum wanita di dalam pasar tenaga kerja dan di dalam rumah tangga atau keluarga bertalian sangat erat satu sama lain, dan merupakan bagian yang tidak terpisahkan dari keseluruhan sistem sosial di mana wanita memiliki kedudukan yang lebih rendah dihadapan laki-laki. Salah satu masalah sangat penting bagi para ahli teori ini adalah alokasi pekerjaan-pekerjaan domestik bagi tenaga kerja wanita. Alokasi pekerjaan-pekerjaan domestik seperti mengasuh anak dan mengurus rumah tangga kepada wanita, bahkan untuk mereka yang bekerja di sektor publik, 5 POPULASI, 1(1), 1990 feminist), merupakan sumber pertama dari mana diskriminasi pembagian kerja seksual berawal. (b). Tingkat pendidikan wanita yang rendah di negara-negara sedang berkembangsebagaiakibat struktur ganjaran ekonomis dan norma- norma masyarakat yang menghalangi keluarga-keluarga di negara-negara itu untuk menginvestasikan sumber daya keluarga bagi pendidikan anak wanita. (b). Tingkat pendidikan wanita yang rendah di negara-negara sedang berkembangsebagaiakibat struktur ganjaran ekonomis dan norma- norma masyarakat yang menghalangi keluarga-keluarga di negara-negara itu untuk menginvestasikan sumber daya keluarga bagi pendidikan anak wanita. 3. Faktor-faktor yang mem- pengaruhi diskriminasi seksual 3. Faktor-faktor yang mem- pengaruhi diskriminasi seksual 3. Faktor-faktor yang mem- pengaruhi diskriminasi seksual (f). Anggapan pengusahabahwawanita lebih cocok untuk melakukan sejumlah pekerjaan-pekerjaan 6 POPULASI, 1(1), 1990 administratif tinggi, oleh karena anggapan populer bahwa wanita tidak memiliki kapasitas untuk melakukan tugas pengawasan atas pekerjaan-pekerjaan oranglain. administratif tinggi, oleh karena anggapan populer bahwa wanita tidak memiliki kapasitas untuk melakukan tugas pengawasan atas pekerjaan-pekerjaan oranglain. tertentu, yang dianggap memiliki tingkat produktivitas dan imbalan ekonomis yang rendah. Diskriminasiseksualyangmerugikan wanita ternyata tidak berhenti hanya sampai pada tingkat persaingan untuk memasuki lapangan kerja, akan tetapi juga sampai pada tingkat pembagian kerja sesudah mereka berhasil memasuki lapangan kerja di sektor modern. Diantara faktor-faktor yang palingkuat mempengaruhi diskriminasi pembagian kerja di dalam sektor modern adalah: (d) (d).Keengganan pengusaha untuk mempekerjakan wanita untuk pekerjaan-pekerjaan yang secara konvensional sudah didominasi oleh pekerja-pekerja laki-laki. (e). Keyakinan-keyakinan pengusaha bahwa wanita lebih cocok untuk tugas-tugas rendahan (di mana kelembutan dan kepatuhan dianggap merupakan faktor sangat penting bagi produktivitas kerja yang tinggi dan tidak menimbulkan banyak kesulitan kerja) dan untuk sejumlah tugas-tugas tertentu yang memerlukan daya tarik seksual wanita untuk menarik konsumen atau langganan. (e). Keyakinan-keyakinan pengusaha bahwa wanita lebih cocok untuk tugas-tugas rendahan (di mana kelembutan dan kepatuhan dianggap merupakan faktor sangat penting bagi produktivitas kerja yang tinggi dan tidak menimbulkan banyak kesulitan kerja) dan untuk sejumlah tugas-tugas tertentu yang memerlukan daya tarik seksual wanita untuk menarik konsumen atau langganan. (e). Keyakinan-keyakinan pengusaha bahwa wanita lebih cocok untuk tugas-tugas rendahan (di mana kelembutan dan kepatuhan dianggap merupakan faktor sangat penting bagi produktivitas kerja yang tinggi dan tidak menimbulkan banyak kesulitan kerja) dan untuk sejumlah tugas-tugas tertentu yang memerlukan daya tarik seksual wanita untuk menarik konsumen atau langganan. (a). Sistem pendidikan dan latihan fokasional (didalam maupundiluar rumah tangga) yang melatih wanita untuk melakukan "pekerjaan- pekerjaan wanita" dan menghalangi mereka untuk memperoleh pendidikan dan latihan bagi sejumlah "pekerjaan-pekerjaan laki-laki". (f). (f). Kesediaan (atau, lebih tepat, keterpaksaan) wanita untuk menerima pekerjaan-pekerjaan yang memiliki imbalan yang rendah dan tidak akan dipilih oleh pekerja laki-laki. (b). Pembatasan-pembatasan atau pengecualian-pengecualian bagi wanita untukmelakukan pekerjaan- pekerjaan tertentu berdasarkan legislasi yang justru dimaksudkan untuk melindungi kaum wanita (misalnya, larangan untuk mempekerjakanwanita padamalam hari, di dalam jenis-jenis pekerjaan yang berat atau berbahaya, atau legislasi yang mengharuskan penyediaan fasilitas-fasilitas khusus untuk wanita). Pembatasan- pembatasan legislatif seperti itu bukan hanya membatasi kesediaan pengusaha untuk mempekerjakan wanita, akan tetapi juga membatasi peluangwanita untuk dipekerjakan di bidang-bidang pekerjaan yang memperoleh imbalan ekonomis yang tinggi. 3. Faktor-faktor yang mem- pengaruhi diskriminasi seksual Bekerjanya faktor-faktor diatas, —membatasi masuknyawanita ke dalam pekerjaan-pekerjaan di sektor modern, dan bagi mereka yang berhasil memasukinya secara seksual mendiskriminasikan mereka di dalam sistem pembagian kerja masyarakat—, kedudukanwanitayangdidalambanyak masyarakat tradisional dijumpai cukup sentral seringkali justru semakin tersudut ke pinggiran struktur sosial masyarakat-masyarakat modern (Boserup, 1970). Sungguh menarik bahwa dampak modernisasi atau pembangunan terhadap peran wanita kadang-kadang memang ironis. Efisiensi, rasionalitas, dan nilai-nilai kemanusaianyangmerupakanbeberapa (c). Pembatasan wanita dari posisi-posisi pengawasan dan 7 POPULASI, 1(1), 1990 memperoleh prioritas istimewa. Program-program pendidikan dan pelatihan fokasional khusus bagi kaum wanita adalah satu diantaranya. Kaum wanita di negara-negara sedang berkembang, misalnya, perlu didorong dan diberi peluang lebih luas untuk memperoleh pendidikan dan latihan di dalam bidang-bidang keahlian yang selama ini menjadi monopoli laki-laki. Dalam hubungan itu adalah sangat penting bagi pemerintah negara-negara sedang berkembang untuk menyusun perencanaan dan program-program intervensi yang tajam untuk mengikis sikap-sikap yang mendiskriminasikan wanita dari kemungkinan untuk memperoleh "keahlian-keahlian laki-laki", serta menjamin kesamaan peluangkerjabagimerekayangmemiliki keahlian-keahlian tersebut. ethos paling esensial dari modemisasi dan mendasari perjuangan peningkatan peran wanita, di dalam pengalaman banyak masyarakat temyata tidak selalu memilikiwajahyangjelita. 4. ImplikasiKebijaksanaan Mengingat akar posisi sosial wanita yang kurangberuntung di dalam sistem pembagian kerja seksual tertanam kuat di dalam berbagai domein yang saling berkaitan satu sama lain (kualitas humancapita/yangrendah, segmentasi pasar tenaga kerja, dan norma-norma masyarakat mengenai status dan peranan wanita), implikasi kebijaksanaan yang dapat diupayakan untuk mengangkat harkat kaum wanita jelas tidak dapat ditemukan di dalam suatu kebijaksanaan yang tunggal. Sebaliknya, kebijaksanaan yang efektif untuk mengangkat peranan wanita di dalammasyarakat harus secara simultan dilakukan di berbagai bidang. hukum, pendidikan, perilaku, struktur ekonomi, dan berbagai kebijaksanaan sosial yang lain. Kebijaksanaan untuk mengangkat peran dan harkat kaum wanita juga tidak dapat hanya dilaksanakan pada tingkat pemerintah nasional akan tetapi harus dilaksanakan pula pada tingkat pemerintah lokal, organisasi-organisasi karyawan, pengusaha, organisasi wanita, dan tidak kalah pentingnya bahkan pada tingkat kelompok-kelompok agama dan kultural. Pada tingkat lain, pemerintah negara-negara sedang berkembang dapat memainkan peranan yang sangat menentukan di dalam mendorong persamaandiantaralaki-lakidanwanita, bukan hanya melalui pengendalian fasilitas-fasilitas pendidikan dan latihan fokasional akan tetapi juga melalui pemanfaatan media massa dan kebijaksanaan-kebijaksanaan pengangkatan dan promosi wanita di dalam sektor publik. Ini sangat penting mengingat bukti-bukti yang terkumpul dari pengalaman banyak negara sedang berkembang bahwa peluang-peluang bagi wanita cenderung lebih besar dan diskriminasi terhadap mereka lebih rendah di sektor publik daripada di sektor swasta. Hasil-hasil penelitian dari berbagai masyarakat membuktikanhalitu (Anker dan Hein, 1986: 46). Meskipun demikian, beberapa kawasan kebijaksanaan intervensi yang dengan mobilisasi dana yang terbatas dapat menghasilkan cost effectiveness yang tinggi untuk meningkatkan peran dan harkat wanita memang perlu Kebijaksanaan untuk mendorong perkembangan organisasi-organisasi serikat buruh wanita dan organisasi- organisasi wanita lain merupakan kawasan kebijaksanaan intervensi lain yang penting pula. Sebagaimana kita 8 POPULASI, 1(1),1990 POPULASI, 1(1),1990 diterima oleh buruhlaki-laki. Ironisnya, beberapa legislasi yang dimaksudkan untuk melindungi kepentingan kaum wanita, seringkali justru merugikan kepentingan para pekerja wanita. Di dalam hubungan ini, adalah sangat menarik untuk mengkaji sejauh mana kebijaksanaan Keluarga Berencana memiliki dampak peningkatan peran dan harkat wanita. ketahui, berbagai faktor yang berkaitan dengan diskriminasi seksual di dalam pelatihan fokasional buruh, jam kerja, pemekerjaan dan pemecatan, sistem promosi, tingkat upah, dan legislasi perlindungan buruh, di mana-mana senantiasa merupakan perhatian perjuanganorganisasiburuh. Sayangnya keterlibatanwanita di dalam organisasi- organisasi buruh di kebanyakan negara-negarasedangberkembangpada umumnya adalah sangat rendah. Dalam pada itu, berbagai organisasi wanita yang lain di negara-negara tersebut (seperti Dharma Wanita dan PKK di Indonesia) pada umumnya tidak terutama didorong perkembangannya untuk perjuangan hak-hak wanita melainkan lebih dikembangkan untuk mobilisasi dukungan terhadap kekuatan-kekuatansosialyangdominan. 4. ImplikasiKebijaksanaan Olehkarenaitu,jikalau perandanharkat wanita benar-benar harus ditingkatkan, pilihan kebijaksanaan intervensi untuk mendorong perkembangan organisasi buruh pada umumnya dan organisasi buruh wanita pada khususnya serta untuk meningkatkan perhatian organisasi-organisasi wanita pada perbaikan nasib buruh wanita tidak bolehdipandang sebelah mata. Pengasuhan anak merupakan kawasan pilihan kebijaksanaan intervensi lain untuk mengurangi diskriminasi seksual di dalam pasar tenaga kerja dan pembagian kerja di sektor-sektor pekerjaan modern. Dalam hal ini, peningkatan peluang wanita untuk memasuki pasar tenaga kerja dan bersaing dengan laki-laki di dalam pembagian kerja di sektor modern, untuk sebagian dapat dicapai melalui reorganisasi pembagian kerja kemasyarakatan di dalam fungsi pengasuhan anak: yakni, melalui pembagian kerjayang seimbang antara laki-laki dan wanita di dalam fungsi pengasuhan anak, atau pengalihan fungsi tersebut dari tangan ibu rumah tangga ke tangan berbagai organisasi kemasyarakatan tertentu (seperti PKK, Dharma Wanita, Karang Taruna, dan organisasi-organisasi khusus yang dimaksudkan untuk itu). Kebijaksanaan intervensi untuk mengatasi dampak negatif peran reproduktif wanita di dalam pasar tenagakerjamerupakanpilihanstrategis lain untuk meningkatkan harkat kaum wanita. Siapapun mengetahui bahwa peran reproduktif wanita merupakan salah satu kendala paling besar yang dihadapi wanita untuk bersaingdengan kekuatan yang seimbang dengan laki-laki di pasar tenaga kerja, dan bagi mereka yang bekerja merupakan kendala yang sama besamya di dalam persaingan untuk memperoleh perlakuan yang sama dengan yang Tidak dapat diabaikan,jikalau bukan sama pentingnyadengan pilihan-pilihan kebijaksanaan yang lain, adalah perlunya kebijaksanaan diseminasi informasi mengenai tempat wanita di dalam organisasi ekonomi, politik, dan sosial kultural. Sangat strategis, misalnya, adalah perlunya penyebar- luasan informasi mengenai ragam keahlian dan prestasi kerja wanita, peran dan kontribusi wanita di dalam ekonomi keluarga dan masyarakat, dan peranmereka di dalam berbagai bidang 9 POPULAS1, 1(1), 1990 hubungan antara berbagai faktor yang menjadi sumber dan penawar diskriminasi seksual di dalam pasar tenaga kerja dan masyarakat. Untuk itu suatu agenda penelitianyang sistematis dan semakin menukik ke dalam pemahaman mengenai determinan- determinan kesenjangan seksual di dalam masyarakat perlu terus-menerus dirumuskan sejalan dengan dan mengikuti semakin menumpuknya hasil-hasil penelitian. Berbagai penelitianyangselamainiterlalubanyak difokuskan untuk membuat deskripsi- deskripsi mengenai situasi ketimpangan seksual di dalam masyarakat perlu semakin diarahkan untuk mengungkap- kan pemahaman-pemahaman eksplanatoris yang lebih mapan dan andal. kehidupan masyarakat. Kecuali apabila badan-badan internasional, pemerintah-pemerintah nasional, organisasi-organisasi buruh, para pengusaha, dan masyarakat menyadari peran wanita yang sebenarnya di dalam masyarakat dan ekonomi, kebijaksanaan-kebijaksanaan untuk mengurangi diskriminasi seksual di dalam masyarakat hanya akan merupakan slogan saja. 5. Penutup Sebagai kesimpulan, ditinjau dari perspektif teoritis maupun dari bukti-bukti empiris diskriminasi seksual terhadap wanita yang nyaris kita temukan di dalam semua masyarakat yangpemahkitakenal,memilikisumber dalam akar-akar budaya (gender ideology) yang menempatkan wanita di pinggiran struktur sosial masyarakat. Manifestasinyamenyatakandiridi dalam rantai hubungan antara kecenderungan keluarga dan masyarakat men- diskriminasikan investasi sumber dayanya bagi pendidikan anak-anak wanita, --yang pada gilirannya menyebabkan wanita hanya dapat menjadi human capital yang berkualitas rendah-, dan diskriminasi seksual di dalam pasar tenaga kerja. Kendati demikian, hal itu tidak berarti bahwa peluang untuk meningkatkan peran dan harkat kaum wanita sama sekali tertutup. Sebagaimana dipaparkandiatas, denganperencanaan yang sungguh-sungguh dan komitmen yang mendalam sejumlah pilihan kebijaksanaan strategis untuk mengurangi diskriminasi seksual di dalam masyarakat bukan tidak mungkin dilaksanakan. Yang masih harus banyak diupayakan adalah penguasaan pengetahuan empiris yang andal mengenai mekanisme-mekanisme 10 POPULASI, 1(1), 1990 DAFTAR PUSTAKA ANKER, Richard dan Catherine Hein 1986 "Introduction and overview", dalam Sex inequalities in urban employment in the third world. London, MacMilian Press BENNHOLDT-THOMPSON, Veronika 1984 "Toward a theory of the sexual division of labor", dalam Joan Smith, et al., ed Householdand the world economy. NewYork, Sage Publications. BOSERUP, Ester 1970 Women's role ineconomic development. NewYork, St.Martins Press. MIES, Maria 1982 "The dynamics of the sexual division of labor and integration of rural women into the world market", dalam Lourdes Beneria, ed, Women anddevelopment: the sexual division of labor in rural societies. New York, Praeger Publishers. Hal.: 1-28. WOLF, Diane Lauren 1986 Factory daughters, their families, and rural industrialization. Tesis, PhD., Cornell University, NewYork. 11
https://openalex.org/W4289753846
https://ccsenet.org/journal/index.php/ijsp/article/download/0/0/39033/40095
English
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An Improved Bound for Security in an Identity Disclosure Problem
arXiv (Cornell University)
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cc-by
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1. Introduction Many agencies release data to motivate statistical research and industrial work. But often these data-sets carry some information which may be sensitive to the individual bearing it. Erasing the name or some identity number associated with an individual may not always be sufficient to hide the identity of the individual. For example, imagine a situation where a data-set of p variables corresponding to n individuals are released and among these p variables there is a variable named “pin-code”( sometimes called zip-code). Now “pin-code” is not supposed to be a sensitive variable, but it may happen that the intruder, who is trying to identify some individual in the data-set, has an idea about where the individual lives and thus can guess his “pin-code”. In this case, if in the data-set there is no other individual having the same “pin- code”, he can directly guess from this information which row in the data-set corresponds to the individual and thus the identity is revealed. Hence, suppressing identity numbers or names is not always sufficient to prevent identity disclosure. Sometimes, some attributes, that may reveal the identity of the individual, also called the identifying attribute, may result in identity disclosure of the individual. Moreover, if an attribute value corresponds to a very few individuals in a data-set, it is usually easy for the intruder to identify the individual. For example, if the “pin-code” value corresponds to one or two individuals in the data-set, then the intruder can guess which row in the data-set corresponds to the individual with high probability. But, if the value corresponds to twenty individuals, then the intruder now has to guess from these twenty rows which one belongs to his target individual. The identification risk is thus low for high frequency cells. Various articles including Bethelem et al. (1990), Trabelski et al. (2009), Nayak et al. (2016) have discussed this problem and have proposed different risk measures to evaluate the security in the released data, i.e., to check if an intruder can identify the row of his target unit from the released data. However, here we follow the framework of Nayak et al. (2016). The intruder here has a knowledge of the variable category X(B) corresponding to his target unit B. If the variable X has k categories c1, c2, . . . An Improved Bound for Security in an Identity Disclosure Problem Debolina Ghatak1 & Bimal K Roy2 1 Visiting Scientist, Applied Statistics Unit Indian Statistical Institute, Indian 2 Professor, Applied Statistics Unit Indian Statistical Institute, Indian Correspondence: Debolina Ghatak, Indian Statistical Institute, 203 B. T. Road, Kolkata 700108, India Received: February 17, 2019 Accepted: March 19, 2019 Online Published: April 3, 2019 doi:10.5539/ijsp.v8n3p24 URL: https://doi.org/10.5539/ijsp.v8n3p24 Received: February 17, 2019 Accepted: March 19, 2019 Online Published: April 3, 2019 doi:10.5539/ijsp.v8n3p24 URL: https://doi.org/10.5539/ijsp.v8n3p24 Abstract Identity disclosure of an individual from a released data is a matter of concern especially if it belongs to a category with low frequency in the data-set. Nayak et al. (2016) discussed this problem vividly in a census report and suggested a method of obfuscation, which would ensure that the probability of correctly identifying a unit from released data, would not exceed ξ for some 1 3 < ξ < 1. However, we observe that for the above method the level of security could be extended under certain conditions. In this paper, we discuss some conditions under which one can achieve a security for any 0 < ξ < 1. Keywords: data obfuscation, identity disclosure problem, categorical data security International Journal of Statistics and Probability International Journal of Statistics and Probability Vol. 8, No. 3; 2019 http://ijsp.ccsenet.org individual. (1998) defined a post randomization method to be an invariant PRAM if P satisfies Equation (2). The error due to estimation after post randomization was studied in the literature by various authors including Nayak et al. (2015). Gouweleeuw et al. (1998) defined a post randomization method to be an invariant PRAM if P satisfies Equation (2). The error due to estimation after post randomization was studied in the literature by various authors including Nayak et al. (2015). One of the common techniques to achieve an invariant PRAM is to use an Inverse Frequency Post Randomization (IFPR) block diagonal matrix, in which the entire data-set is partitioned into few groups and within each group, categories are interchanged. If it is not desirable to change the category of some variable, it can be made to form its own block. Thus, if there are m groups, given by {c1, c2, · · · , ck1}, {ck1+1, ck1+2, · · · , ck1+k2}, . . ., {ckm−1+1, ckm−1+2, · · · , ckm−1+km}, where k1 + k2 + · · · + km = k, then pij > 0 if cj and ci fall into the same group and pij = 0 if cj and ci fall into different groups. Within each group, pij is given by,  pij =  1 −θ/Ti if i = j θ (k′−1)Ti if i , j , (3) (3) (3) where 0 < θ < 1 and k′ > 1 is the block size of the group that i and j fall into. However, the parameter θ of the model should be carefully chosen to ensure that the perturbed data is secured from the intruder, at least, up to a certain extent. To measure the risk of disclosure, Nayak et al. (2016) suggested checking whether the probability of correctly identifying an individual given any structure of T and any value of S 1 is bounded by some specified quantity 0 < ξ < 1. Moreover, they showed that there exists a θ⋆, where 0 < θ⋆< 1 which gives the transition matrix, P(θ⋆) = ((p⋆ i j))1≤i≤k,1≤j≤k where p⋆ ij is chosen according to Equation (3) with θ = θ⋆for each i, j = 1, 2, · · · , k1 and k1 is the block size of the group c1 belongs to. individual. Without loss of generality, we assume the block c1 belongs to is the first block. This matrix P(θ⋆) when used to post randomize X, (4) P[ CM | S 1 = a, T = t] ≤ξ ∀a ≥0, ∀t, P[ CM | S 1 = a, T = t] ≤ξ ∀a ≥0, ∀t, (4) for any 1 3 ≤ξ < 1, where CM denotes “Correct Match”. Our aim in this paper is to check whether the security can be extended from 1 3 ≤ξ < 1 to any 0 < ξ < 1. We observed that, if we can extend the search range of θ from 0 < θ < 1 to 0 < θ < T1 and can find all categories in the first block that satisfy T j ≥T1 for all j , 1, then the level of security can be extended to any 0 < ξ < 1. Note that, under this definition, there is no harm in the range of the probabilities, ( given in Equation (3)) as they certainly lie between 0 and 1. However, smaller the value of ξ, larger the block size will be required. This is due to the methodology described in Section 2. Therefore we can extend the security as far as the frequency distribution permits. individual. Let {X1, X2, · · · , Xn} be the original data-set which is assumed to be a collection of i.i.d. random variables and X is a random variable from the common distribution of {X1, X2, · · · , Xn}. If it is an identifying attribute, i.e., release of it in its raw form can reveal the identity of the individuals, then it is perturbed to {Z1, Z2, · · · , Zn} before releasing and Z follows the same distribution as {Z1, Z2, · · · , Zn}. However, this change may cause a loss of information to the data-set. To minimize this loss, the data is perturbed such that the probability of each X being perturbed to Z is given by the transition matrix P = ((pi j)), where, pij = P[Z = cj|X = ci] , i, j = 1, 2, · · · k. (1) (1) pij = P[Z = cj|X = ci] , i, j = 1, 2, · · · k. This matrix is not released and is unknown to the statistician. The method of obfuscation is known as the post-randomization method (PRAM). If we assume the frequencies of {X1, X2, · · · , Xn} to be T = (T1, T2, · · · , Tk) for the categories {c1, c2, · · · , ck and T ∼Multinomial(Π1, Π2, · · · , Πk), then after transformation of X to Z, S = (S 1, S 2, · · · , S k) are the frequencies of the corresponding class in the perturbed data. Here, S ∼Multinomial(Λ1, Λ2, · · · , Λk), where Λ = PΠ (Λ := (Λ1, Λ2, · · · , Λk), Π := (Π1, Π2, · · · , Πk)). If we want to treat Z as the original data, we must have Π = Λ = PΠ. But Π is generally unknown to the one, who is masking the data. However, one can estimate Π from the original data with T/n where n is the total sample size. If we want S/n to be an unbiased estimator of Π, we must have, due to Equation (1), (2) E[S | T] = T/n , or equivalently, PT = T. E[S | T] = T/n , or equivalently, PT = T. (2) Gouweleeuw et al. 1. Introduction , ck, then we assume without loss of generality X(B) = c1 and the frequencies of the categories in the data-set are T1, T2, . . . , Tk respectively. If T1 = 1, i.e., only X(B) has category c1, the intruder can guess the row of his target unit with certainty. If T1 is small, the intruder knows that his target unit is definitely one of the T1 many units and then taking into consideration other information, he may successfully identify the row of his target unit or make a correct guess. Thus, in this case, the variable information must be suppressed before releasing the data. One way to do that is to completely erase the variable but that is not desirable to the statistician. In case there exists some identifying attribute in a data-set, it is perturbed before releasing publicly. Bethelem et al. (1990), Trabelski et al. (2009), Nayak et al. (2016) have discussed different ways of perturbing such attributes. However, the most common practise of perturbing discrete data is the post randomisation method which will be discussed in the following paragraphs. The aim of the research problem is to find an ideal way of perturbing such attribute values that may result in least possible loss of information, provided the data is secured, i.e., looking at the released data, the intruder cannot guess the row of his target 24 International Journal of Statistics and Probability Vol. 8, No. 3; 2019 http://ijsp.ccsenet.org 3. Model, Assumptions and Results As discussed earlier, the goal of the paper is to find out a method by which a data can be perturbed ensuring as much se- curity as possible. Since security is an abstract term, we limit ourselves to ensure that the measure, given by Equation (4)) holds for low values of ξ. Smaller the value of ξ, better the security of the data. Let us denote, by R1(a, t), the probability of correctly identifying the individual from released data given S 1 = a and the frequency distribution of X given by t := (t1, t2, · · · , tk). In other words, R1(a, t) = P[CM | S 1 = a, T = t], a ≥0, t ∈Rk. (5) (5) If R1(a, t) is bounded by ξ for any t, then note that R1(a) = P[CM | S 1 = a], R1(a) = P[CM | S 1 = a], (6) (6) is bounded by ξ for any a ≥0, which signifies that the probability of correctly identifying an individual is less than ξ, no matter how small or large the frequency of category c1 is, in the released data. R1(a, t) is used instead of R1(a) because it is hard to calculate the probability if t is not known. Note that, CM stands for “Correct Match” in the above equations (5) (6). Recall that if we use, IFPR block diagonal matrix to perturb X, the category c1 may get changed to one of {c1, c2, . . . , ck1}, k1 ≥2 with positive probability. let us denote αi = p1i, βi = αi 1−αi for i ∈{1, 2, . . . , k1}. Observe that, R1(a, t) can be re-written as R1(a, t) = P[CM | S 1 = a, Z(B) = c1, T = t]P[Z(B) = c1 | S 1 = a, T = t] +P[CM | S 1 = a, Z(B) , c1, T = t]P[Z(B) , c1 | S 1 = a, T = t]. By our assumption, since the intruder searches his target unit B among the ones with category c1, P[CM | S 1 = a, Z(B) , c1, T = t] = 0. Again, since, the intruder is assumed to choose randomly one unit among a units to be B, P[CM | S 1 = a, Z(B) = c1, T = t] = 1 a for any t. 2. Our Approach As mentioned earlier, our framework is similar to that of Nayak et al. (2016). From the intruder’s point of view, we assume that as he gets access of the released data {Z1, Z2, · · · , Zn}, he checks the rows for which Zi = c1 for {i = 1, 2, · · · , n}. Let S 1 be the total number of units having class c1. If S 1 = 0, intruder stops searching for his target unit B in the data-set. If S 1 = a for some a > 0, he selects one unit randomly among these a individuals and concludes that to be his target unit B. Under this assumption, we discuss how to choose the parameter θ of the IFPR block diagonal matrix ( See Equation (3)), depending on T1, so that the probability of correctly identifying unit B is less than some specified 0 < ξ < 1. Our method is described in the following paragraph. 25 International Journal of Statistics and Probability Vol. 8, No. 3; 2019 http://ijsp.ccsenet.org Fix a 0 < ξ < 1. Note that, if T1 > 1 ξ , then there is no need for obfuscation as the intruder can choose one unit randomly and conclude it as his target unit B. Since, in the original data, the probability of correctly identifying B is 1/T1, if T1 > 1 ξ , the probability is less than ξ. This is quite intuitive since identification risk is a problem associated with low-frequency classes. If T1 ≤1 ξ , then we find k1 = K1(ξ, T1) classes ( where the function K1 is discussed in Sec. 3 ) such that for each of these classes {c1, c2, · · · , ck1}, T j ≥T1 for each j ∈{1, 2, · · · , k1}. Such an event is usually feasible for moderate values of ξ as T1 usually has small values. If such classes are available, we can have any desired level of security, i.e., for any fixed 0 < ξ < 1, there exists a corresponding θ⋆such that if the data is perturbed with matrix P(θ⋆), Equation (4) holds. The choice of θ⋆is discussed in Section 3. If, however, such classes are not available, we can find the integer n⋆such that 1 n⋆≤ξ < 1 n⋆−1. 2. Our Approach Since k1 classes are not available such that T j ≥T1 for each j ∈{1, 2, · · · , k1}, we now set ξ1 = 1 n⋆−1 and try to find k1 1 = K1(T1, ξ1) classes such that T j ≥T1 for each j ∈{1, 2, · · · , k1 1}. If we fail, we next try for ξ2 = 1 n⋆−2 and so on until we get a success for some ξl = 1 n⋆−l. Since for ξ = 1 n⋆−l, there exists kl 1 = K1(T1, ξl) classes such that T j ≥T1 for each j ∈{1, 2, · · · , kl 1}, we can now find a θ⋆, such that if the data is perturbed with P(θ⋆), then Equation (4) is satisfied for any 1 n⋆−l < ξ < 1. According to Nayak et al. (2016), there is always a solution for ξ ≥1 3 which implies there exists a solution for 1 3 ≤ξ < 1 2, i.e., n⋆can take a minimum value 3. However, n⋆can take higher values in many cases. 2. Our Approach Since k1 classes are not available such that T j ≥T1 for each j ∈{1, 2, · · · , k1}, we now set ξ1 = 1 n⋆−1 and try to find k1 1 = K1(T1, ξ1) classes such that T j ≥T1 for each j ∈{1, 2, · · · , k1 1}. If we fail, we next try for ξ2 = 1 n⋆−2 and so on until we get a success for some ξl = 1 n⋆−l. Since for ξ = 1 n⋆−l, there exists kl 1 = K1(T1, ξl) classes such that T j ≥T1 for each j ∈{1, 2, · · · , kl 1}, we can now find a θ⋆, such that if the data is perturbed with P(θ⋆), then Equation (4) is satisfied for any 1 n⋆−l < ξ < 1. According to Nayak et al. (2016), there is always a solution for ξ ≥1 3 which implies there exists a solution for 1 3 ≤ξ < 1 2, i.e., n⋆can take a minimum value 3. However, n⋆can take higher values in many cases. Fix a 0 < ξ < 1. Note that, if T1 > 1 ξ , then there is no need for obfuscation as the intruder can choose one unit randomly and conclude it as his target unit B. Since, in the original data, the probability of correctly identifying B is 1/T1, if T1 > 1 ξ , the probability is less than ξ. This is quite intuitive since identification risk is a problem associated with low-frequency classes. If T1 ≤1 ξ , then we find k1 = K1(ξ, T1) classes ( where the function K1 is discussed in Sec. 3 ) such that for each of these classes {c1, c2, · · · , ck1}, T j ≥T1 for each j ∈{1, 2, · · · , k1}. Such an event is usually feasible for moderate values of ξ as T1 usually has small values. If such classes are available, we can have any desired level of security, i.e., for any fixed 0 < ξ < 1, there exists a corresponding θ⋆such that if the data is perturbed with matrix P(θ⋆), Equation (4) holds. The choice of θ⋆is discussed in Section 3. If, however, such classes are not available, we can find the integer n⋆such that 1 n⋆≤ξ < 1 n⋆−1. 3. Model, Assumptions and Results Thus, R1(a, t) = 1 aP[Z(B) = c1 | S 1 = a, T = t]. (7) (7) Again, we have, P[Z(B) = c1, S 1 = a, | T = t] = α1 ∑k1 ∏ i=1 (T ⋆ i ai ) αai i (1 −αi)T ⋆ i −ai = α1[ k1 ∏ i=1 (1 −αi)T ⋆ i ] ∑k1 ∏ i=1 (T ⋆ i ai ) βai i (8) (8) where T ⋆ 1 = T1 −1, T ⋆ i = Ti, i ≥2 and the sum is over all integer-valued a1, a2, · · · ak1 such that 0 ≤ai ≤T ⋆ i and where T ⋆ 1 = T1 −1, T ⋆ i = Ti, i ≥2 and the sum is over all integer-valued a1, a2, · · · ak1 such that 0 ≤ai ≤T ⋆ i and 26 International Journal of Statistics and Probability Vol. 8, No. 3; 2019 International Journal of Statistics and Probability Vol. 8, No. 3; 2019 http://ijsp.ccsenet.org ∑ai = a −1. We denote the sum by Σa−1 ∑ai = a −1. 3. Model, Assumptions and Results If α1 ≥α j, i.e., β1 ≥βj for any j = 1, 2, · · · , k1, then R1(1, t) ≥R1(a, t) for any t, a > 1, where R1(a, t) is given by Equation (10). Assuming Theorem 3.1 holds, proving Equation (4) is equivalent to prove that R1(1, t) ≤ξ for any t. For this condition to hold, we must carefully choose the parameter θ in (1). Due to Nayak et. al. (2016), we have, R1(1, T) = T1 + θ T1 −θ k1 ∑ i=2 θTi (k1 −1)Ti −θ  −1 = (T1 −θ) T1(T1 −θ) + θ2 k1 ∑ i=2 Ti (k1 −1)Ti −θ  −1 ≤ T1 −θ T1(T1 −θ) + θ2 = ψ(T1, θ) (11) (11) To proceed further we also need the following lemma, proof of which is deferred in Appendix Section. Lemma 3.2. For any fixed 0 < ξ < 1, there exists a θ⋆∈(0, T1) such that ψ(θ, T1) ≤ξ. Lemma 3.2. For any fixed 0 < ξ < 1, there exists a θ⋆∈(0, T1) such that ψ(θ, T1) ≤ξ. Lemma 3.2. For any fixed 0 < ξ < 1, there exists a θ⋆∈(0, T1) such that ψ(θ, T1) ≤ξ. For Theorem 3.1 to hold, in an IFPR block diagonal matrix, we must have T1−θ θ ≥ θ (k1−θ)T1−θ which leads to the condition, θ ≤ T1 1+ T1 T j(k1−1) , i.e., k1 −1 ≥ θ T1−θ T1 T j . Note that, if k1 −1 ≥ θ T1−θ, and T1 T j ≤1, k1 −1 ≥ θ T1−θ T1 T j . Hence, it is enough to find K(θ, T1) = 1 + θ T1−θ = T1 T1−θ for Theorem 3.1 to hold. Again, θ is chosen by solving ψ(θ, T1) = ξ. Thus, for fixed ξ and T1 we have a θ and a corresponding K1(ξ, T1) which is the largest integer contained in K(θ, T1). K1(ξ, T1) is the minimum number of categories required to form the block containing c1. For some possible choices of ξ and some possible values of T1, the value of K1(ξ, T1) is calculated and given in Table 1. While choosing the block size, one must note that the block size k1 must be larger than or at least equal to K1(ξ, T1) to ensure Equation (4). 3. Model, Assumptions and Results We denote the sum by Σa−1 P[Z(B) , c1, S 1 = a, | T = t] = (1 −α1)[ k1 ∏ i=1 (1 −αi)T ⋆ i ]Σa (9) Equation (8) and (9) implies that P[S 1 = a | T = t] = k1 ∏ i=1 (1 −αi)T ⋆ i (α1Σa−1 + (1 −α1)Σa) and since P[Z(B) = c1 | S 1 = a, T = t] = P[Z(B) = c1, S 1 = a | T = t] P[S 1 = a | T = t] f E i (7) fi ll h P[Z(B) , c1, S 1 = a, | T = t] = (1 −α1)[ k1 ∏ i=1 (1 −αi)T ⋆ i ]Σa (9) (9) [ (B) 1, 1 , | ] ( 1)[ ∏ i=1 ( i) ] a ( ) Equation (8) and (9) implies that k1 ∏ T ⋆ Equation (8) and (9) implies that Equation (8) and (9) implies that P[S 1 = a | T = t] = k1 ∏ i=1 (1 −αi)T ⋆ i (α1Σa−1 + (1 −α1)Σa) and since P[Z(B) = c1 | S 1 = a, T = t] = P[Z(B) = c1, S 1 = a | T = t] P[S 1 = a | T = t] from Equation (7) , we finally have, R1(a, t) = 1 a [ α1Σa−1 α1Σa−1+(1−α1)Σa ] = 1 a [ 1 + 1 β1 Σa Σa−1 ]−1 . (10) R1(a, t) = 1 a [ α1Σa−1 α1Σa−1+(1−α1)Σa ] = 1 a [ 1 + 1 β1 Σa Σa−1 ]−1 . (10) Nayak et al.(2016) observed that although it seems intuitive that R1(1, t) ≥R1(a, t) for any t, a > 1 there are certain cases it does not hold true. However, they proved that if α1 ≥α j, i.e., β1 ≥βj for all j = 1, 2, · · · , k1, then R1(1, t) ≥R1(2, t) for any t. Intuitively, if β1 is highest, i.e., the odds that c1 goes to any category other than c1, then the risk of disclosure should be maximum if a = 1. We checked that this is quite true which leads us to our first result, stated in the following theorem and the proof is given in Appendix Section. Theorem 3.1. To illustrate the process, we simulate a sample of size n = 2000 from k = 8 categories such that the probability of falling into a category is given by the vector Π = (0.001, 0.1, 0.2, 0.05, 0.12, 0.13, 0.301, 0.098). The sample has frequency distribution given by Table 2. Table 2. Table showing frequencies of Categories for True Data from Simulated data-set Category T 1 2 2 205 3 431 4 106 5 230 6 221 7 611 8 194 Two units in the data-set have Category 1, one of which is unit B = 780. Since T1 = 2, the probability of Correct Match from true data is 0.5 which is very high. We want this probability to be lower, say below ξ = 0.1. So, we transform the data to Z using the IPRAM method with a transition matrix P. To choose an ideal P we apply the procedure of this paper. From Table 1, we get the required block size is 6. So, we would apply transition to the first k1 = 6 categories with the lowest probability of occurrence and do not alter the categories for the rest 2 categories. To solve for h(θ) = ξ, we have θ⋆= 1.656854 which gives the transition matrix, P =  0.172 0.166 0 0.166 0.166 0.166 0 0.166 0.002 0.992 0 0.002 0.002 0.002 0 0.002 0 0 1 0 0 0 0 0 0.003 0.003 0 0.984 0.003 0.003 0 0.003 0.001 0.001 0 0.001 0.993 0.001 0 0.001 0.001 0.001 0 0.001 0.001 0.993 0 0.001 0 0 0 0 0 0 1 0 0.002 0.002 0 0.002 0.002 0.002 0 0.991  Using this transition matrix we ran 1000 simulations to get 1000 different Zs. The mean squared estimation error for each category is given by E = (4.9350·10−07, 7.6125·10−07, 0.0000, 7.4300·10−07, 8.8550·10−07, 7.8375·10−07, 0.0000, 8.5550· 10−07) which is quite low and the average probability of correct match in 1000 simulations is 0.07639286 < 0.1. The process thus seems to work well for simulated data. Using this transition matrix we ran 1000 simulations to get 1000 different Zs. The mean squared estimation error for each category is given by E = (4.9350·10−07, 7.6125·10−07, 0.0000, 7.4300·10−07, 8.8550·10−07, 7.8375·10−07, 0.0000, 8.5550· 10−07) which is quite low and the average probability of correct match in 1000 simulations is 0.07639286 < 0.1. The process thus seems to work well for simulated data 4. Simulation Results To illustrate the process, we simulate a sample of size n = 2000 from k = 8 categories such that the probability of falling into a category is given by the vector Π = (0.001, 0.1, 0.2, 0.05, 0.12, 0.13, 0.301, 0.098). The sample has frequency distribution given by Table 2. 27 International Journal of Statistics and Probability Vol. 8, No. 3; 2019 http://ijsp.ccsenet.org Table 1. Showing minimum block size required for some possible choices of security level ξ and some possible values of class frequency T1 HHHHH T1 ξ 0.1 0.125 0.15 0.175 0.2 0.25 0.3 1 11 9 8 7 6 5 5 2 6 5 5 4 4 3 3 3 5 4 3 3 3 2 2 4 4 3 3 2 2 2 2 5 3 3 2 2 2 2 2 6 3 2 2 2 2 2 2 7 2 2 2 2 2 2 2 8 2 2 2 2 2 2 2 9 2 2 2 2 2 2 2 10 2 2 2 2 2 2 2 HHHHH T1 ξ 0.1 0.125 0.15 0.175 0.2 0.25 0.3 1 11 9 8 7 6 5 5 2 6 5 5 4 4 3 3 3 5 4 3 3 3 2 2 4 4 3 3 2 2 2 2 5 3 3 2 2 2 2 2 6 3 2 2 2 2 2 2 7 2 2 2 2 2 2 2 8 2 2 2 2 2 2 2 9 2 2 2 2 2 2 2 10 2 2 2 2 2 2 2 HHHHH T1 ξ 0.1 0.125 0.15 0.175 0.2 0.25 0.3 1 11 9 8 7 6 5 5 2 6 5 5 4 4 3 3 3 5 4 3 3 3 2 2 4 4 3 3 2 2 2 2 5 3 3 2 2 2 2 2 6 3 2 2 2 2 2 2 7 2 2 2 2 2 2 2 8 2 2 2 2 2 2 2 9 2 2 2 2 2 2 2 10 2 2 2 2 2 2 2 Table 2. Table showing frequencies of Categories for True Data from Simulated data-set References Bethlehem, J. G., & Keller, W. J., & Pannekoek, J. (1990). Disclosure Control of Microdata, Journal of American Statistical Association. Fuller, W. A. (1993). Masking Procedures for Microdata Disclosure Limitation Journal of Official Statistics (pp. 383-406). Gouweleeuw, J. M., & Kooiman, P., & de Wolf, P. P. (1998). Post Randomisation for Statistical Disclosure Control: Theory and Implementation, Journal Of Official Statistics. Nayak, T. K., & Adeshiyan, S. A. (2015). On invariant Post Randomization for Statistical Dislosure Control, International Statistical Review. Nayak, T. K., & Adeshiyan, S. A., & Zhang, C. (2016). A Concise Theory of Randomized Response Techniques for Priva- cy and Confidentiality Protection. Handbook of Statistics, 34, 273-286. https://doi.org/10.1016/bs.host.2016.01.015 Nayak, T. K., & Zhang, C., & You, J. (2016). Measuring Identification Risk in Microdata Release and Its Control by Post-Randomization, Center for Disclosure Avoidance Research U.S. Census Bureau Washington DC 20233. Trabelsi, S., & Salzgeber, V., & Bezzi, M., & Montagnon, G. (2009) Data Disclosure Risk Evaluation, IEEE Xplore. https://doi.org/10.1109/CRISIS.2009.5411979 Appendix Proof to Theorem 3.1 5. Conclusion The method works fine in most practical cases, because, in general, since we want to obfuscate categories with low frequency, there will be sufficient number of categories with higher frequency values than them. Accordingly, the security level can be increased. However, the greatest drawback of this method of obfuscation is that we have assumed the game of the intruder, i.e., it 28 International Journal of Statistics and Probability http://ijsp.ccsenet.org Vol. 8, No. 3; 2019 selects one of the units with the desired categorical value randomly looking at the obfuscated data. But this is not expected to happen since in most cases there will be many regressive variables associated and the selection will not be, in general, random. This problem was also discussed in Trabelski et al. (2009). selects one of the units with the desired categorical value randomly looking at the obfuscated data. But this is not expected to happen since in most cases there will be many regressive variables associated and the selection will not be, in general, random. This problem was also discussed in Trabelski et al. (2009). However, if the model assumptions hold true, the discussed method is successful in giving a better security. Proof to Theorem 3.1 To prove the result, we need to show R1(a + 1, T) ≤R1(1, T), i.e., 1 a+1(1 + Σa+1 Σaβ1 )−1 ≤(1 + Σ β1 )−1 which leads us to check an equivalent statement, ˜Σa+1 −Σ˜Σa + β1 ˜Σa ≥0 (12) To prove the result, we need to show R1(a + 1, T) ≤R1(1, T), i.e., 1 a+1(1 + Σa+1 Σaβ1 )−1 ≤(1 + Σ β1 )−1 which leads us to check an equivalent statement, ˜Σ 1 −Σ˜Σ + β1 ˜Σ ≥0 (12) ˜Σa+1 −Σ˜Σa + β1 ˜Σa ≥0 (12) (12) where ˜Σa = a!Σa (Σa as defined in Equation (8) and (9)). Thus, we will need to check if 12 holds for all a and all k1 to prove Theorem 3.1. We will prove this result by a two dimensional induction procedure. First, we show that the statement is true for k1 = 2 for all a ∈N, then we show that if the statement is true for k1 = k10, then it is true for k1 = k10 + 1 for all a. Since, Σ1 = ∑T ⋆ i βi and Case: k1 = 2: Since, Σ1 = ∑T ⋆ i βi and Case: k1 = 2: Since, Σ1 = ∑T ⋆ i βi and Case: k1 = 2: Since, Σ1 = ∑T ⋆ i βi and ˜Σa = a ∑ s=0 (a s ) ∑ i1,i2 T ⋆ i1 (T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)T ⋆ i2 (T ⋆ i2 −1) · · · (T ⋆ i2 −a −s)βs i1βa−s i2 We have, We have, ˜ΣaΣ1 = ∑a s=0 (a s ) ∑ i1,i2 T ⋆ i1 2(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)T ⋆ i2 (T ⋆ i2 −1) · · · (T ⋆ i2 −a −s)βs+1 i1 βa−s i2 + ∑a s=0 (a s ) ∑ i1,i2 T ⋆ i1 (T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)T ⋆ i2 2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s)βs i1βa−s+1 i2 Writing Σa+1 similarly, we note that there are a + 2 terms in the expansion of ˜Σa+1 −Σ˜Σa + β1 ˜Σa. Proof to Theorem 3.1 First term = (a+1 0 ) ∑k1 i2=1 T ⋆ i2 (T ⋆ i2 −1) · · · (T ⋆ i2 −a)βa+1 i2 − (a 0 ) ∑k1 i2=1 T ⋆ i2 2(T ⋆ i2 −1) · · · (T ⋆ i2 −a + 1)βa+1 i2 +a (a 0 ) ∑k1 i2=1 T ⋆ i2 (T ⋆ i2 −1) · · · (T ⋆ i2 −a + 1)βa i2β1 = a ∑k1 i2=1 T ⋆ i2 (T ⋆ i2 −1) · · · (T ⋆ i2 −a + 1)βa i2(β1 −βi2) ( 0 ) ∑ i2=1 i2 ( i2 ) ( i2 )βi2 ( 0 ) ∑ i2=1 i2 ( i2 ) ( i2 )βi2 +a (a 0 ) ∑k1 i2=1 T ⋆ i2 (T ⋆ i2 −1) · · · (T ⋆ i2 −a + 1)βa i2β1 = a ∑k1 i2=1 T ⋆ i2 (T ⋆ i2 −1) · · · (T ⋆ i2 −a + 1)βa i2(β1 −βi2) ( 0 ) ∑ i2=1 i2 ( i2 ) ( i2 )βi2β = a ∑k1 i2=1 T ⋆ i2 (T ⋆ i2 −1) · · · (T ⋆ i2 −a + 1)βa i2(β1 −βi2) For s = 1, 2, · · · a, For s = 1, 2, · · · a, 29 Vol. 8, No. Proof to Theorem 3.1 3; 2019 International Journal of Statistics and Probability http://ijsp.ccsenet.org = (a+1 s ) ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)T ⋆ i2(T ⋆ i2 −1) · · · (T ⋆ i2 −a + 1 −s + 1)βs i1βa+1−s i2 − (a s ) ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)T ⋆ i2 2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s + 1)βs i1βa+1−s i2 − ( a s−1 ) ∑ i1,i2 T ⋆ i1 2(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 2)T ⋆ i2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s)βs i1βa+1−s i2 +aβ1 (a s ) ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)T ⋆ i2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s + 1)βs i1βa−s i2 = (a s ) ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)T ⋆ i2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s + 1)(−a −s)βs i1βa+1−s i2 − ( a s−1 ) (s −1) ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 2)T ⋆ i2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s)βs i1βa+1−s i2 +aβ1 (a s ) ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)T ⋆ i2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s + 1)βs i1βa−s i2 [Using Pascal’s rule (a+1 s ) = (a s ) + ( a s−1 ) ] [Using Pascal’s rule (a+1 s ) = (a s ) + ( a s−1 ) ] = a (a s ) ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)T ⋆ i2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s + 1)βs i1βa−s i2 (β1 −βi2) + (a s ) sβ1 ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)T ⋆ i2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s + 1)βs i1βa−s i2 − ( a s−1 ) (s −1) ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 2)T ⋆ i2 2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s)βs i1βa+1−s i2 ≥ (a s ) sβ1 ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)T ⋆ i2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s + 1)βs i1βa−s i2 − ( a s−1 ) (s −1) ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s −1 + 1)T ⋆ i2 2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −(s −1) + 1)βs i1βa−(s−1) i2 [Since, β1 ≥βi∀i] = a (a s ) ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)T ⋆ i2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s + 1)βs i1βa−s i2 (β1 −βi2) + (a s ) sβ1 ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)T ⋆ i2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s + 1)βs i1βa−s i2 − ( a s−1 ) (s −1) ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 2)T ⋆ i2 2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s)βs i1βa+1−s i2 ≥ (a s ) sβ1 ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)T ⋆ i2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −s + 1)βs i1βa−s i2 − ( a s−1 ) (s −1) ∑ i1,i2 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s −1 + 1)T ⋆ i2 2(T ⋆ i2 −1) · · · (T ⋆ i2 −a −(s −1) + 1)βs i1βa−(s−1) i2 [Since, β1 ≥βi∀i] In the last expression, let us denote the first term by Term(s, β1) and the second term by Term(s −1, β). (s + 1)th term Proof to Theorem 3.1 Since for any {a1, a2, · · · , ak1 ≥0, ∑k1 i=1 ai = a : a! a1!a2!···ak1! = a! a!!(a−a1)! (a−a1)! a2!a3!···ak1!}, we can write, ˜Σa = a ∑ s=0 a! s!(a −s)! k1 ∑ i1=1 T ⋆ i1 (T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1 ˜Σ(a−s,k10) ˜Σa = ∑ a1+a2+···+ak1=a a! a1! · · · ak1! ∑ i1,i2···,ik1 T ⋆ i1 . . . (T ⋆ i1 −a1 + 1) . . . T ⋆ ik1 . . . (T ⋆ ik1 −ak1 + 1)βa1 i1 βa2 i2 . . . β ak ik Since for any {a1, a2, · · · , ak1 ≥0, ∑k1 i=1 ai = a : a! a1!a2!···ak1! = a! a!!(a−a1)! (a−a1)! a2!a3!···ak1!}, we can write, ˜Σa = ∑ a1+a2+···+ak1=a a! a1! · · · ak1! ∑ i1,i2···,ik1 T ⋆ i1 . . . (T ⋆ i1 −a1 + 1) . . . T ⋆ ik1 . . . (T ⋆ ik1 −ak1 + 1)βa1 i1 βa2 i2 . . . β ak1 ik1 Since for any {a1, a2, · · · , ak1 ≥0, ∑k1 i=1 ai = a : a! a1!a2!···ak1! = a! a!!(a−a1)! (a−a1)! a2!a3!···ak1!}, we can write, ˜Σa = a ∑ s=0 a! s!(a −s)! k1 ∑ i1=1 T ⋆ i1 (T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1 ˜Σ(a−s,k10) where ˜Σ(s,k10) = s!Σs for k10 categories instead of k1 = k10 + 1 categories. Like before, we write down the terms of ˜Σa+1 −Σ˜Σa + β1 ˜Σa. where ˜Σ(s,k10) = s!Σs for k10 categories instead of k1 = k10 + 1 categories. Like before, we write down the terms of ˜Σa+1 −Σ˜Σa + β1 ˜Σa. Proof to Theorem 3.1 Note that since β1 ≥βi∀i Term(s, β1) −Term(s, β) ≥0. In the last expression, let us denote the first term by Term(s, β1) and the second term by Term(s −1, β). Note that since β1 ≥βi∀i Term(s, β1) −Term(s, β) ≥0. (a + 2)th term = (a + 1 a + 1 ) k1 ∑ i1=1 T ⋆ i1 (T ⋆ i1 −1) · · · (T ⋆ i1 −a)βa+1 i1 − (a a ) k1 ∑ i1=1 T ⋆ i1 2(T ⋆ i1 −1) · · · (T ⋆ i1 −a + 1)βa+1 i1 Thus, it can be clearly seen that, ˜Σa+1 −Σ1 ˜Σa + β1 ˜Σa Σa+1 −Σ1Σa + β1Σa ≥Term(1, β1) + Term(2, β1) −Term(1, β) + Term(3, β1) −Term(2, β) + · · · + Term(a, β1) −Term(a −1, β) + ( (a+1 a+1 ) ∑k1 i1=1 T ⋆ i1 (T ⋆ i1 −1) · · · (T ⋆ i1 −a)βa+1 i1 ) −Term(a, β) ≥0 Hence, (12) is true for k1 = 2 for any a. Now, let it be true for some k1 = k10 , k10 ∈{2, 3, . . . }. We will show then that (12) is true for k1 = k10 + 1. Hence, (12) is true for k1 = 2 for any a. Now, let it be true for some k1 = k10 , k10 ∈{2, 3, . . . }. We will show then that (12) is true for k1 = k10 + 1. Case: k1 = k10 + 1: The general expression for ˜Σa can be given by the following expression. Case: k1 = k10 + 1: The general expression for ˜Σa can be given by the following expression. ˜Σa = ∑ a1+a2+···+ak1=a a! a1! · · · ak1! ∑ i1,i2···,ik1 T ⋆ i1 . . . (T ⋆ i1 −a1 + 1) . . . T ⋆ ik1 . . . (T ⋆ ik1 −ak1 + 1)β Since for any {a1, a2, · · · , ak1 ≥0, ∑k1 i=1 ai = a : a! a1!a2!···ak1! = a! a!!(a−a1)! (a−a1)! a2!a3!···ak1!}, we can write, k ˜Σa = ∑ a1+a2+···+ak1=a a! a1! · · · ak1! ∑ i1,i2···,ik1 T ⋆ i1 . . . (T ⋆ i1 −a1 + 1) . . . T ⋆ ik1 . . . (T ⋆ ik1 −ak1 + 1)βa1 i1 βa2 i2 . . . Proof to Lemma 3.2 For T1 ≥2, ψ(1, θ) = ψ(T1, θ) iff 1−θ 1−θ+θ2 = T1−θ T1(T1−θ)+θ2 , i.e., θ = T1 T1+1. Consider, h(θ) =  ψ(1, θ) , if θ < T1 T1+1 ψ(T1, θ) , if θ ≥ T1 T1+1 For T1 ≥2, ψ(1, θ) = ψ(T1, θ) iff 1−θ 1−θ+θ2 = T1−θ T1(T1−θ)+θ2 , i.e., θ = T1 T1+1. Consider, For T1 ≥2, ψ(1, θ) = ψ(T1, θ) iff 1−θ 1−θ+θ2 = T1−θ T1(T1−θ)+θ2 , i.e., θ = T1 T1+1. Consider, For T1 ≥2, ψ(1, θ) = ψ(T1, θ) iff 1−θ 1−θ+θ2 = T1−θ T1(T1−θ)+θ2 , i.e., θ = T1 T1+1. Consider, h(θ) = ψ(1, θ) , if θ < T1 T1+1 h(θ) =  ψ(1, θ) , if θ < T1 T1+1 ψ(T1, θ) , if θ ≥ T1 T1+1 Note that, h(θ) is continuous and strictly decreasing in θ ∈(0, 1) with h(0) = 1, h(T1) = 0. By Mean Value Theorem, there must exist a θ⋆∈(0, T1) such that h(θ⋆) = ξ for 0 < ξ < 1. Note that, h(θ) is continuous and strictly decreasing in θ ∈(0, 1) with h(0) = 1, h(T1) = 0. By Mean Value Theorem, there must exist a θ⋆∈(0, T1) such that h(θ⋆) = ξ for 0 < ξ < 1. Proof to Theorem 3.1 Thus, we see (12) always holds and hence the proof. Thus the statement is true for k1 = k10 + 1 if true for k10 for any a ≥1. Thus, we see (12) always proof. Proof to Theorem 3.1 ∑k1 i1=1 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1 ˜Σ(a−s,k10 ) = (a s ) ∑k1 i1=1 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1(˜Σ(a+1−s,k10 ) −˜Σ(a−s,k10 ) ˜Σ(1,k10 ) + (a −s)β1 ˜Σ(a−s,k10 )) + ( a s−1 ) ∑k1 i1=1 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1(˜Σ(a−s−1,k10 ) − (a s ) ∑k1 i1=1 T ⋆ i1 2(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs+1 i1 (˜Σ(a−s,k10 ) +sβ1 (a s ) ∑k1 i1=1 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1(˜Σ(a−s,k10 ) [ Using Pascal’s rule] = (a s ) ∑k1 i1=1 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1(˜Σ(a+1−s,k10 ) −˜Σ(a−s,k10 ) ˜Σ(1,k10 ) + (a −s)β1 ˜Σ(a−s,k10 )) + ( a s−1 ) ∑k1 i1=1 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1(˜Σ(a−s−1,k10 ) − (a s ) ∑k1 i1=1 T ⋆ i1 2(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs+1 i1 (˜Σ(a−s,k10 ) +sβ1 (a s ) ∑k1 i1=1 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1(˜Σ(a−s,k10 ) [ Using Pascal’s rule] ≥+ ( a s−1 ) ∑k1 i1=1 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1(˜Σ(a−s−1,k10 ) − (a s ) ∑k1 i1=1 T ⋆ i1 2(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs+1 i1 (˜Σ(a−s,k10 ) +sβ1 (a s ) ∑k1 i1=1 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1(˜Σ(a−s,k10 ) [ Using Assumption on size k10] (a + 2)th term = k1 ∑ i1=1 T ⋆ i1 (T ⋆ i1 −1) · · · (T ⋆ i1 −a)βa+1 i1 ≥+ ( a s−1 ) ∑k1 i1=1 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1(˜Σ(a−s−1,k10 ) − (a s ) ∑k1 i1=1 T ⋆ i1 2(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs+1 i1 (˜Σ(a−s,k10 ) +sβ1 (a s ) ∑k1 i1=1 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1(˜Σ(a−s,k10 ) [ Using Assumption on size k10] (a + 2)th term = k1 ∑ i1=1 T ⋆ i1 (T ⋆ i1 −1) · · · (T ⋆ i1 −a)βa+1 i1 Summing all the elements we get, Summing all the elements we get, Σa+1 −ΣΣa + β1Σa = (a 1 ) ∑k1 i1=1 T ⋆ i1 βi1 ˜Σ(a−1,k10)(β1 −βi1) + (a 2 ) ∑k1 i1=1 T ⋆ i1 (T ⋆ i1 −1)β2 i1 ˜Σ(a−2,k10)(β1 −βi1) + · · · + (a a ) ∑k1 i1=1 T ⋆ i1 (T ⋆ i1 −1) · · · (T ⋆ i1 −a + 1)βa i1(β1 −βi1) ≥0 [ Since β1 ≥βi∀i] Thus the statement is true for k1 = k10 + 1 if true for k10 for any a ≥1. Proof to Theorem 3.1 First term = ˜Σ(a+1,k10) −∑k1 i1=1 T ⋆ i1 βi1 ˜Σ(a,k10) −˜Σ(a,k10) ˜Σ(1,k10) + aβ1 ˜Σ(a,k10) = (˜Σ(a+1,k10) −˜Σ(a,k10) ˜Σ(1,k10) + aβ1 ˜Σ(a,k10)) −∑k1 i1=1 T ⋆ i1 βi1 ˜Σ(a,k10) ≥−∑k1 i1=1 T ⋆ i1 βi1 ˜Σ(a,k10) [by Assumption over size k10] = ˜Σ(a+1,k10) −∑k1 i1=1 T ⋆ i1 βi1 ˜Σ(a,k10) −˜Σ(a,k10) ˜Σ(1,k10) + aβ1 ˜Σ(a,k10) = (˜Σ(a+1,k10) −˜Σ(a,k10) ˜Σ(1,k10) + aβ1 ˜Σ(a,k10)) −∑k1 i1=1 T ⋆ i1 βi1 ˜Σ(a,k10) ≥−∑k1 i1=1 T ⋆ i1 βi1 ˜Σ(a,k10) [by Assumption over size k10] ( (a+1,k10) (a,k10) (1,k10) β1 (a,k10)) ∑ i1=1 i1 βi1 (a,k10) ≥−∑k1 i1=1 T ⋆ i1 βi1 ˜Σ(a,k10) [by Assumption over size k10] For s = 1, 2, · · · a, For s = 1, 2, · · · a, 30 International Journal of Statistics and Probability Vol. 8, No. 3; 2019 International Journal of Statistics and Probability Vol. 8, No. 3; 2019 http://ijsp.ccsenet.org = (a+1)! s!(a−s+1)! ∑k1 i1=1 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1 ˜Σ(a−s+1,k10 ) − a! s!(a−s)! ∑k1 i1=1 T ⋆ i1 2(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs+1 i1 ˜Σ(a−s,k10 ) −( a! s!(a−s)! ∑k1 i1=1 T ⋆ i1(T ⋆ i1 −1) · · · (T ⋆ i1 −s + 1)βs i1 ˜Σ(a−s,k10 ))(˜Σ(1,k10 )) +aβ1 a! s!(a−s)! 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Habitat fragmentation and anthropogenic factors affect wildcat Felis silvestris silvestris occupancy and detectability on Mt Etna
Wildlife biology
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To cite this version: Stefano Anile, Sébastien Devillard, Bernardino Ragni, Francesco Rovero, Federica Mattucci, et al.. Habitat fragmentation and anthropogenic factors affect wildcat Felis silvestris silvestris occupancy and detectability on Mt Etna. Wildlife Biology, 2019, 2019 (1), pp.00561. ￿10.2981/wlb.00561￿. ￿hal- 02308851￿ Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use HAL Id: hal-02308851 https://hal.science/hal-02308851v1 Submitted on 8 Oct 2019 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Stefano Anile, Sebastien Devillard, Bernardino Ragni, Francesco Rovero, Federica Mattucci and Mario Lo Valvo Stefano Anile, (https://orcid.org/0000-0001-8871-9615) ✉ (stefanoanile@yahoo.it), Cooperative Wildlife Research Laboratory, Southern Illinois Univ., Carbondale, IL 62901, USA. – S. Devillard, Univ Lyon, Université Claude Bernard Lyon, CNRS, Laboratoire de Biométrie et Biologie Evolutive, Villeurbanne, France. – B. Ragni, Dipto di Chimica, Biologia e Biotecnologie, Univ. degli Studi di Perugia, Perugia, Italy. – F. Rovero, Sezione di Biodiversità Tropicale, MUSE – Museo delle Scienze di Trento, Trento, Italy. – F. Mattucci, Laboratorio di Genetica, Istituto Superiore per la Protezione e la Ricerca Ambientale (ISPRA), Bologna, Italy. – M. Lo Valvo, Dipto STEBICEF, Univ. di Palermo, Palermo, Italy. Knowledge of patterns of occupancy is crucial for planning sound biological management and for identifying areas which require paramount conservation attention. The European wildcat Felis silvestris is an elusive carnivore and is classified as ‘least concern’ on the IUCN red list, but with a decreasing population trend in some areas. Sicily hosts a peculiar wildcat population, which deserves conservation and management actions, due to its isolation from the mainland. Patterns of occupancy for wildcats are unknown in Italy, and especially in Sicily. We aimed to identify which ecological drivers deter- mined wildcat occurrence on Mt Etna and to provide conservation actions to promote the wildcats’ long-term survival in this peculiar environment. The genetic identity of the wildcat population was confirmed through a scat-collection which detected 22 different wildcat individuals. We analysed wildcat detections collected by 91 cameras using an occupancy frame work to assess which covariates influenced the detection (p) and the occupancy (ψ) estimates. We recorded 70 detections of the target species from 38 cameras within 3377 trap-days. Wildcat detection was positively influenced by the distance to the major paved roads and negatively affected by the presence of humans. Wildcat occupancy was positively associated with mixed forest and negatively influenced by pine forest, fragmentation of mixed forest and altitude. A spatially explicit pre- dicted occupancy map, validated using an independent dataset of wildcat presence records, showed that higher occupancy estimates were scattered, mainly located on the north face and at lower altitude. Habitat fragmentation has been claimed as a significant threat for the wildcat and this is the first study that has ascertained this as a limiting factor for wildcat occur- rence. Conservation actions should promote interconnectivity between areas with high predicted wildcat occupancy while minimising the loss of habitat. Habitat fragmentation and anthropogenic factors affect wildcat Felis silvestris silvestris occupancy and detectability on Mt Etna Authors: Stefano Anile, Sebastien Devillard, Bernardino Ragni, Francesco Rove Federica Mattucci, et. al. Source: Wildlife Biology, 2019(1) Published By: Nordic Board for Wildlife Research URL: https://doi.org/10.2981/wlb.00561 BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Complete website, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/terms-of-use. Usage of BioOne Complete content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use Wildlife Biology 2019: wlb.00561 doi: 10.2981/wlb.00561 © 2019 The Authors. This is an Open Access article Subject Editor: Luc Wauters. Editor-in-Chief: Ilse Storch. Accepted 15 May 2019 Wildlife Biology 2019: wlb.00561 doi: 10.2981/wlb.00561 © 2019 The Authors. This is an Open Access article Subject Editor: Luc Wauters. Editor-in-Chief: Ilse Storch. Accepted 15 May 2019 Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use Habitat fragmentation and anthropogenic factors affect wildcat Felis silvestris silvestris occupancy and detectability on Mt Etna nile, Sebastien Devillard, Bernardino Ragni, Francesco Rovero, Federica Mattucci o Lo Valvo Stefano Anile, Sebastien Devillard, Bernardino Ragni, Francesco Rovero, Federica Mattucci and Mario Lo Valvo Stefano Anile, Sebastien Devillard, Bernardino Ragni, Francesco Rovero, Federica Mattucci and Mario Lo Valvo 2006, Apostolico et al. 2015). However, the role of the fragmentation of preferred habitats on the popu- lation dynamics and population density, or at least on the occurrence of the wildcat, remains understudied. In addi- tion, habitat fragmentation might also have an effect on the degree of hybridization with domestic cats, which may have strong potential effects on wild carnivores (Morin et al. 2018). Hybridization with the domestic cat is indeed a direct threat to wildcat populations (Nowell and Jackson 1996, Macdonald and Loveridge 2010), that has been widely detected in Europe (Nowell and Jackson 1996, Steyer et al. 2018). Interestingly, a recent study (Beugin et al. 2018) sug- gests that in fragmented habitat, hybridization should be more common due to the higher presence of domestic cats when compared with continuous and large habitats. This is also supported by the findings of Nussberger et al. (2018) that hybrids were located more frequently at the periphery of the wildcat distribution range where forested habitat might be of lower quality. In this study, we used camera-trapping data from Mt Across Europe five distinct biogeographic groups of wildcat populations were identified (Mattucci et al. 2016) and likely originated during the protracted isolation which occurred in the late Pleistocene–early Holocene due to cli- mate fluctuations. A similar evolutionary patterns explained the main genetic/geographic substructure observed in Italy, with the exception two subpopulations found in the cen- tral Italian peninsula that likely resulted from a more recent adaptive processes (Mattucci et al. 2013). Human induced habitat fragmentation, instead, mainly conditioned the genetic clustering of the subspecies in France (Say  et  al. 2012) and Germany (Steyer et al. 2016) where wildcats split into two main groups during the last century.h The Sicilian wildcat population is therefore considered a distinct unit of conservation (Mattucci et al. 2013) and deserves particular management conservation actions since being isolated from the Italian peninsula precludes any immigration/recolonization process, decreasing the likeli- hood of long-term persistence (Anile et al. 2014). The main threats to the long-term survival of wildcat populations are loss and fragmentation of suitable habitat, hybridization with domestic cats Felis catus (Nowell and Jackson 1996, Macdonald and Loveridge 2010) and human-induced mor- tality (mainly road kills and poaching; Krone et al. 2008, Devillard et al. 2013, Falsone et al. 2014). Wildcat populations are usually found at low densities (less than 1/km2; Can et al. Stefano Anile, Sebastien Devillard, Bernardino Ragni, Francesco Rovero, Federica Mattucci and Mario Lo Valvo 2006; % of area occupied while accounting for an imperfect detection process) and to elucidate the role played by environmental variables in determining wildcat occupancy (Kilshaw et al. 2016). Knowing spatial variations in density and occurrence might help in deciphering the impacts of major threats to wildcat populations over their geographical distribution. The European wildcat Felis silvestris is a small (body mass = 2.4–6.2 kg) (Nowell and Jackson 1996, Macdonald and Loveridge 2010) carnivorous mammal currently listed in the HABITATS directive (European Council Directive 92/43/EEC, Appendix IV of 21 May 1992) and classified by the IUCN as ‘Least Concern’, but notably with a decreas- ing population trend (Yamaguchi et al. 2015) throughout most of its range, although wildcat distribution is currently expanding in some countries (Nussberger et al. 2018).i g g p Habitat fragmentation has been frequently considered (Nowell and Jackson 1996, Macdonald and Loveridge 2010) as a direct threat to wildcat populations in Europe, but its effect has not been investigated properly (Zanin et al. 2014). The current scientific knowledge on wildcat habitat require- ments is mainly based on radio-tracking studies conducted in Germany (Klar et al. 2008, Jerosch et al. 2010, 2017), Spain (Sarmento  et  al. 2006, Soto and Palomares 2013), Portugal (Monterroso et al. 2009), France (Germain et al. 2008, Beugin  et  al. 2016) and Scotland (Daniels  et  al. 2001). Radio-tracking is relatively expensive and invasive as it requires the live-trapping of wildcats (Bizzarri et al. 2010), therefore radio-tracking studies in Italy are rare (Anile et al. 2017) and have not addressed wildcat habitat requirements. According to radio-tracking studies, wildcats prefer forest habitats (Nowell and Jackson 1996, Daniels  et  al. 2001, Wittmer 2001), mainly deciduous (but see Klar et al. 2008, Lozano 2010). In Mediterranean areas shrub vegetation can also play an important role in the ecology of this spe- cies (Lozano et al. 2003, Monterroso et al. 2009). Moreover, open meadows can also be used by wildcats as hunting areas due to the abundance of suitable prey (Silva et al. 2012). Some studies have found that agricultural area can be used by wildcats (Lozano 2010), as long as these areas are linked to forest habitat (Jerosch et al. 2017). Overall, the wildcat is adapted to a wide range of different ecological scenarios and this remarkable capacity for adaptation is also reflected in the plasticity of its feeding behaviour (Malo et al. 2004, Lozano et al. Stefano Anile, Sebastien Devillard, Bernardino Ragni, Francesco Rovero, Federica Mattucci and Mario Lo Valvo planning sound biological management and for identifying areas which require paramount conservation attention for a given species. In the current, unprecedented extinction crisis, carnivorans and especially felids, are facing the highest extinction risks among mammals (Cardillo  et  al. 2004). Threats are even more important for populations on islands (Purvis  et  al. 2000), that likely suffer from a lack of connectivity with larger mainland source populations. Habitat destruction and fragmentation are the major causes of biodiversity loss worldwide (Krauss et al. 2010). In addition, an increasing human population can severely interfere with and disturb ecological processes (Cardillo et al. 2008, Pimm et al. 2014). In such a context of habitat change, knowledge of patterns of occupancy, and their ecological correlates are crucial for Within the family Felidae, the smallest species are understudied (Brodie 2009, Zanin  et  al. 2014, Anile and Devillard 2015), but efforts to correct this bias are increas- ing (Macdonald  et  al. 2010, Anile and Devillard 2018, Ruiz-Olmo et al. 2018). This lack of knowledge about the ecology of small felids might result in overlooking their eco- logical and conservation requirements, hence leading to an enhanced risk of extinction in the short term. Luckily, with the advent of camera-trapping (O’Connell et al. 2010) an increasing number of studies has been run worldwide to identify environmental and anthropogenic variables critical for determining the occurrence of a variety of felid species (Schuette et al. 2013, Andresen et al. 2014, Lesmeister et al. 2015, Hemami et al. 2018, Penjor et al. 2018, Rovero et al. 2018, Wang et al. 2018), including small ones (Galvez et al. This work is licensed under the terms of a Creative Commons Attribution 4.0 International License (CC-BY) < http:// creativecommons.org/licenses/by/4.0/ >. The license permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1 1 Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use 2013, Singh et al. 2014, Jennings et al. 2015). This approach has improved our knowledge of the ecology of felid species, enabling researchers to identify or predict areas of particular value for conservation purposes, and hence to provide sound management actions with the ultimate goal of increasing the likelihood of the long-term survival of these carnivorous spe- cies. has also been used in Scotland to estimate wildcat occupancy (McKenzie et al. Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use Stefano Anile, Sebastien Devillard, Bernardino Ragni, Francesco Rovero, Federica Mattucci and Mario Lo Valvo 2009, Soto and Palomares 2013, Anile et al. 2014, Gil-Sanchez et al. 2015), most of their activ- ity is nocturnal (Daniels et al. 2001, Germain et al. 2008) and they typically display elusive behaviour (i.e. human avoidance) (Piñeiro et al. 2012). However, the application of camera-trapping to study wildcats has overcome these con- straints (Anile et al. 2009), and camera-trapping surveys for wildcats have been used extensively in Spain (Sarmento et al. 2009, Soto and Palomares 2013, Gil-Sanchez et al. 2015), Scotland (Kilshaw and Macdonald 2011, Kilshaw et al. 2014, Littlewood et al. 2014), Turkey (Can et al. 2009), Germany (Beutel et al. 2017), the Netherlands (Canters et al. 2005) and Italy (Velli et al. 2015). Camera-trapping data have been analysed using capture–recapture (CR) (Can  et  al. 2009, Anile et al. 2012a) and spatially explicit capture–recapture (SECR) models (Anile et al. 2014, Kilshaw et al. 2014, Gil- Sanchez et al. 2015) to estimate wildcat population densities in numerous environments. Interestingly, camera-trapping In this study, we used camera-trapping data from Mt Etna (Sicily, Italy) to 1) identify ecological drivers and human-induced determinants to estimate wildcat occupancy (ψ) and detection (p); 2) develop a spatially explicit wild- cat occupancy model for the entire study area; 3) test the 2 robustness of this model against an independent dataset of wildcat records and 4) use the results to propose sound con- servation actions for the long-term persistence of this unique insular wildcat population in the peculiar ecosystem of the highest active volcano in Europe. Our a priori expectations, based on scientific literature and preliminary observations of wildcat records collected within our study area, were that 1) mixed forest should promote wildcat occupancy whereas pine forest are used less frequently; and 2) fragmentation of the preferred habitat should impact occupancy negatively. Additionally, we also expected that 3) human disturbance (in term of distance to major roads and human presence) should negatively affect wildcat detectability. and usually abundant at higher altitudes. Wildcat refuges are widely available in the form of cavities, characteristic of the volcanic soil, which also represent (due to water condensa- tion on the cavity walls) the only available source of water in summer. The park is divided into four main management units (zone A–D) according to different level of protection/activi- ties. In zone A vehicle access is subject to permission and restricted to unpaved roads used for forest management, sheep farming and tourism. Stefano Anile, Sebastien Devillard, Bernardino Ragni, Francesco Rovero, Federica Mattucci and Mario Lo Valvo ‘Integral reserve’ – zone A is subject to more constraints in order to ensure maximum protection of plant and animal species. ‘General reserve’ – zone B where educational activities and regular excursions are permitted. ‘Protection area’ – zone C, located close to population centres, where eco-friendly activities are encour- aged. ‘Pre-park area’ – zone D where the landscape is domi- nated by medium-sized traditional agricultural fields, mixed with wooded areas and human activities, should comply with the general purpose of the park. The most remote areas (zone A) of Mt Etna have been declared a World Heritage Site by UNESCO in 2013 (< https://whc.unesco.org/en/ list/1427 >). The most congested roads within the park serve to reach the two skiing centres located on the southern and north-eastern slope (SP. 92 and ‘Mareneve’, respectively). Other heavily congested roads, located on the north-west- ern slope of Mt Etna, are the SS. 284 which connects the town of Bronte with that of Randazzo, the SS. 120 which connects the town of Randazzo with that of Maniace, and the ‘Quota Mille’ which intersects the park at 1000 m a.s.l. on the north-eastern slope (Fig. 1). Currently Mt Etna is almost totally surrounded by unsuitable wildcat habitat with widespread towns and villages, in addition to the close pres- ence of a metropolitan city (Catania). Only a narrow strip (~30 km) of the northern slope is adjacent to the Nebrodi Park (~855 km2) where suitable wildcat habitat is present and wildcats are historically widespread. Unfortunately, this strip is intersected by congested roads where evidence of wildcat Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use Study area The Etna Regional Park (~590 km2) comprises the ecosys- tem of Mt Etna, Italy, with an altitude range from 550 up to ~3360 m a.s.l. The landscape is characterized by recent large lava flows and inactive secondary cones of different ages, intermixed with areas dominated by trees (Corsican pine Pinus laricio, different species of oak Quercus pubescens, Quercus ilex, chestnut Castanea sativa, aspen Populus tremu- lus, European beech Fagus sylvatica and Mt Etna broom Genista etniensis). Forest cover usually lies between 1000 and 2000 m a.s.l. and areas at higher altitude are characterized by low shrub vegetation. The most widespread habitat within the forest cover range consists of large woodland patches intermingled with relatively small open fields, sometimes surrounded or interrupted by lava flows of variable extents (Fig. 1). The climate is typically Mediterranean at the lower altitudes, with warm springs and hot dry summers. Rain- fall is concentrated during autumn and winter with, a yearly mean of 1000–1400 mm. Snow cover is common in winter Figure 1. Approximate distribution ranges of the European wildcat Felis silvestris for western Europe (A) and Etna Park (Sicily, Italy) (B), where we conducted the camera-trapping study in 2015–2016. Major paved roads (white lines) across the Etna Park and habitat vegetation classes (areamf = mixed forest; areapf = pine forest; areame = meadow; areash = shrub; arealf = lava flow; areaal = agricultural) are indicated. Figure 1. Approximate distribution ranges of the European wildcat Felis silvestris for western Europe (A) and Etna Park (Sicily, Italy) (B), where we conducted the camera-trapping study in 2015–2016. Major paved roads (white lines) across the Etna Park and habitat vegetation classes (areamf = mixed forest; areapf = pine forest; areame = meadow; areash = shrub; arealf = lava flow; areaal = agricultural) are indicated. Figure 1. Approximate distribution ranges of the European wildcat Felis silvestris for western Europe (A) and Etna Park (Sicily, Italy) (B), where we conducted the camera-trapping study in 2015–2016. Major paved roads (white lines) across the Etna Park and habitat vegetation classes (areamf = mixed forest; areapf = pine forest; areame = meadow; areash = shrub; arealf = lava flow; areaal = agricultural) are indicated. 3 3 Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use We used 18 digital cameras. Study area We angled cameras slightly downwards (15 ± 3°) to obtain a better view of the wildcat’s norma dorsalis (part of the coat that is useful for identifying individuals (Anile et al. 2014). Each camera was accommo- dated in an iron box, locked with a padlock and then tied to a tree (at 50 ± 10 cm from the ground) with a chain. We set cameras with a delay time of 10 min between successive bursts (n = 3) of photos. No lure or bait was used as an attrac- tant. For each camera we measured the width (width) of the road/trail where cameras were placed using a tape measure and the elevation (elevation) by using a GPS. Wildcat iden- tification was conducted according to the coat-colour and marking-system of the European wildcat developed by Ragni and Possenti (1996) and used in previous camera-trapping studies for the same study area (Anile et al. 2014). Follow- ing Anile  et  al. (2014), we also walked transects (n = 97; 1.23 ± 0.06 km mean ± SE; range = 402–2742 m) four times on a weekly basis between cameras to collect putative wild- cat scats (Fig. 2). For each fresh ‘putative’ wildcat scat, a small portion (~40 µg) was scratched from the surface and immediately stored in a small pipe (capacity ~12 ml) filled with 7 ml of 95% ethanol. Soon after the collection, the scat and the pipe were both stored in a thermic bag filled with replaceable ice. Finally, scat samples were stored at −20°C within four hours of finding. Samples were then genotyped at eleven feline unlinked autosomal microsatellites to ensure the genetic identity of the wildcat population sampled using cameras (Supplementary material Appendix 1). road-kills (Falsone et al. 2014) have been found, probably reducing dispersal movements of the species between the two parks. Camera trapping and scat-collection We deployed seven arrays of camera traps across Mt Etna from 2 July 2015 to 22 December 2015 (3 arrays with 15 cameras and 1 array with 12 cameras) and from 1 April 2016 to 1 July 2016 (2 arrays with 12 cameras and 1 array with 10 cameras), with the aim of surveying the widest presumed suitable area for wildcats (i.e. <2000 m a.s.l.) within the park (Fig. 2), as is usually done in camera trapping surveys on this and other felids (Anile and Devillard 2015, 2018). The dis- crepancy in the number of cameras used per array is because four cameras (array no. 4) were stolen during the study. However data from two cameras were partially retrieved before the theft. Cameras were located within public areas of the park (i.e. not on private property). We designed in GIS a grid with a cell size of 1 km that we randomly laid over the study area. Within each grid cell in each array, we then placed cameras as close as possible to the cell centroid on suitable sites for wildcat movements, i.e. along unpaved roads (>2 m) and trails (<2 m). This resulted in a mean spac- ing of 759.9 ± 24.5 m (± SE; range = 273–1663) between adjacent cameras, and at a mean elevation of 1415 ± 24.5 m a.s.l. (± SE; range = 1075–1874). Cameras were checked twice per week for camera functioning, batteries and data download. We aimed to collate at least 30 functional days at each camera; when cameras were not properly working (e.g. batteries to replace and human alteration such as covering or moving the camera), we accordingly accommodated the ‘capture’ matrix. Covariates 2018); 4) distanceMR (the straight-line distance between each camera and the closest major paved road): wildcat detections might decrease for those cam- eras located closer to the major roads. Indeed, it has been shown that wildcat ranging behaviour is seriously affected by the presence of such human structures (Klar et al. 2009, Mata et al. 2017, Planillo et al. 2018); 5) RAIh (the sum of all events with humans such as bikers, hikers, forest workers and mushroom collectors): wildcat detections can be nega- tively influenced by persistent use of the trails by humans (Piñeiro et al. 2012). to promoting wildcat occurrence in Mediterranean habitat (Lozano 2010). Additionally, meadows (areame; 29 km2 – 4.9%) can also be important for wildcats as hunting areas (Klar et al. 2008, Kilshaw et al. 2016). The Etna ecosystem is also characterized by the presence of extensive lava flows (arealf; 152 km2 – 26.1%) at higher altitudes (>2000) which become irregularly sparse at lower altitudes. Small agricul- tural lands (areaal; 97 km2 – 16.6%) can be tolerated by wildcats (Jerosch et al. 2017), whereas lands with persistent human presence (areahl; 3.5 km2 – 0.5%) are usually avoided (Klar et al. 2008, Silva et al. 2012). In our analysis we did not consider areahl and arearv (river vegetation; 0.5 km2 – 0.08%) as these habitat classes were not represented within our sampling area. p g For each camera (Supplementary material Appendix 3) we created a circular buffer with a radius of 750 m (hence we covered an area of ~1.7 km2 which corresponds to the minimum home-range for a wildcat in a Mediterranean habitat (Klar et al. 2008, Monterroso et al. 2009) and for each buffer/camera we extracted the following variables: 1) the area (% of km2) of each habitat class (areamf, areapf, areash, areame, arealf, areaal); 2) the number of the habi- tat classes (nclass) as a proxy for habitat structural complex- ity; 3) the number of patches per habitat class (npatchesmf, npatchespf, npatchessh, npatchesme, npatcheslf, npatchesal) as a proxy for fragmentation for each habitat class; 4) the num- ber of all patches npatchestot as a proxy of overall habitat fragmentation; 5) the length of major paved roads lengthMR (km) located within the circular buffer (Galvez et al. 2013); 6) distanceMR (km, the straight-line distance between each camera and the nearest major roads). Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use Covariates Spatial analysis of habitat covariates were performed using the software QGIS ver. 2.8 Wien (< www.qgis.org >) and its Figure 2. Results of combined sampling, using camera-trapping and scat-collection along transects (black lines), of European wildcat Felis silvestris on Mt Etna during 2015–2016. Black points = cameras positive for wildcat; white points = cameras negative for wildcats; num- bers = individual genotype detected. Figure 2. Results of combined sampling, using camera-trapping and scat-collection along transects (black lines), of European wildcat Felis silvestris on Mt Etna during 2015–2016. Black points = cameras positive for wildcat; white points = cameras negative for wildcats; num- bers = individual genotype detected. Figure 2. Results of combined sampling, using camera-trapping and scat-collection along transects (black lines), of European wildcat Felis silvestris on Mt Etna during 2015–2016. Black points = cameras positive for wildcat; white points = cameras negative for wildcats; num- bers = individual genotype detected. 4 Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use Table 1. Description of covariates used to model the detection (p) and the occupancy (ψ) process of the European wildcat Felis silvestris on Mt Etna (Sicily, Italy) from data collected using camera traps in 2015–2016. Model component Covariates Description Detection (p) width width of the road/trail where cameras were placed days camera trap effective working days array categorical factor indicating each array (1–7) distanceMR straight distance between each camera and the closest major road RAIh sum of all events with humans such as bikers, trekkers, forest workers and mushroom collectors Occupancy (ψ) areamf, areapf, areash, areame, arealf, areaal the area (% of km2) of each habitat class within each circular buffer nclass number of habitat class npatchesmf, npatchespf, npatchessh, npatchesme, npatcheslf, npatchesal number of patches per habitat class npatchestot sum of all patches lengthMR length of the major roads located within the circular buffer distanceMR straight distance between each camera and the closest major road elevation altitude of each camera plugins. We expected detection probability (p) to be influ- enced by the following covariates (Table 1): 1) width (width of the road/trail where cameras were placed): wildcat detec- tion on narrower trails might be enhanced (i.e. Covariates a wildcat moving through a smaller detection zone is more likely to be detected than when passing through a larger detection zone (Clare et al. 2015, Bahaa-el-din et al. 2016); 2) days (camera- trap days, i.e. from detection to retrieval or to last photo taken in case the camera malfunctioned): wildcat detections can decrease along with an increase in the effort if a trap-shy response occurs (Wegge et al. 2004); 3) array (a categori- cal factor created for each array of traps (1–7)): given that we used seven non simultaneous arrays, we expected that all parameters of interest for the detection process could also vary by arrays, hence this detection covariate was acting as a ‘random site effect’ (Harihar and Pandav 2012, Tan et al. 2017, Penjor et al. 2018); 4) distanceMR (the straight-line distance between each camera and the closest major paved road): wildcat detections might decrease for those cam- eras located closer to the major roads. Indeed, it has been shown that wildcat ranging behaviour is seriously affected by the presence of such human structures (Klar et al. 2009, Mata et al. 2017, Planillo et al. 2018); 5) RAIh (the sum of all events with humans such as bikers, hikers, forest workers and mushroom collectors): wildcat detections can be nega- tively influenced by persistent use of the trails by humans (Piñeiro et al. 2012). plugins. We expected detection probability (p) to be influ- enced by the following covariates (Table 1): 1) width (width of the road/trail where cameras were placed): wildcat detec- tion on narrower trails might be enhanced (i.e. a wildcat moving through a smaller detection zone is more likely to be detected than when passing through a larger detection zone (Clare et al. 2015, Bahaa-el-din et al. 2016); 2) days (camera- trap days, i.e. from detection to retrieval or to last photo taken in case the camera malfunctioned): wildcat detections can decrease along with an increase in the effort if a trap-shy response occurs (Wegge et al. 2004); 3) array (a categori- cal factor created for each array of traps (1–7)): given that we used seven non simultaneous arrays, we expected that all parameters of interest for the detection process could also vary by arrays, hence this detection covariate was acting as a ‘random site effect’ (Harihar and Pandav 2012, Tan et al. 2017, Penjor et al. Testing predictions We also created a grid of 1300 × 1300 m (1.69 km2), which is approximately the minimum home range for a wildcat in a Mediterranean habitat (Monterroso et al. 2009) over the entire study area (grid cells = 402), populated with the covariates included in the top model (the model with the minimum AIC), to spatially predict the wildcat occu- pancy across Mt Etna. Elevation data were downloaded as GMTED (< https://earthexplorer.usgs.gov >) and the mean elevation value was extracted for each grid cell. We then tested our wildcat occupancy predictions (predicted occu- pancy and 95% CI) against an independent (i.e. one record per cell) dataset (Supplementary material Appendix 7) of wildcat presence records to identify which scenario among our predictions was the most congruent with actual data. This dataset was made up of 26 wildcat presence locations (cameras record = 7, dead wildcat = 6, opportunistic wild- cat pictures = 10 and wildcat sightings observed by the first author of this study = 3) collected from 2005 to 2019. There- fore, we used the continuous Boyce index (hereafter, CBI) to compare the predicted values of wildcat occupancy for the entire study area against the predicted values assigned for any wildcat presence location by our top ‘best’ model (Klar et al. 2008, Clare et al. 2015). This index ranges from −1 to +1 with values close to 0 indicating a random asso- ciation between predicted occupancy and verified presence locations, whereas positive values describing a useful model (i.e. good agreement between predicted values and verified presence locations) and negative values indicating a model with fewer predicted values than verified presence locations (Hirzel et al. 2006). All results are presented as mean plus standard errors, unless explicitly stated. Although our survey was split into two non-consecutive periods, we used a single-season occupancy model as adult wildcats (we detected only adults) show high home-range fidelity (Daniels  et  al. 2001), therefore shifts in individ- ual home-ranges between years can be consider minimal (Wang et al. 2018) and changes in occupancy between these two periods were assumed negligible (assumption 1). We pooled five consecutive sampling days into one ‘capture’ occasion to minimize the risk of non-independence between detections at each site (assumption 2), whereas detections of different individuals at the same site and detections of the same individual at neighbouring cameras (assumption 2) were rarely observed within our dataset. Covariates As lengthMR and dis- tanceMR are both proxy for disturbance, these two covariates were independently included in occupancy models. Ecological covariates to model wildcat occupancy (ψ) (Table 1) were derived from the land map ‘Nature map of the Sicilian region’ at the scale of 1:50 000 created in 2008 with a resolution of 1 ha. Map accuracy was screened through extensive ground-truthing. The map is originally composed of 41 layers which we merged into eight main habitat classes (Supplementary material Appendix 2). Spe- cifically, layers were combined into habitat classes accord- ing to the current knowledge of wildcat ecology. Previous studies have shown that mixed forest (areamf; 170 km2 – 29.3%), mostly composed by deciduous trees, are pre- ferred by wildcats to pine forests (areapf; 42 km2 – 7.2%) (Silva et al. 2012, Kilshaw et al. 2016), whereas shrub vege- tation (areash; 88 km2 – 15.1%) has been found to be crucial Lastly, we note that our camera trapping sites sampled the habitat classes suitable for wildcat occurrence proportionally to their availability in the whole study area, as their extents within the circular buffers and across the area were correlated significantly (Pearson’s correlation coefficient = 0.64; Supple- mentary material Appendix 4). Candidate covariates for occupancy and detection were screened for collinearity with a threshold for the Pearson’s correlation >|0.7| (Silva et al. 5 2012, Kilshaw et al. 2016); when two covariates were found to be collinear, we included only one covariate of the pairs within occupancy models. For easier model computations covariates were normalized to have mean = 0 and SD = 1. wildcat occupancy in the study area. Owing to sparse data and to facilitate numerical computations, occupancy models were constrained to a maximum of 6 covariates and with only additive terms. All models with convergence issues were discarded (Supplementary material Appendix 6). Goodness- of-fit for the best model was checked with the function mb. gof.test (500 replicates) within the R package AICcmodavg (Mazerolle 2016) to test for overdispersion. The relative importance of covariates (ԐAICw) was calculated for each covariate included in those models within the cumulative AIC weight of 0.95 (Kilshaw et al. 2016). Occupancy modelling We used the R package camtrapR (Niedballa et al. 2016) to manage the data (camera records and camera setting deploy- ments), to prepare the ‘capture’ matrix (i.e. detection 1 ver- sus no detection 0) and the ‘occasion’ matrix (0 = camera not operational or malfunctioned; 1 = camera operational and working). We then ran single-species, single-season occu- pancy analysis with the R package unmarked (Fiske and Chandler 2011) in the R environment (< www.r-project. org >). Each occupancy model represented a set of ecologi- cal hypotheses that can be tested for the detection (p) and the occupancy (ψ) process, given that model assumptions are respected (Rovero and Zimmermann 2016). Model assump- tions for single-season, single species occupancy models are: 1) occupancy is assumed to be constant throughout the sur- vey; 2) detections and sites are independent 3) occupancy and detectability are constant among sites or they can be modelled using covariates. The detection process (p) and the occupancy process (ψ) are then estimated, using maximum likelihood estimations in our case, from a ‘capture’ matrix, where detection (1) or non-detection (0) of a given species at each site for each occasion, is reported, resulting in an occupancy estimate corrected for imperfect detection (McK- enzie et al. 2006). Testing predictions However, we still checked for a spatial autocorrelation signal in wildcat detec- tions by running the Moran’s I test (Silva et al. 2012), which may have inflated our occupancy model. l In our first step, we modelled detection (p) while occu- pancy (ψ) was over-parametrized (e.g. an occupancy model formulation with all habitat classes included as predictors). Therefore, detection was investigated by building all the possible combinations (n = 31) for the above mentioned six covariates) (Karanth et al. 2011) (Table 1). We included a null model p(.)ψ(areamf+areapf+areame+areash+arealf+are aal) which represents the null hypothesis (e.g. detection is constant and not influenced by any predictors). We selected the best fitting models among this first set of models using AIC information criteria with a ΔAIC < 2. In our a second step, we ran each of the best detection models (i.e. those ranked within ΔAIC<2) in conjunction with 42 occu- pancy models (Supplementary material Appendix 5) which reflected a priori, plausible ecological hypotheses about Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use Results In contrast, distanceMR two detections from one camera (6 N) showing a black cat, presumably the same individual. Removing these two detections and the relative camera did not significantly alter our results; further research is needed to classify the identity of this cat (Supplementary material Appendix 8). The Moran’s I test did not suggest any spatial autocor- relation signal for wildcat detections (observed = 0.02; expected = -0.01; SD = 0.02; p = 0.10). Collinearity was detected for npatchestot and npatchesmf (Pearson’s correla- tion coefficient = 0.71), which therefore were independently included in our occupancy models. Time-to-first wildcat detection was 17 ± 1.23 days after camera deployment and 28, 60 and 87% of the detections occurred within the first 10, 20 and 30 days after camera deployment, respectively. The first step of model selection identified 4 best detection models (Table 2), which were then combined with the 42 a priori occupancy models. After discarding 123 models due to lack of convergence we successfully ran 45 models (Sup- plementary material Appendix 6). Of these, only one model was strongly supported (Table 3); p(array+distanceMR+RAI h)ψ(areamf+areapf+npatchesmf+elevation). This model didn’t show signs of overdispersion (ĉ = 0.7; p = 0.834): mixed forest (areamf) had a positive effect on wildcat occupancy (ԐAICw = 0.95), whereas altitude (elevation), number of patches of mixed forest (npatchesmf) and pine forest (areapf) negatively affected wildcat occupancy (ԐAICw = 0.90; ԐAICw = 0.86, ԐAICw = 0.80; respectively). The remaining covariates (arealf, areash, areame and lengthMR) included in those models within 0.95 AIC cumulative weight poorly influenced wildcat occupancy (ԐAICw < 0.02). Results of the best model (i.e. the model with the minimum AIC) are shown in Table 4. Although not significant (p = 0.09), RAIh was retained within the most supported model and negatively influenced detection. In contrast, distanceMR Results The CBI clearly indicated that the lower confidence limit of our occupancy model was the most congruent scenario for fitting the independent dataset of wildcat presence data (CBI = 0.93), whereas the average occupancy scenario and the upper confidence limit scenario were less well-supported (CBI = 0.70 and CBI = 0.52 respec- tively). Spatially-explicit predictions of wildcat occupancy for Mt Etna (Fig. 4) for the most congruent scenario (lower confidence limit) showed that the distribution of cells with high predicted wildcat occupancy was highly fragmented, with the majority of these cells located at lower altitudes and mostly on the northern slope of Mt Etna. two detections from one camera (6 N) showing a black cat, presumably the same individual. Removing these two detections and the relative camera did not significantly alter our results; further research is needed to classify the identity of this cat (Supplementary material Appendix 8). The Moran’s I test did not suggest any spatial autocor- relation signal for wildcat detections (observed = 0.02; expected = -0.01; SD = 0.02; p = 0.10). Collinearity was detected for npatchestot and npatchesmf (Pearson’s correla- tion coefficient = 0.71), which therefore were independently included in our occupancy models. Time-to-first wildcat detection was 17 ± 1.23 days after camera deployment and 28, 60 and 87% of the detections occurred within the first 10, 20 and 30 days after camera deployment, respectively. The first step of model selection identified 4 best detection models (Table 2), which were then combined with the 42 a priori occupancy models. After discarding 123 models due to lack of convergence we successfully ran 45 models (Sup- plementary material Appendix 6). Of these, only one model was strongly supported (Table 3); p(array+distanceMR+RAI h)ψ(areamf+areapf+npatchesmf+elevation). This model didn’t show signs of overdispersion (ĉ = 0.7; p = 0.834): mixed forest (areamf) had a positive effect on wildcat occupancy (ԐAICw = 0.95), whereas altitude (elevation), number of patches of mixed forest (npatchesmf) and pine forest (areapf) negatively affected wildcat occupancy (ԐAICw = 0.90; ԐAICw = 0.86, ԐAICw = 0.80; respectively). The remaining covariates (arealf, areash, areame and lengthMR) included in those models within 0.95 AIC cumulative weight poorly influenced wildcat occupancy (ԐAICw < 0.02). Results of the best model (i.e. the model with the minimum AIC) are shown in Table 4. Although not significant (p = 0.09), RAIh was retained within the most supported model and negatively influenced detection. Results We collected 41 putative wildcat scats after walking 478.8 km. Of these, 27 (66%) were successfully genotyped, identifying 22 different wildcats (one wildcat was detected three times, three wildcats were detected twice and the rest once each (Supplementary material Appendix 1 Fig. A1, A2). We accumulated 3377 camera-trap days (37.1 ± 0.48) from 91 camera traps that worked effectively (Supplemen- tary material Appendix 3) over an area of 62.2 km2 (the cumulative sum of the MPC for each array). We obtained 70 wildcat detections from 38 cameras located across the seven arrays (naïve occupancy = 0.41; trap-rate = 2.07 number of detections/100 trap-days) (Fig. 2), including 6 Table 2. Model selection for determining the best detection process (p) for European wildcat Felis silvestris on Mt Etna (Sicily, Italy) from data collected using camera traps in 2015–2016 while the occupancy process (ψ) is kept constant and over parametrized. First four models shown along with the null model for the detection process. Models within 2 AIC are in bold. Table 2. Model selection for determining the best detection process (p) for European wildcat Felis silvestris on Mt Etna (Sicily, Italy) from data collected using camera traps in 2015–2016 while the occupancy process (ψ) is kept constant and over parametrized. First four models shown along with the null model for the detection process. Models within 2 AIC are in bold. Detection process nPars AIC Delta p(array+distanceMR+RAIh) ψ(areamf+areapf+areame+areash+arealf+areaal) 16 408.04 0.00 p(days+array+distanceMR+RAIh) ψ(areamf+areapf+areame+areash+areal+areaal) 17 408.94 0.90 p(array+distanceMR) ψ(areamf+areapf+areame+areash+arealf+areaal) 15 409.69 1.66 p(array+RAIh) ψ(areamf+areapf+areame+areash+arealf+areaal) 17 410.04 2.00 p(.) ψ(areamf+areapf+areame+areash+arealf+areaal) 8 414.65 6.61 positively influenced detection. Estimated values for p and ψ from the best occupancy model were 0.016 ± 0.009 and 0.74 ± 0.03 respectively, so when imperfect detection was not taken into account, naïve occupancy (0.41) was greatly underestimated (45% less than the estimated occupancy). When plotting univariate responses for wildcat occupancy ψ, we found that it steadily increased when areamf reached the threshold of 15% and decreased when areapf reached the threshold of 50% (Fig. 3). Moreover, wildcat occupancy ψ decreased when npatchesmf approached the value of 9 and the same was observed for elevation at the threshold value of ~1400 m a.s.l. (Fig. 3). Discussion In our study wildcat presence was confirmed on Mt Etna by DNA analysis, therefore subsequent phenotypic identi- fications on pictures can be considered reliable. Addition- ally, by using camera-trapping and an occupancy modelling frame-work, we documented how wildcat occupancy on Mt Etna was driven by a combination of habitat structure and vegetation features. Our basic expectations of the role Table 3. Model selection results testing for habitat covariates on European wildcat Felis silvestris occupancy on Mt Etna (Sicily, Italy) from data collected using camera traps in 2015–2016. First four models shown and models within 2 AIC are in bold. Full model selection is reported in Supplementary material Appendix 6. Formula nPars AIC Delta AICwt CumltvWt Rsq p(array+distanceMR+RAIh) ψ(areamf+areapf+elevation+npatchesmf) 14 394.86 0.00 0.55 0.55 0.50 p(array+distanceMR) ψ(areamf+areapf+elevation+npatchesmf) 15 396.85 1.99 0.20 0.76 0.50 p(width+array+distanceMR+RAIh) ψ(areamf+areapf+elevation+npatchesmf) 13 399.84 4.98 0.04 0.80 0.46 p(array+distanceMR+RAIh) ψ(areamf+areapf+elevation+nclass) 14 399.95 5.09 0.04 0.85 0.48 7 Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use Pattern of detectability Model component β SE z p(>|z|) Detection parameters (p)   Intercept −4.102 0.589 −6.97 0.0000   Array 2 3.329 0.880 3.78 0.0001   Array 3 1.597 0.672 2.38 0.0173   Array 4 1.411 0.637 2.22 0.0267   Array 5 2.288 0.626 3.65 0.0002   Array 6 2.770 0.722 3.84 0.0001   Array 7 3.309 0.905 3.66 0.0002   distanceMR 0.732 0.288 2.54 0.0109   RAIh −0.506 0.303 −1.67 0.0951 Occupancy parameters (ψ)   Intercept 10.57 5.06 2.09 0.0368   areamf 7.70 3.94 1.95 0.0509   areapf −2.88 1.61 −1.79 0.0737   npatchesmf −4.20 2.04 −2.06 0.0394   elevation −4.14 2.08 −1.99 0.0469 Model component β SE z p(>|z|) Detection parameters (p)   Intercept −4.102 0.589 −6.97 0.0000   Array 2 3.329 0.880 3.78 0.0001   Array 3 1.597 0.672 2.38 0.0173   Array 4 1.411 0.637 2.22 0.0267   Array 5 2.288 0.626 3.65 0.0002   Array 6 2.770 0.722 3.84 0.0001   Array 7 3.309 0.905 3.66 0.0002   distanceMR 0.732 0.288 2.54 0.0109   RAIh −0.506 0.303 −1.67 0.0951 Occupancy parameters (ψ)   Intercept 10.57 5.06 2.09 0.0368   areamf 7.70 3.94 1.95 0.0509   areapf −2.88 1.61 −1.79 0.0737   npatchesmf −4.20 2.04 −2.06 0.0394   elevation −4.14 2.08 −1.99 0.0469 ariables were supported as mix d to wildcat occupancy positive ontributed negatively along wi st importantly, we also found th erred habitat (npatchesmf) neg occupancy. The considerable d upancy and estimated occupan s almost twice the naïve occ se detection probability was qui mera-trapping surveys of felid sp ndresen et al. 2014, Penjor et  8). 0.589 −6.97 0.0000 9 0.880 3.78 0.0001 7 0.672 2.38 0.0173 0.637 2.22 0.0267 8 0.626 3.65 0.0002 0 0.722 3.84 0.0001 9 0.905 3.66 0.0002 0.288 2.54 0.0109 6 0.303 −1.67 0.0951 5.06 2.09 0.0368 3.94 1.95 0.0509 1.61 −1.79 0.0737 2.04 −2.06 0.0394 2.08 −1.99 0.0469 Pattern of detectability Table 4. Results of the top ‘best’ model. p(array+distanceMR+RAIh) ψ(areamf+areapf+elevation+npatchesmf) for describing European wildcat Felis silvestris occupancy on Mt Etna (Sicily, Italy) from data collected using camera traps in 2015–2016. Distance to major roads (distanceMR) clearly influenced wildcat detectability, making wildcats more detectable in remote areas far away from major roads, but the effect of this covariate on wildcat occupancy was not found. We argue that, rather than the distance from major roads per se, the level of traffic might be a better proxy for disturbance (Klar et al. 2009). Although the effect was not statistically significant (p = 0.09), the detection covariate RAIh (a mea- sure of human disturbance) was retained within the most supported model and negatively influenced wildcat detect- ability. Wildcats are mainly nocturnal (Daniels et al. 2001, Wittmer 2001, Germain et al. 2008), but daylight move- ments can also occur (Daniels et al. 2001, Germain et al. 2008) and the contemporary presence of humans along the trails might have induced wildcats to be more ‘cautious’ when human presence was ‘massive’ (Piñeiro et al. 2012), similar to bobcats (George and Crooks 2006, Clare et al. 2015), leopards Panthera pardus (Carter  et  al. 2015) and tigers (Carter et al. 2012). played by environmental variables were supported as mixed forest (areamf) contributed to wildcat occupancy positively and pine forest (areapf) contributed negatively along with the altitude (elevation). Most importantly, we also found that fragmentation of the preferred habitat (npatchesmf) nega- tively influenced wildcat occupancy. The considerable dif- ference, between naïve occupancy and estimated occupancy (estimated occupancy was almost twice the naïve occu- pancy), was expected because detection probability was quite low as usually found in camera-trapping surveys of felid spe- cies (Galvez et al. 2013, Andresen et al. 2014, Penjor et al. 2018, Strampelli et al. 2018). Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use Pattern of occupancy 2017), or at least, used less frequently as they rep- resent a less suitable habitat in terms of abundance of prey (Lozano and Malo 2012). Wildcat occupancy was also nega- tively affected by altitude (elevation) and, congruently with Say et al. (2012), in our study wildcat occupancy levelled off at ~1800 m a.s.l. probably because long-lasting thick snow cover may have hindered wildcat movements (Mermod and Liberek 2002) and hence habitat above this altitude was less suitable for wildcats. ) The impact of habitat fragmentation on felid species has often been claimed, although direct evidence of its effects is rare (Zanin et al. 2014, Galvez et al. 2017). Habitat frag- mentation is considered one of the most severe threats for wildcat (Macdonald and Loveridge 2010, Lozano and Malo 2012) and our study, to the best of our knowledge, is the first to find direct evidence that fragmentation (npatchesmf) of the preferred habitat (areamf) negatively affects wild- cat occupancy. Moreover, as fragmentation of mixed for- est (npatchesmf) also negatively impacts wildcat occupancy, our study confirms the pivotal role of mixed forest (mainly deciduous such as Quercus sp., Castanea sativa and Fagus sylvatica, but also the particular local habitat of wooded patches populated by Genista etniensis in our case) as even a minimal amount of this habitat promoted wildcat occu- pancy on Mt Etna. The critical role of habitat fragmenta- tion for wildcat conservation also emerged when considering that hybrids were mostly located at the periphery of the local wildcat distribution area where, reasonably, habitat frag- mentation should be more pronounced and thus wildcats were less abundant (Nussberger et al. 2018). Additionally, female wildcats prefer to establish their home ranges inside forest patches rather than at the periphery as did males (Beu- gin et al. 2016, Oliveira et al. 2018), thus displaying avoid- ance of fragmented habitats, similar to that of female jaguars (Conde et al. 2010). Considering this recent evidence in its Despite being a Mediterranean area, the importance of scrub (Lozano et al. 2003, Monterroso et al. 2009, Lozano 2010) for wildcats was not supported by our data. It is likely that the shelter provided by this habitat (Lozano et al. 2003) is largely overwhelmed by the almost ubiquitous natural cav- ities typical of the volcanic soil, which offer a safe shelter for wildcats throughout our study area. Likewise, meadows did not promote wildcat occupancy in our study area. Pattern of occupancy played by environmental variables were supported as mixed forest (areamf) contributed to wildcat occupancy positively and pine forest (areapf) contributed negatively along with the altitude (elevation). Most importantly, we also found that fragmentation of the preferred habitat (npatchesmf) nega- tively influenced wildcat occupancy. The considerable dif- ference, between naïve occupancy and estimated occupancy (estimated occupancy was almost twice the naïve occu- pancy), was expected because detection probability was quite low as usually found in camera-trapping surveys of felid spe- cies (Galvez et al. 2013, Andresen et al. 2014, Penjor et al. 2018, Strampelli et al. 2018). We found that mixed forest (areamf) promoted wildcat occupancy, as found for the wildcat population in Scotland (Kilshaw et al. 2016). Forest cover was important for other small felid species, such as bobcats (Clare et al. 2015), kod- kods Leopardus guigna (Galvez et al. 2013, Schuttler et al. 2016) and caracals Caracal caracal (Singh et al. 2014). Forest cover has been frequently found to be an important habi- tat feature for wildcats (Klar et al. 2008, 2012, Jerosch et al. 2010, Lozano 2010, Beugin et al. 2016, 2018, Beutel et al. 2017), although it seems that when rabbits Oryctolagus cunic- ulus are present, wildcats tend to be less forest-dependent Figure  3. Predictions from the ‘best’ model p(array+distanceMR+RAIh)ψ(areamf+areapf+elevation+npatchesmf) for describing European wildcat Felis silvestris occupancy on Mt Etna (Sicily, Italy) from data collected using camera traps in 2015–2016. Top-left panel shows the relationship between wildcat detectability and distance from main road (distanceMR). The other panels show the relationship between wildcat occupancy and the covariates areamf, areapf, elevation and npatchesmf (the other covariates in the model where held at their mean values). Figure  3. Predictions from the ‘best’ model p(array+distanceMR+RAIh)ψ(areamf+areapf+elevation+npatchesmf) for describing European wildcat Felis silvestris occupancy on Mt Etna (Sicily, Italy) from data collected using camera traps in 2015–2016. Top-left panel shows the relationship between wildcat detectability and distance from main road (distanceMR). The other panels show the relationship between wildcat occupancy and the covariates areamf, areapf, elevation and npatchesmf (the other covariates in the model where held at their mean values). 8 Figure 4. Spatially explicit predictions from the ‘best’ model. Pattern of occupancy p(array+distanceMR+RAIh)ψ(areamf+areapf+elevation+npatchesmf) for describ- ing European wildcat Felis silvestris occupancy on Mt Etna (Sicily, Italy) from data collected using camera traps in 2015–2016 and from the most congruent scenario (lower estimates) for fitting the independent dataset of European wildcat presence (black square = camera-trapping records; black points = dead wildcats; black triangle = opportunistic photographs; black diamonds = sightings) collected on Mt Etna. Figure 4. Spatially explicit predictions from the ‘best’ model. p(array+distanceMR+RAIh)ψ(areamf+areapf+elevation+npatchesmf) for describ- ing European wildcat Felis silvestris occupancy on Mt Etna (Sicily, Italy) from data collected using camera traps in 2015–2016 and from the most congruent scenario (lower estimates) for fitting the independent dataset of European wildcat presence (black square = camera-trapping records; black points = dead wildcats; black triangle = opportunistic photographs; black diamonds = sightings) collected on Mt Etna. (Monterroso  et  al. 2009, Lozano 2010, Silva  et  al. 2012, Lozano et al. 2013). (Monterroso  et  al. 2009, Lozano 2010, Silva  et  al. 2012, Lozano et al. 2013).h entirety, we suggest more studies are needed to better under- stand the effects of habitat fragmentation on wildcat ecology. entirety, we suggest more studies are needed to better under- stand the effects of habitat fragmentation on wildcat ecology. In line with our basic expectations, wildcat occupancy decreased in pine forests (areapf), especially when this habi- tat was the most predominant, but see Klar et al. (2008) and Lozano (2010) for contrasting results. Pine forests are usually avoided by wildcats (Sarmento et al. 2006, Martin- Diaz et al. 2017), or at least, used less frequently as they rep- resent a less suitable habitat in terms of abundance of prey (Lozano and Malo 2012). Wildcat occupancy was also nega- tively affected by altitude (elevation) and, congruently with Say et al. (2012), in our study wildcat occupancy levelled off at ~1800 m a.s.l. probably because long-lasting thick snow cover may have hindered wildcat movements (Mermod and Liberek 2002) and hence habitat above this altitude was less suitable for wildcats. stand the effects of habitat fragmentation on wildcat ecology. In line with our basic expectations, wildcat occupancy decreased in pine forests (areapf), especially when this habi- tat was the most predominant, but see Klar et al. (2008) and Lozano (2010) for contrasting results. Pine forests are usually avoided by wildcats (Sarmento et al. 2006, Martin- Diaz et al. Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use Comparison with other camera-trapping studies We suggest that the maintenance of large patches of mixed forest would be a good practice to promote long-term population viability of wildcats on Mt Etna. Additionally, improving the connectivity between the largest patches of mixed forest (Klar et al. 2012) and reducing the impact of traffic on critical roads by employing appropriate wildcat mitigation structures, such as fences, tunnels and viaducts (Klar  et  al. 2009), are management actions that should favour wildcat survival. During our survey four cameras were stolen and this prompted us to reduce our camera- trapping effort. We also detected other illegal activities like unauthorized vehicle accesses and a large presence of feral pigs in some areas of the park. Moreover, the numerous illegal human-caused fires that afflict our study area dur- ing summer are likely to be a major threat for the wildcat as they increase the loss and the fragmentation of suitable wildcat habitat. A radical change in forest fire prevention, along with more stringent and concrete surveillance of this protected area is therefore needed to ensure the viable per- sistence of this wildcat population. Moreover, cooperation between adjacent natural parks (e.g. Nebrodi Park, Alcan- tara Park and the Etna Park) to sustain wildcat conserva- tion actions at the regional level is strongly recommended. A camera-trapping project across the largest regional parks (e.g. Etna, Nebrodi and Madonie) and suitable connective corridors would be a step forward for the conservation of the Sicilian wildcat population. Although we recognized that comparisons among raw met- rics, such as naïve occupancy and trap-rate, can be biased (Sollmann et al. 2013, Anile and Devillard 2015) and thus have to be taken with great caution, we observed a general decrease (range naïve occupancy = 0.66–0.88; range trap- rate = 2.9–6.48) when comparing our results to those of previous surveys from the same study area (range naïve occu- pancy = 0.66–0.88; range trap-rate = 2.9–6.48) (Anile et al. 2009, 2010, 2012a, b, 2014) (Table 5). Even if a different sampling design likely affected this comparison as previous studies used fewer camera locations (range = 11–18) to cover smaller areas (range = 6.6–10.9 km2) and with less effort (range trap-days = 671–1080), in the current study we also sampled the majority of these previous camera locations and obtained only a few detections (n = 8 at only four cameras). Pattern of occupancy Meadows (as depicted on the map we used to derive our occupancy covariates) were usually very small (0.11 ± 0.02 km2), not very widespread (39 cameras out of 91 hosted this habitat class) and isolated (npatchesme per camera = 1.23 ± 0.14), 9 Table 5. List of camera-trapping studies on the European wildcat Felis silvestris across Europe. * indicates this study. Trap-rate is expressed as number of detections for 100 trap-days. Table 5. List of camera-trapping studies on the European wildcat Felis silvestris across Europe. * indicates this study. Trap-rate is expressed as number of detections for 100 trap-days. Study Country Naive occupancy No. of #cameras Trap-rate Trap-days Baited/not baited Anile et al. 2009 Italy (Mt Etna) 0.66 18 2.9 824 baited and not baited Anile et al. 2010 Italy (Mt Etna) 0.63 11 4.02 671 not baited Anile et al. 2012a, b Italy (Mt Etna) 0.66 18 2.9 1080 not baited Anile et al. 2012b Italy (Mt Etna) 0.72 18 4.07 1080 baited Anile et al. 2014 Italy (Mt Etna) 0.88 18 6.48 1080 not baited Anile and Devillard 2018* Italy (Mt Etna) 0.41 91 2.07 3377 not baited Velli et al. 2015 Italy 0.5 20 3.1 819 baited Can et al. 2009 Turkey 0.3 40 1.8 1200 not baited Beutel et al. 2017 Germany 0.34 46 0.06 31107 not baited Sarmento et al. 2009 Spain 0.01 186 0.02 6127 baited Soto and Palomares 2013 Spain 0.07 166 0.4 5761 baited Gil-Sanchez et al. 2015 Spain 0.2 64 1.23 4278 baited Kilshaw and MacDonald 2011 Scotland 0.55 20 0.5 1460 baited and not baited Littlewood et al. 2014 Scotland 0.04 356 0.28 7499 baited Kilshaw et al. 2016 Scotland 0.04 526 0.17 49031 baited of both naïve occupancy and trap-rate (Table 5). From the current study Mt Etna is still confirmed a stronghold for the Sicilian wildcat population, despite the fact that a decreasing trend in population density might have occurred. of both naïve occupancy and trap-rate (Table 5). From the current study Mt Etna is still confirmed a stronghold for the Sicilian wildcat population, despite the fact that a decreasing trend in population density might have occurred. so it is very likely this habitat did not influence wildcat occupancy in our study area. Pattern of occupancy Future studies on wildcats in the unique ecosystem of Mt Etna are needed; in particu- lar, home-range estimation through the use of GPS collars would better elucidate wildcat habitat requirements. Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use Acknowledgements – We thank all the people who kindly shared with us their wildcat pictures shot on Mt Etna. Alisia Schiff kindly and Clay Nielsen reviewed and edited the English of this manuscript. During the writing of this study, Prof. Bernardino Ragni passed away. He was the supervisor of SA during his PhD-project on wildcats and an always present mentor during the successive years of collaboration. This study is dedicated to him, without his References Wildcat population density on the Etna vol- cano, Italy: a comparison of density estimation methods. – J. Zool. 293: 252–261. George, S. L. and Crooks, K. R. 2006. Recreation and large mam- mal activity in an urban nature reserve. – Biol. Conserv. 133: 107–111. Anile, S. et  al. 2017. Home-range size of the European wildcat (Felis silvestris silvestris): a report from two areas in central Italy. – Mammalia 82: 1–11. Germain, E. et  al. 2008. Spatio-temporal sharing between the European wildcat, the domestic cat and their hybrids. – J. Zool. 276: 195–203. Apostolico, F. et al. 2015. 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Forest cover outside protected areas plays an important role in the conservation of the vulnerable guina Leopardus guigna. – Oryx 47: 251–258. Anile, S. et  al. 2012b. A non-invasive monitoring on European wildcat (Felis silvestris silvestris Schreber, 1777) in Sicily using hair trapping and camera trapping: does scented lure work? – Hystrix Ital. J. Mammal. 23: 45–50. Galvez, N. et al. 2017. A spatially integrated framework for assess- ing socioecological drivers of carnivore decline. – J. Appl. Ecol. 55: 1393–1405. Anile, S. et al. 2014. Comparison with other camera-trapping studies SA also thanks Dr Luigi Piccinini and Maurizio Pennisi from the ‘Ripartizione Faunistico Venatoria di Catania’ for providing the cameras used in this study. We also thank Subject Editor Dr Luc Wauters for the helpful suggestions which greatly improved this manuscript. Lastly, we all thank Marisa Mazzaglia (former president of Etna park) for supporting this wildcat research without any hesitation.h Cardillo, M. et. al. 2008. The predictability of extinction: biolog- ical and external correlates of decline in mammals. – Proc. R. Soc. B 275: 1441–1448. Carter, N. H. et al. 2012. Coexistence between wildlife and humans at fine spatial scales. – Proc. Natl Acad. Sci. USA 109: 1–6. i Carter, N. et al. 2015. Impacts of people and tigers on leopard spatiotemporal activity patterns in a global biodiversity hot- spot. – Global Ecol. Conserv. 3: 149–162. Funding – This research was funded by the Etna Park. Funding – This research was funded by the Etna Park. Clare, J. D. J. et al. 2015. Predicting bobcat abundance at a land- scape scale and evaluating occupancy as a density index in central Wisconsin. – J. Wildl. Manage. 3: 469–480. Comparison with other camera-trapping studies We suspect this decreasing trend in either naïve occupancy and trap-rate might truly corresponded to a decrease in wild- cat population density, at least compared to the last survey performed in 2010. Although previous data are not avail- able, rabbits were much more widespread and abundant in the recent past than when we observed them during the current study (29 detections at only 3 cameras). This likely reduction in the abundance of rabbits (the wildcat’s preferred prey) (Malo et al. 2004, Lozano et al. 2006) in the entire Etna Park, as well as in the smaller study area sampled during previous surveys, has probably driven this decrease in wildcat detection (Lozano and Malo 2012, Littlewood et al. 2014). Future analyses are required to evaluate whether a decrease in wildcat population density has really occurred and so our findings here should be considered preliminary. With regards to other similar camera-trapping studies, although again different sampling designs have to be taken into con- sideration (i.e. in the current study we did not use any lure or bait), our results were generally higher or similar in terms i along with more stringent and concrete surveillance of this protected area is therefore needed to ensure the viable per- sistence of this wildcat population. Moreover, cooperation between adjacent natural parks (e.g. Nebrodi Park, Alcan- tara Park and the Etna Park) to sustain wildcat conserva- tion actions at the regional level is strongly recommended. A camera-trapping project across the largest regional parks (e.g. Etna, Nebrodi and Madonie) and suitable connective corridors would be a step forward for the conservation of the Sicilian wildcat population. Acknowledgements – We thank all the people who kindly shared with us their wildcat pictures shot on Mt Etna. Alisia Schiff kindly and Clay Nielsen reviewed and edited the English of this manuscript. During the writing of this study, Prof. Bernardino Ragni passed away. He was the supervisor of SA during his PhD-project on wildcats and an always present mentor during the successive years of collaboration. This study is dedicated to him, without his 10 Cardillo, M. et al. 2004. Human population density and extinction risk in the world’s carnivores. – PLoS Biol. 2: 909–914.h support this study (as well as previous ones) would not have been possible. SA thanks all the officers of Etna Park (particularly Dr Michele Leonardi and Dr Rosa Spampinato), their hard work made this study feasible. Downloaded From: https://bioone.org/journals/Wildlife-Biology on 04 Oct 2019 Terms of Use: https://bioone.org/terms-of-use References Conserv. 15: e00413 Klar, N. et  al. 2012. Between ecological theory and planning practice: (re-)connecting forest patches for the wildcat in lower Saxony, Germany. – Landsc. 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https://openalex.org/W2110622122
https://europepmc.org/articles/pmc4253083?pdf=render
English
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Washoff of cypermethrin residues from slabs of external building material surfaces using simulated rainfall
Environmental toxicology and chemistry
2,013
cc-by
6,151
All Supplemental Data may be found in the online version of this article. * Address correspondence to traskj@waterborne-env.com. Published online 15 October 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.2432 INTRODUCTION have focused on hardscape materials found in urban areas and examined the potential impact of runoff from these materials. Weston et al. [3] found pyrethroid residues in runoff from residential neighborhoods around Sacramento, California, USA. Although the source was thought to be primarily professional pest controllers, they concluded that use by homeowners may also contribute. Jorgenson and Young [7] and Jorgenson et al. [8] examined washoff from concrete surfaces and concluded that washoff is a function of the product formulation. A study by Jiang et al. [9] examined the effects of time on desorption following application of 4 pyrethroids to small concrete blocks in the laboratory. They concluded that formulation and persistence of the chemicals on the concrete surface over time may be a factor in washoff potential. In another study by Jiang et al. [10], 2 pyrethroids were tested under field conditions in California and demonstrated similar behaviors as observed in the laboratory study. They also investigated the effects of surface conditions (i.e., sealing, acid washing) on potential runoff of pyrethroids and found only minor differences yet suggested that surface roughness may play a role in reducing pyrethroid transport. The use of pyrethroids has increased since the 1990s as a result of the phase-out of the organophosphate insecticides in both the agricultural and urban environments. In California, USA, for example, pyrethroid sales for use in agricultural and urban areas increased from 2000 to 2006 according to the California Department of Pesticide Regulation, although sales in California have shown a downward trend since 2007 [1]. Recent studies have documented detection of pyrethroids in various stream environments across the United States [2–5]. These pesticides are commonly used by professionals and homeowners for treatments around structures (e.g., house foundations), perimeters, and landscapes. In California, >95% of the reported urban use for major pyrethroid chemicals is for structural pest control [1]. Work performed by Greenberg et al. [6] has shown pyrethroid residues moving to the street in runoff as a result of applications around the border of residential structures. Weston and Lydy [2] also found pyrethroid residues in runoff entering various California urban streams; however, specific sources were not identified. Given their wide distribution of use, specific sources of pyrethroids that have the potential to contribute residues to urban streams have been a challenge to characterize. INTRODUCTION The purpose of the present study was to determine differences in washoff of cypermethrin applied to various building material surfaces. The present study also included 2 classes of residential insecticide formulations (emulsifiable concentrate [EC] and wet- table powder [WP]) to compare washoff between formulations. Although sources of residues from pyrethroids are fairly well understood in agricultural environments, knowledge about urban sources (i.e., urban landscapes) that may contribute pyrethroid residues to nearby water bodies is limited. Few studies JENNIFER R. TRASK,*y CHRISTOPHER M. HARBOURT,y PAUL MILLER,y MEGAN COX,y RUSSELL JONES,z PAUL HENDLEY,x and CHUNG LAMz yWaterborne Environmental, Leesburg, Virginia, USA zBayer CropScience, Research Triangle Park, North Carolina, USA xSyngenta Crop Protection, Greensboro, North Carolina, USA JENNIFER R. TRASK,*y CHRISTOPHER M. HARBOURT,y PAUL MILLER,y MEGAN COX,y RUSSELL JONES,z PAUL HENDLEY,x and CHUNG LAMz yWaterborne Environmental, Leesburg, Virginia, USA zBayer CropScience, Research Triangle Park, North Carolina, USA xSyngenta Crop Protection, Greensboro, North Carolina, USA (Submitted 18 January 2013; Returned for Revision 25 March 2013; Accepted 9 October 2013) Abstract: The use of pesticides by homeowners or pest-control operators in urban settings is common, yet contributions of washoff from these materials are not easily understood. In the present study, cypermethrin, formulated as Cynoff EC (emulsifiable concentrate) and Cynoff WP (wettable powder) insecticides, was applied at typical rates to 10 different building material surfaces to examine its washoff potential from each surface. Using an indoor rainfall simulator, a 1-h rainfall event was generated and washoff samples were collected from 3 replicates of each surface type. Washoff was analyzed for cypermethrin using gas chromatography-negative chemical ionization mass spectrometry. An analysis of variance for a split-plot design was performed. Many building materials had similar water runoff masses, but asphalt resulted in significantly reduced average water runoff masses (73% less). The Cynoff WP formulation generally produced greater cypermethrin washoff than the Cynoff EC formulation. In addition, results for both the WP and EC formulations indicated that smoother surfaces such as vinyl and aluminum siding had higher washoff (1.0–14.1% mean percentage of applied mass). Cypermethrin washoff from rough absorptive surfaces like concrete and stucco was lower and ranged from 0.1 to 1.3% and from 0 to 0.2%, respectively, mean percentage of applied mass. Both building material surface and formulation play a significant role in cypermethrin washoff. Environ Toxicol Chem 2014;33:302–307. # 2013 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited. Keywords: Washoff Pyrethroid Simulated rainfall Building material Keywords: Washoff Pyrethroid Simulated rainfall Building material Environmental Toxicology and Chemistry, Vol. 33, No. 2, pp. 302–307, 2014 # 2013 SETAC Printed in the USA Environmental Toxicology and Chemistry, Vol. 33, No. 2, pp. 302–307, 2014 # 2013 SETAC Printed in the USA Environmental Toxicology and Chemistry, Vol. 33, No. 2, pp. 302–307, 2014 # 2013 SETAC Printed in the USA Test materials The building materials selected include those found on surface areas of structures (e.g., driveways, foundations) that may receive applications of pyrethroids. In the present study, 6 302 Washoff of cypermethrin residues 303 Environ Toxicol Chem 33, 2014 The simulator contains 2 emitter modules, each containing 5 oscillating nozzles located 10 m above the test floor. This distance allows the simulated raindrops to attain terminal velocity upon impact with the simulator floor, with a droplet size, speed, and energy representative of natural rainfall [13]. The rainfall simulator’s uniform test floor is 1.82 m  4.65 m (6 ft  15 ft), making room for 11 test slab locations during a rainfall event. Details regarding the mechanics and operations of the simulator have been well documented ([13]; J. R. Trask, 2002, Master’s thesis, University of Illinois at Urbana- Champaign, Urbana, IL, USA). The simulator contains 2 emitter modules, each containing 5 oscillating nozzles located 10 m above the test floor. This distance allows the simulated raindrops to attain terminal velocity upon impact with the simulator floor, with a droplet size, speed, and energy representative of natural rainfall [13]. The rainfall simulator’s uniform test floor is 1.82 m  4.65 m (6 ft  15 ft), making room for 11 test slab locations during a rainfall event. Details regarding the mechanics and operations of the simulator have been well documented ([13]; J. R. Trask, 2002, Master’s thesis, University of Illinois at Urbana- Champaign, Urbana, IL, USA). different building materials of varying surface finishes were tested. These included clean unpainted and painted concrete, clean unpainted and painted stucco, clean aluminum siding, clean vinyl siding, clean asphalt, and clean unpainted and painted wood. A dusty painted wood surface was also chosen to see if particulate materials affect pyrethroid washoff. Each test slab was approximately 23 cm  61 cm (nominal 9 in  24 in), but of varying thicknesses (3.9–11.4 cm) depending upon the material. Latex exterior paint was used on all painted surfaces. Additional slabs made of clean aluminum siding were used as field blanks (i.e., untreated slabs). For each building material surface (e.g., painted concrete), 6 slabs were constructed to provide 3 replicates per pyrethroid formulation. Each replicate group was subjected to a single rainfall event for 1 h at a rate of 2.54 cm/h (1 in/h). A field blank was included with each rainfall event. Chemical analysis Sample analyses were performed at Bayer CropScience laboratories in Stilwell, Kansas, USA. A validated analytical method was used to analyze the following matrices: water samples, emptied bottles, filter papers, and spray solutions. The method employed a liquid–liquid partition followed by gas chromatography with negative chemical ionization mass spectrometric detection. The limit of quantitation in water as specified for the method was 1.03 mg/L. This limit of quantification value represents 0.01% of applied cypermethrin (assumes an average sample volume of 1.7 L and a target mass applied of cypermethrin as Cynoff EC insecticide of approxi- mately 26.5 mg a.i. per slab or approximately 28.1 mg a.i. per slab as Cynoff WP insecticide, based on product label information). Although recent studies involving pyrethroids have used limits of quantification as low as 1 part per trillion [16], the limits of quantification set for these analyses are adequate considering the application rates and the range of concentrations determined in the washoff samples. Calibration standard solutions were prepared using a primary stock standard of cypermethrin (purity 93.9%) and an internal standard of cyfluthrin-methyl-d6 dissolved in acetonitrile (standard K-939, 16% by wt). A 5-point standard calibration curve was prepared as described in the method and consisted of the following amounts: 7.23 mg/L, 20.7 mg/L, 51.7 mg/L, 103 mg/L, and 207 mg/L. This calibration curve range (7.23–207 mg/L) was equivalent to a sample concentration range of 0.36 mg/L to 10.4 mg/L in the washoff samples. Procedural recoveries at a concentration of 10.3 mg/L were run with each set of analyses, and the average recovery of the 9 procedural recovery samples was 92% with a relative standard deviation of 1%. Prior to extraction, runoff samples were allowed to come to room Test materials A test stand was constructed to hold the test slabs at a 60-degree angle from the horizontal. This angle was chosen to simulate wind causing rainfall to impact a vertical surface at a similar angle in the environment [14,15]. Stainless steel flashing and collection devices were attached to the sides and the downslope face of the slab, respectively (Supplemental Data, Figure S2). The design ensured that rainwater flowed down the length of the slab surface and into the collection device while minimizing losses to splashing. Aluminum rain shields were attached to the top of the collection device to prevent any direct rainfall (i.e., rainfall not in contact with the test slab) from being collected in the sample bottle. Runoff from each slab was collected in a precleaned 2-L amber glass bottle and preserved with sufficient 10% formic acid to lower the pH between 5 and 7, maximizing pyrethroid chemical stability. Rainfall simulation timings were approximately 24 h after test substance application. Additional details regarding sample collection and test slab placement are provided in the Supplemental Data. Prior to application, each building material surface was washed and rinsed, with the exception of dusty painted wood. Unpainted concrete slabs were soaked for at least 2 d to remove any substances from the surface that might affect the pH. This was important for the concrete slabs because freshly poured concrete can have a pH greater than 10 [11], and pyrethroids can hydrolyze more quickly under alkaline conditions [7,12], which might have biased results for substrates that can generate local high pH environments (e.g., concrete). Small amounts of muriatic acid (31.45% hydrochloric acid) were added each day to the water during the soaking process to aid in neutralizing the surface of the unpainted concrete slabs. Dusty painted wood surfaces were created by pouring and rubbing soil (obtained from the Central Valley of California) onto the painted surface after it was cleaned, rinsed, and dried. The soil obtained was analyzed for cypermethrin, which was <0.1 mg/L. Municipal water from the city of Urbana, Illinois, USA, was used for washing, soaking, and rainfall simulation events, which had a pH between 8.5 and 9.0 (Illinois American Water, personal communication). More information regarding the test materials is available in the Supplemental Data. Test substance application Two commercial cypermethrin formulations were selected for the present study: Cynoff EC insecticide (24.8% cypermeth- rin) and Cynoff WP insecticide (40% cypermethrin). Certified test materials were obtained from FMC Corporation and stored at room temperature. The 60 building material test slabs were divided into 6 groups of 10, with 3 replicate groups per formulation. Cypermethrin was applied using a laboratory track sprayer at the University of Illinois at Urbana-Champaign in Urbana, Illinois, USA (Supplemental Data, Figure S1). The system was calibrated to make applications to the test slab area at the recommended maximum label rate. This corresponded to 26.5 mg and 28.1 mg active ingredient (a.i.) per slab for Cynoff EC and Cynoff WP insecticides, respectively. To quantify the mass of test substance being applied to the slabs, application monitoring samples (filter paper samples) were collected every fifth application, resulting in 2 filter paper samples per replicate group. Samples were collected using 15- cm–inner diameter glass Petri dishes, the lids of which were fitted with Whatman 15-cm-diameter filter paper. Immediately following applications, test slabs were removed from the booth and placed into individual opaque storage containers for drying and storage until rainfall simulation. Additional details about the track sprayer equipment and application are available in the Supplemental Data. Simulated rainfall events Data analysis An analysis of variance (ANOVA) for a split-plot design was performed using the Statistical Analysis Software package (SAS Institute) [17] to investigate the following hypotheses: there is no difference in pyrethroid washoff from different building materials, and there is no difference in pyrethroid washoff between different formulations of cypermethrin. This mixed- model analysis was used to determine whether or not building material, formulation, and the interaction between these variables were significant contributors in the model for water runoff mass and the percentage of washoff of cypermethrin. Tukey’s multiple comparison adjustment was used to determine significant differences (a < 0.05) between the building materials and the 2 formulation types. The split-plot analysis using SAS software (mixed-models procedure) [17] was used to determine if there were significant differences in the collection of runoff between building materials and formulations. The analysis showed that formulation was not a significant factor. There were some differences among building materials in water runoff mass (mean range 1505–1989 g), but asphalt (mean 468 g) was found to be significantly different from all other building materials (p < 0.05). The mechanics of the rainfall simulator limited the number of slabs tested in a single experimental simulated event. In addition, applications had to be grouped by formulation to 1) comply with the application-to-rainfall time constraint, and 2) avoid possible cross-contamination. The split-plot design made it possible to evaluate the influence of the main effects, building material and formulation in regard to the mass of water runoff, and the percentage of washoff of cypermethrin and to evaluate the interaction of the factors. The simulation event was also included in the model. The fixed part of the model consisted of the following form to evaluate the main effects and their interaction: formulation þ building material þ (formulation  building ma- terial). The random part of the model was the interaction of formulation and simulation event. This analysis was followed by comparing differences of means using the Tukey adjustment. The masses for the asphalt slabs ranged between 295 g and 591 g. The low recovered water masses are most likely a result of the material absorbing water during the simulation events. This may be related to the level of compaction of the material during the construction phase or the lack of a sealant, which is typically used on roads to prevent water absorption over time. Simulated rainfall and runoff The rainfall simulator performed consistently with little variability between simulation events. The mean rainfall amount for all events was within 10% of the target rainfall amount (2.54 cm). The mean water runoff masses varied from 468.3 g for asphalt to 1988.9 g for clean unpainted concrete (Supplemental Data, Figure S4). The mean runoff masses were used with the mean rainfall to determine runoff coefficients from each surface; the runoff coefficients ranged between 0.80 to 1.06 with the exception of the asphalt, which was estimated to be approxi- mately 0.25. Typical values for impervious surfaces such as concrete and asphalt range from 0.70 to 0.95 [18], while urban playground and suburban areas are between 0.20 and 0.40 [18]. Clean painted and unpainted concrete and dirty painted wood had runoff coefficients >1; however, the positions of these slabs within the simulator most likely received higher rainfall than the mean rainfall, giving a runoff coefficient closer to 1.0. Building material slab cypermethrin washoff results Masses of cypermethrin in runoff water from the building material slabs were added to the masses recovered from the walls of the washoff sample container. The total mass of cypermethrin that washed off each building material slab was compared with the estimated mean applied mass for each formulation (determined from the application monitoring samples). Data are presented graphically (Figure 1) in terms of percentage of applied cypermethrin for all building materials and formulations. Field blanks (clean aluminum siding) were analyzed, and the runoff contained no detectable residues (less than limit of quantification of 1.03 mg/L). Data analysis In addition, there may have been splash or leakage from the test slabs, which contributed to a lower water runoff mass; however, none of these potential causes were observed. Additional information regard- ing rainfall, water runoff, and transit stability results can be found in the Supplemental Data. Simulated rainfall events Simulated rainfall events were conducted using a 3-story indoor laboratory rainfall simulator at the University of Illinois. Environ Toxicol Chem 33, 2014 304 J.R. Trask et al. J.R. Trask et al. temperature, thoroughly agitated, and then analyzed for cypermethrin in water. Other matrices analyzed were the empty bottles from the washoff samples, the tank mix samples, and the application monitoring samples (filter papers). The empty sample bottles were washed with dichloromethane, and then the rinsate was analyzed to determine the amount of cypermethrin remaining in the sample bottle. The estimated mass of cypermethrin recovered from the empty bottle was added to the amount of cypermethrin found in the water phase of the washoff sample. Application monitoring samples were extracted and washed with an acetonitrile/acetone solution (2:98 by volume) and then analyzed for cypermethrin. More information about sample extraction can be found in the Supplemental Data. estimated mean masses of cypermethrin applied (as opposed to the target values) to the building material slabs for each formulation. Application monitoring samples The application monitoring samples were used to determine the mean mass of cypermethrin applied to the test slabs for each formulation. Using the effective spray interception area per Petri dish (176.4 cm2/dish) and the effective spray interception area per building material slab (1393.5 cm2/slab), the mean mass recovered was determined for each formulation. The recovered mean mass for Cynoff EC insecticide from the Petri dishes, as measured from the laboratory analysis, was 4.27  0.11 mg a.i. (n ¼ 6), while the mean mass recovered for Cynoff WP insecticide was 5.40  0.52 mg a.i. (n ¼ 6). By scaling these masses based on the relative areas of Petri dishes and slabs, the spray solution loadings per slab were 33.8 mg a.i. and 42.7 mg a. i. for the EC insecticides and WP insecticides, respectively. The masses per Petri dish recovered demonstrate that the cyper- methrin application in the present study was higher than the target rates. The final washoff results were based on these The results showed that clean vinyl siding produced the highest mean percentage of washoff, while clean unpainted stucco generated the lowest values for both formulations. With the exception of clean vinyl siding, the Cynoff WP formulation washoff values were higher than for the EC formulation as percentage of applied cypermethrin. The washoff data produced residuals that were not normally distributed, so the data were log-transformed, which produced residuals that satisfied the assumptions of normality and equal variance. Tables 1 and 2 present the washoff means expressed as a percentage of applied cypermethrin for the EC and WP formulations, respectively. The Washoff of cypermethrin residues Environ Toxicol Chem 33, 2014 305 Figure 1. Washoff of cypermethrin as percentage of amount applied for 10 building materials and by formulation (n ¼ 3 per building material/ formulation). ASP ¼ clean asphalt; CPW ¼ clean painted wood; CAL ¼ clean aluminum siding; CPS ¼ clean painted stucco; CVL ¼ clean vinyl siding; DPW ¼ dirty painted wood; CPC ¼ clean painted concrete; CUS ¼ clean unpainted stucco; CUC ¼ clean unpainted concrete; CUW ¼ clean unpainted wood; EC ¼ emulsifiable concentrate; WP ¼ wettable powder. Table 2. Application monitoring samples Cypermethrin washoff of applied mass (means and confidence intervals) and summary of statistical significance (Tukey’s test) for Cynoff WP formulation Building material Cynoff WP insecticide washoff (% of applied mass) Tukey’s groupinga Clean vinyl siding 5.4 (2.9–9.9) A Clean aluminum siding 3.6 (2.0–6.6) A,B Clean unpainted wood 2.7 (1.5–4.9) A,B,C Clean unpainted concrete 1.3 (0.73–2.4) B,C,D Dirty painted wood 0.65 (0.35–1.2) C,D,E Clean asphalt 0.57 (0.31–1.04) C,D,E Clean painted wood 0.42 (0.23–0.77) D,E Clean painted stucco 0.22 (0.12–0.41) E,F Clean painted concrete 0.08 (0.04–0.14) F,G Clean unpainted stucco 0.04 (0.02–0.07) G aTukey’s multiple comparison test evaluates pairwise differences between materials. Materials sharing the same letter have mean percent washoffs of applied mass that are not significantly different. For example,clean painted stucco for the WP is an “E,” which means there is no difference between it and other “E” designations such as clean painted wood; however, clean vinyl siding is an “A,” which means there is a significant difference between it and clean painted stucco. WP ¼ wettable powder. Table 2. Cypermethrin washoff of applied mass (means and confidence intervals) and summary of statistical significance (Tukey’s test) for Cynoff WP formulation Figure 1. Washoff of cypermethrin as percentage of amount applied for 10 building materials and by formulation (n ¼ 3 per building material/ formulation). ASP ¼ clean asphalt; CPW ¼ clean painted wood; CAL ¼ clean aluminum siding; CPS ¼ clean painted stucco; CVL ¼ clean vinyl siding; DPW ¼ dirty painted wood; CPC ¼ clean painted concrete; CUS ¼ clean unpainted stucco; CUC ¼ clean unpainted concrete; CUW ¼ clean unpainted wood; EC ¼ emulsifiable concentrate; WP ¼ wettable powder. and clean aluminum siding (1.04%) were found to be significantly different from textured surfaces such as clean unpainted concrete (0.14%). Dirty painted wood (0.30%) was found to be similar to most materials excluding clean vinyl siding, clean unpainted wood, and clean painted and unpainted stucco. Smoother surfaces produced higher washoff values than textured surfaces when comparing individual building material means. analysis showed that building material, formulation, and their interaction were significant. Building material and formulation (main effects) were most significant (F ¼ 82.53 and 76.64, respectively), while the interaction was <10% of the main effect F values. Therefore, it is reasonable to say that 1 formulation had a far higher percentage overall and the building materials surface did impact cypermethrin washoff. Application monitoring samples Washoff results from the WP formulation, unlike the EC formulation, showed there was no single building material that was significantly different from all others. As seen in Table 2, clean vinyl siding (5.4%) was not significantly different from clean aluminum siding (3.6%) and clean unpainted wood (2.7%) but was different from all other building materials. Clean unpainted stucco (0.04%) was found to be significantly different from all building materials except clean painted concrete (0.08%). The remaining materials showed more similar washoff fractions than the EC formulation. For example, clean aluminum siding was found to be similar to clean vinyl siding, clean unpainted wood, and clean unpainted concrete but significantly different from all others. Dirty painted wood (0.65%) was significantly different from clean vinyl siding, clean aluminum, clean painted concrete, and clean unpainted stucco. Similar results were seen when the comparison of building materials independent of formulation was examined (Table 3). The study suggested that the effects of surface roughness played a significant role in the losses of cypermethrin from building material surfaces. Tukey’s adjustment was used to examine which building materials were significantly different from each formulation. For the EC formulation, many building material surfaces proved to be significantly different (from each other; p < 0.05), which can be seen in Table 1. Clean vinyl siding (14.1%) and clean unpainted stucco (<0.01%) were significantly different from all other building materials, representing the highest and lowest percentage of applied washoff, respectively; the remaining materials were distributed into 3 additional groups. Materials with smoother surfaces such as clean unpainted wood (1.6%) Table 1. Cypermethrin washoff of applied mass (means and 95% confidence intervals) and summary of statistical significance (Tukey’s test) for Cynoff EC formulation Building material Cynoff EC insecticide washoff (% of applied mass) Tukey’s groupinga Clean vinyl siding 14.1 (7.7–25.8) A Clean unpainted wood 1.6 (0.85–2.86) B Clean aluminum siding 1.0 (0.57–1.9) B,C Dirty painted wood 0.30 (0.16–0.55) C,D Clean unpainted concrete 0.14 (0.07–0.25) D,E Clean painted wood 0.10 (0.06–0.19) D,E Clean asphalt 0.06 (0.03–0.12) D,E Clean painted concrete 0.06 (0.03–0.12) D,E Clean painted stucco 0.04 (0.02–0.07) E Clean unpainted stucco <0.01 (0.00–0.01) F aTukey’s multiple comparison test evaluates pairwise differences between materials. Materials sharing the same letter have mean percentage ofwashoffs of applied mass that are not significantly different. Application monitoring samples Materials sharing the same letter have mean percent washoffs of applied mass that are not significantly different. For example,clean painted concrete is an “E,” which means there is no difference between it and other “E” designations such as clean painted stucco; however, clean vinyl siding is an “A,” which means there is a significant difference between it and clean painted concrete. EC ¼ emulsifiable concentrate; WP ¼ wettable powder. Table 3. Cypermethrin washoff of applied mass (means and confidence intervals) and summary of statistical significance (Tukey’s test) of building materials (n ¼ 6) esfenvalerate and lambda-cyhalothrin) than the present results. The differences when compared with Cynoff EC may be attributed to several factors, such as differences in formulation, different slopes, different methods of application, and different experimental designs (number of replicates and replicate variability) and scales. Jorgensen and Young used concrete surfaces that were 4 degrees from the horizontal, while the present study used a greater angle (60 degrees from horizontal); therefore, the lower results seen in the present study may be attributed to a shorter retention time of water on the slab surface. Jorgensen and Young pointed out that their method of using a handheld sprayer may be more crude than more precise application methods yet similar to how pest-control operators would apply products. The study concluded that application rates and differences in the active ingredient’s chemical properties did not significantly explain the differences seen in total mass washoff between the formulated products. However, both the Jorgensen and Young article and the present study conclude that formulation type can be a contributing factor to total washoff of pyrethroids. aAnalysis includes both formulations (EC and WP) in a single group per building material surface,which are compared. bTukey’s multiple comparison test evaluates pairwise differences between materials. Materials sharing the same letter have mean percent washoffs of applied mass that are not significantly different. For example,clean painted concrete is an “E,” which means there is no difference between it and other “E” designations such as clean painted stucco; however, clean vinyl siding is an “A,” which means there is a significant difference between it and clean painted concrete. EC l ifibl WP bl d In a more recent study by Jorgensen et al. CONCLUSIONS The present study showed that cypermethrin mass in runoff varied significantly between building material types and formulation. Smoother surfaces produced higher washoff values than textured surfaces, and formulation was found to be significant on surfaces where the roughness was greater. In general, the WP formulation had a higher percentage of washoff overall than the EC formulation. In addition, the present study showed that the amount of water runoff was affected by building material surface and construction. Further work is being conducted to examine the effects of formulation in greater detail. In addition, experiments including larger scales, environmentally weathered material surfaces, and applications under current use practices are needed to determine the relevance of the present results to actual use conditions. Application monitoring samples For example, clean painted stucco for the EC is an “E,” which means there is no difference between it and other “E” designations such as clean painted concrete; however, clean vinyl siding is an “A,” which means there is a significant difference between it and clean painted stucco. EC ¼ emulsifiable concentrate. Table 1. Cypermethrin washoff of applied mass (means and 95% confidence intervals) and summary of statistical significance (Tukey’s test) for Cynoff EC formulation The analysis also showed that formulation was a significant factor in the percentage of washoff. The ANOVA results showed overall that the EC formulation (0.19%) behaved differently from the WP formulation (0.61%). Although the mean percentage of washoff values, when examining individual building materials, showed that greater losses occurred from the WP formulation than the EC formulation except for clean vinyl siding, only 4 building materials showed significant differences (p < 0.05) in washoff percentage between the EC and WP formulations, based on Tukey’s adjusted p values. These building materials—asphalt, clean painted and unpainted stucco, and clean unpainted concrete—had lower washoff than most of the other building materials, excluding clean unpainted concrete. aTukey’s multiple comparison test evaluates pairwise differences between materials. Materials sharing the same letter have mean percentage ofwashoffs of applied mass that are not significantly different. For example, clean painted stucco for the EC is an “E,” which means there is no difference between it and other “E” designations such as clean painted concrete; however, clean vinyl siding is an “A,” which means there is a significant difference between it and clean painted stucco. EC ¼ emulsifiable concentrate Environ Toxicol Chem 33, 2014 J.R. Trask et al. 306 J.R. Trask et al. Table 3. Cypermethrin washoff of applied mass (means and confidence intervals) and summary of statistical significance (Tukey’s test) of building materials (n ¼ 6) Building material Cypermethrin washoff (% of applied mass)a Tukey’s groupingb Clean vinyl siding 8.7 (5.7–13.4) A Clean unpainted wood 2.0 (1.3–3.1) B Clean aluminum siding 1.9 (1.3–3.0) B Dirty painted wood 0.44 (0.29–0.68) C Clean unpainted concrete 0.42 (0.28–0.66) C Clean painted wood 0.21 (0.14–0.32) C,D Clean asphalt 0.19 (0.12–0.30) C,D,E Clean painted stucco 0.09 (0.06–0.14) D,E Clean painted concrete 0.07 (0.05–0.11) E Clean unpainted stucco 0.01 (0.01–0.02) F aAnalysis includes both formulations (EC and WP) in a single group per building material surface,which are compared. bTukey’s multiple comparison test evaluates pairwise differences between materials. Application monitoring samples [8], washoff as percentage of applied mass for EC formulations from concrete surfaces were similar to the results presented in the present study (1.2–2.4% washoff), although different active ingredients and a smaller slope (4 degrees) were used. However, the suspension concentrate formulation showed much greater losses, indicating again that formulation is an important factor in washoff results. This suggested, in general, that as the surface of the material became smoother there was less impact of formulation on the resulting washoff characteristics from the treated building material surfaces. For example, clean painted concrete was the least significant of the materials in terms of formulation differences (p ¼ 1.0), followed by clean unpainted wood (p ¼ 0.99). Clean vinyl siding (p ¼ 0.74) produced the highest percentage of applied washoff (Table 3) and was significant when comparing building materials, but it was not significantly different in terms of formulation. This may be because of the particle size of the applied formulation versus the available surface area coverage with respect to the depth of coverage. It may also be a function of the final prepared solution and particle size (i.e., colloidal vs dissolved particle). The track sprayer was able to deliver a uniform application, so it is unlikely that the differences were caused by differences in application techni- ques. The hypothesis that there is no difference in pyrethroid washoff between different formulations of cypermethrin was rejected based on the analysis. However, one must consider that the results for the WP formulation exhibited increased variability (as measured by the nontransformed data standard deviation) between replicates compared with the same building material surfaces with the EC formulation. The present study demon- strates that the type and texture of the building material surface in combination with the formulation applied result in varying losses of cypermethrin. The present study also demonstrated that the surface texture of the material plays a role in the potential transport of pyrethroids in washoff. A similar conclusion was made by Jiang et al. [10] after testing 3 types of surface conditions (stamped, acid wash, and silicone sealant) on concrete slabs. The stamped concrete generally produced less pyrethroids in runoff water than the other surface types. The authors also concluded that the effects of surface conditions on pyrethroid washoff may be increased at a lower rainfall intensity than used in the present study (26.2 mm/h). DISCUSSION The present study shows that washoff of pyrethroids is significantly affected by the specific building material to which it has been applied. Other studies in the published literature have examined washoff from concrete surfaces, turf, and bare soil, so the results from the present study are unique in examining the potential for pyrethroids to wash off from other typical building surfaces. In comparing concrete surface results, Jorgensen and Young [7] reported a 10- to 20-fold higher mass washoff as percentage of applied active ingredient under similar experi- mental conditions (2.5 cm/h rainfall for 1 h, 24-h delay between application and rainfall) with EC pyrethroid formulations (a.i. SUPPLEMENTAL DATA Sections S1-S8 Tables S1-S4 Figure S1-S4. (294 KB DOCX). Acknowledgment—The authors thank the Pyrethroid Working Group for support and financing throughout the study. The authors also thank FMC Corporation for supplying test materials and support during the study. Great appreciation goes to the University of Illinois at Urbana-Champaign, 307 Environ Toxicol Chem 33, 2014 Washoff of cypermethrin residues 8. Jorgenson BC, Wissel-Tyson C, Young TM. 2012. Factors contributing to the off-target transport of pyrethroid insecticides from urban surfaces. J Agric Food Chem 60:7333–7340. Department of Agricultural and Biological Engineering and Department of Natural Resources and Environmental Sciences, for use of their facilities to conduct the study. The authors also thank S. Aref for her expertise and assistance with the statistical analysis. Finally, the authors also extend their appreciation to numerous reviewers for their time and efforts spent to make this research publishable. 9. Jiang W, Lin K, Haver D, Qin S, Ayre G, Spurlock F, Gan J. 2010. Wash-off potential of urban use insecticides on concrete surfaces. Environ Toxicol Chem 29:1203–1208. 10. Jiang W, Haver D, Rust M, Gan J. 2012. Runoff of pyrethroid insecticides from concrete surfaces following simulated and natural rainfalls. Water Res 46:645–652. REFERENCES 11. Grubb JA, Limaye HS, Kakade AM. 2007. Testing pH of concrete: Need for a standard procedure. Concrete International 29:78–83. 1. Moran KM. 2010. Pesticides in urban runoff, wastewater, and surface water. Annual Urban Pesticide Use Data Report 2010. San Francisco Estuary Partnership, San Mateo, CA, USA. 12. Laskowski DA. 2002. Physical and chemical properties of pyrethroids. Rev Environ Contam Toxicol 174:49–170. 13. Hirschi MC, Mitchell JK, Feezor DR, Lesikar BJ. 1990. Microcomputer- controlled laboratory rainfall simulator. Trans Am Soc Agric Eng 33:1950–1953. 2. Weston DP, Lydy MJ. 2010. Urban and agricultural sources of pyrethroid insecticides to the Sacramento–San Joaquin delta of California. Environ Sci Technol 44:1833–1840. 14. Galitz CL, Whitlock AR. 1999. Simulating design storms with water chamber testing. In Boyd JA, Scheffler MJ, eds, Water Problems in Building Exterior Walls: Evaluation, Prevention, and Repair, Vol 3. American Society for Testing and Materials West Conshohocken, PA, USA, pp 276–289. 3. Weston DP, Holmes RW, Lydy MJ. 2008. Residential runoff as a source of pyrethroid pesticides to urban creeks. Environ Pollut 157:287–294. 4. Ding Y, Harwood AD, Foslund HM, Lydy MJ. 2009. Distribution and toxicity of sediment-associated pesticides in urban and agricultural waterways from Illinois, USA. Environ Toxicol Chem 29:149–157. 15. Erpul G, Norton LD, Gabriels D. 2006. Erosion by wind-driven rain. In Lal R, ed. Encyclopedia of Soil Science, Vol 1. CRC Press, Boca Raton, FL, USA, pp 624–627. 5. Schmidt C. 2010. Sediment pyrethroid sampling in irrigation canal/ditch system, Missoula, Montana. Montana Department of Agriculture. Helena, MT, USA. 16. Hanzas JP, Jones RL, White JW. 2011. Runoff transport of pyrethroids from a residential lawn in central California. J Environ Qual 40:587–597. 6. Greenberg L, Rust M, Klotz JH, Haver D, Kabashima JN, Bondarenko S, Gan J. 2010. Impact of ant control technologies on insecticide runoff and efficacy. Pest Manag Sci 66:980–987. 17. SAS Institute. 2012. Users’s Guide: Statistics Ver 9. Cary, NC, USA. 18. Schwab GO, Fangmeier DD, Elliot WJ, Frevert RK. 1993. Soil and Water Conservation Engineering. 4th ed. John Wiley & Sons, New York, NY, USA. 7. Jorgenson BC, Young TM. 2010. Formulation effects and the off-target transport of pyrethroid insecticides from urban hard surfaces. Environ Sci Technol 44:4951–4957.
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The role of modelling and analytics in South African COVID-19 planning and budgeting
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PLOS GLOBAL PUBLIC HEALTH PLOS GLOBAL PUBLIC HEALTH OPEN ACCESS Citation: Meyer-Rath G, Hounsell RA, Pulliam JRC, Jamieson L, Nichols BE, Moultrie H, et al. (2023) The role of modelling and analytics in South African COVID-19 planning and budgeting. PLOS Glob Public Health 3(7): e0001063. https://doi.org/ 10.1371/journal.pgph.0001063 Methods Our tools included epidemic projection models, several cost and budget impact models, and online dashboards to help government and the public visualise our projections, track case development and forecast hospital admissions. Information on new variants, including Delta and Omicron, were incorporated in real time to allow the shifting of scarce resources when necessary. Copyright: © 2023 Meyer-Rath et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Eunha Shim, Soongsil University, KOREA, REPUBLIC OF Received: August 22, 2022 Accepted: May 24, 2023 Published: July 3, 2023 Received: August 22, 2022 Accepted: May 24, 2023 Published: July 3, 2023 Copyright: © 2023 Meyer-Rath et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE The role of modelling and analytics in South African COVID-19 planning and budgeting Gesine Meyer-RathID1,2,3*, Rachel A. Hounsell4,5, Juliet RC PulliamID3, Lise JamiesonID1,3,6, Brooke E. NicholsID1,2,6,7, Harry MoultrieID8, Sheetal P. SilalID4,5 1 Faculty of Health Sciences, Health Economics and Epidemiology Research Office, University of the Witwatersrand, Johannesburg, South Africa, 2 Department of Global Health, School of Public Health, Boston University, Boston, Massachusetts, United States of America, 3 South African DSI-NRF Centre of Excellence in Epidemiological Modelling and Analysis (SACEMA), Stellenbosch University, Stellenbosch, South Africa, 4 Department of Statistical Sciences, Modelling and Simulation Hub, Africa, University of Cape Town, Cape Town, South Africa, 5 Nuffield Department of Medicine, Centre for Tropical Medicine and Global Health, University of Oxford, Oxford, United Kingdom, 6 Department of Medical Microbiology, Amsterdam University Medical Center, Amsterdam, the Netherlands, 7 Foundation for Innovative New Diagnostics, Geneva, Switzerland, 8 National Institute for Communicable Diseases (NICD), a division of the National Health Laboratory Service, Johannesburg, South Africa a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * gesine@bu.edu Background The South African COVID-19 Modelling Consortium (SACMC) was established in late March 2020 to support planning and budgeting for COVID-19 related healthcare in South Africa. We developed several tools in response to the needs of decision makers in the different stages of the epidemic, allowing the South African government to plan several months ahead. Editor: Eunha Shim, Soongsil University, KOREA, REPUBLIC OF Conclusion The SACMC’s models, developed rapidly in an emergency setting and regularly updated with local data, supported national and provincial government to plan several months ahead, expand hospital capacity when needed, allocate budgets and procure additional resources where possible. Across four waves of COVID-19 cases, the SACMC continued to serve the planning needs of the government, tracking waves and supporting the national vaccine rollout. PLOS GLOBAL PUBLIC HEALTH PLOS GLOBAL PUBLIC HEALTH The role of modelling and analytics in South African COVID-19 planning and budgeting capacity. Finally, real-time analyses of the most important characteristics of the Omicron variant first identified in South Africa in November 2021 allowed us to advise policymakers early in the fourth wave that a relatively lower admission rate was likely. authors and do not necessarily reflect the views of PEPFAR, USAID or the United States Government. SPS, RAH and the development of the SACMC dashboards are funded by the Wellcome Trust (GN: 2114236/Z/18Z) and the Clinton Health Access Initiative. JRCP is supported by the Department of Science and Innovation and the National Research Foundation. Any opinion, finding, and conclusion or recommendation expressed in this material is that of the authors, and the NRF does not accept any liability in this regard. The SACMC’s work is also supported by the Bill & Melinda Gates Foundation under Investment INV-035464. The views and opinions expressed in this report do however not necessarily reflect the positions or policies of the Bill & Melinda Gates Foundation. Introduction Competing interests: The authors have declared that no competing interests exist. As of May 2022, South Africa has experienced four waves of COVID-19, with an official tally of more than 3,711,000 cases and 101,000 reported deaths [1]. The country reported its first imported COVID-19 case on 5 March 2020, with subsequent rapid spread into all districts in the country. In response, the South African government implemented a five-level COVID-19 alert system, beginning with a full lockdown (Level 5) from late March 2020 [2]. The alert levels determined the extent of restrictions to be applied during the national state of disaster, which was initiated in March 2020 and ended two years later, in April 2022 [2]. The risk-adjusted approach was guided by several criteria, including numbers of infections and rate of transmis- sion, health facility capacity, the extent of the implementation of public health and social mea- sures (PHSM), as well as the economic and social impact of continued restrictions [2]. The South African COVID-19 Modelling Consortium (SACMC) was established at the end of March 2020 in response to a request by the South African National Department of Health (NDOH) to project the spread of the disease to support policy and planning in South Africa over the course of the epidemic. The consortium developed two models to project incidence, deaths, need for hospital beds at different levels of care and the corresponding resources required. The models incorporated available data on COVID-19 cases, severity and mortality, and served to inform the public and a range of decision makers, including the Ministerial Advisory Committee on COVID-19 (MAC) advising the Minister of Health, staff in the NDOH and National Treasury, officials in the provincial departments of health and the private health sector. This paper describes the 2-year process of continuously updating the models while collabo- rating with the diverse partners. The paper further provides an overview of the model results and the consortium’s recommendations at the different stages of the South African COVID-19 epidemic. The aim is to highlight the dynamic, multidisciplinary nature of policy-driven modelling of an emergency in a country with severely constrained resources. A full description of the models developed during wave 1 is provided in [3], and our analysis of COVID-19 related hospitalisations in [4]. Results Data Availability Statement: All data are available in publicly accessible reports under https:// sacmcepidemicexplorer.co.za/. Given the rapidly changing nature of the outbreak globally and in South Africa, the model projections were updated regularly. The updates reflected 1) the changing policy priorities over the course of the epidemic; 2) the availability of new data from South African data sys- tems; and 3) the evolving response to COVID-19 in South Africa, such as changes in lock- down levels and ensuing mobility and contact rates, testing and contact tracing strategies and hospitalisation criteria. Insights into population behaviour required updates by incorpo- rating notions of behavioural heterogeneity and behavioural responses to observed changes in mortality. We incorporated these aspects into developing scenarios for the third wave and developed additional methodology that allowed us to forecast required inpatient Funding: The work of GMR and LJ on the SACMC has been made possible by the generous support of the American People and the President’s Emergency Plan for AIDS Relief (PEPFAR) through the United States Agency for International Development (USAID) under the terms of Cooperative Agreement 72067419CA00004 to HE2RO. The contents are the responsibility of the 1 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 Policy-driven modelling Since its establishment in March 2020, the SACMC has provided policy-driven modelling and analytics support in response to the evolving priorities of decision makers across the different stages of the epidemic. Several tools were developed and adapted over time to meet these needs. Taken together, these tools supported the South African government at national and provincial levels to conduct timely resource planning, shift scarce resources and implement appropriate PHSM. At the start of the epidemic, the most pressing need was for short- and long-term projec- tions of COVID-19 cases, including the number of severe and critical cases requiring hospital admission, and deaths under different PHSM scenarios. To fulfil these needs, the SACMC developed the National COVID-19 Epi Model (NCEM), a compartmental transmission model following a generalised Susceptible-Exposed-Infectious-Removed structure that accounts for disease severity (asymptomatic, mild, severe and critical cases) and treatment pathways (out- patient, inpatient non-ICU and ICU care) [3]. The National COVID-19 Cost Model (NCCM), a companion model, used epidemiological outputs from the NCEM on the number of mild, severe and critical cases. Based on this, it pro- jected total COVID-19 resource needs and the associated impact on the national and provin- cial health budgets by incorporating information on the need for inpatient and outpatient resources (including their baseline availability and how to scale availability with the size of the epidemic). Resource projections covered drugs, diagnostics, ventilators, oxygen supply, field hospitals and other hospital infrastructure, staffing requirements and additional mortuary space. Model extensions included the quantification and cost of vaccines under different vacci- nation scenarios. These projections informed the development of resource quantifications and budgets, allowing timely negotiation with manufacturers and preparation of contracts for the additional resources anticipated based on precise quantifications. The exact volumes required could be regularly updated based on the latest model outputs. As the epidemic progressed, in addition to the ongoing projections described above, prior- ity was placed on resurgence monitoring, estimating the impact of relevant emerging variants of concern and modelling to inform the government’s vaccination procurement and rollout strategy. Between March 2020 and December 2022 in South Africa, apart from the wildtype virus that drove the first case wave, there were three dominant variants of concern: Beta, B.1.351, first detected in South Africa; Delta, B.1.617.2; and Omicron, B.1.1.529, which was again first identified in South Africa. The South African COVID-19 Modelling Consortium The South African COVID-19 Modelling Consortium The SACMC is a group of researchers from academic, non-profit and government institutions across South Africa. Established in late March 2020 by the NDOH, its mandate is to provide, PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 2 / 18 PLOS GLOBAL PUBLIC HEALTH The role of modelling and analytics in South African COVID-19 planning and budgeting assess and validate model projections to be used for planning purposes by the Government of South Africa. The SACMC’s work is coordinated by the South African National Institute for Communicable Diseases (NICD), which maintains the datasets used by the SACMC’s models. In addition to its core group of experienced infectious disease modellers and health econo- mists, the SACMC convenes experts across a range of disciplines to provide insights, guide the selection of appropriate parameter values, ensure a close alignment to current clinical practice and sense-check model outputs. Policy-driven modelling Fig 1 shows the tools developed over the course of the epidemic in South Africa and the timing of their main outputs. Use of modelling The SACMC’s modelling outputs have been used by a range of strategic and operational deci- sion makers (Table 1). Different departments within the NDOH used NCEM outputs for pur- poses ranging from the quantification of drug volumes required for inpatient and outpatient care by the Affordable Medicines Directorate to the estimation of additional mortuary and PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 3 / 18 PLOS GLOBAL PUBLIC HEALTH The role of modelling and analytics in South African COVID-19 planning and budgeting Fig 1. Select SACMC modelling and analysis contributions: Timeline from March 2020 to December 2021. See Table A in S1 Appendix for additional information. Fig 1. Select SACMC modelling and analysis contributions: Timeline from March 2020 to December 2021. See Table A in S1 Appendix for additional i f ti Fig 1. Select SACMC modelling and analysis contributions: Timeline from March 2020 to December 2021. See Table A in S1 Appendix for additional information Fig 1. Select SACMC modelling and analysis contributions: Timeline from March 2020 to December 2021. See Table A information. https://doi.org/10.1371/journal.pgph.0001063.g001 https://doi.org/10.1371/journal.pgph.0001063.g001 https://doi.org/10.1371/journal.pgph.0001063.g001 graveyard spaces by the Environmental Health Directorate. Private sector initiatives such as the National Ventilator Project, which sourced ventilation equipment for public and private hospitals in South Africa, also used our model outputs. In addition, analysts coordinated by the Reserve Bank, the South African central bank, used SACMC model outputs to predict the macro-economic impact of the epidemic under different scenarios. The resurgence monitoring tool was used by planners and technical advisory teams to declare a resurgence of cases and respond according to the guidelines detailed in the NDOH Resurgence Plan [5]. The third and fourth wave scenario modelling and short-term forecasts were primarily used to estimate hospital admissions ahead of a third wave driven by the Delta variant in June 2021, and a fourth wave driven by the Omicron variant in November 2021. Outputs of the NCCM and vaccination cost models were used by the National Treasury to make decisions regarding the additional budget allocation required for COVID-19. Model out- puts, in part, informed a budget allocation of 1.42 billion USD for COVID-19 specific health care announced by the South African president at the end of April 2020 [6], most of which was financed through the reallocation of the existing health budget [7]. PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 Use of modelling Additionally, a group of experienced public finance specialists was trained to work with provincial Departments of Table 1. Uses of NECM and NCCM outputs. User Purpose NDOH: Ministerial Advisory Committee Policy advice Facility Readiness Committee Number of beds and expansion of facilities Human Resources for Health team Staff required per level National Ventilator Project Number of ventilators needed Oxygen planning team Oxygen required Affordable Medicines Directorate Drug quantities National Health Laboratory Service Number of tests and testing priorities Environmental Health Directorate Number of mortuary containers Provincial DOHs Local planning and resource quantification National Treasury COVID-19 health budgets https://doi.org/10.1371/journal.pgph.0001063.t001 Table 1. Uses of NECM and NCCM outputs. 4 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 PLOS GLOBAL PUBLIC HEALTH The role of modelling and analytics in South African COVID-19 planning and budgeting Health to update the model with provincially specific data. This included the baseline availabil- ity of resources, prices and need for resources that were independent of the course of the epi- demic, such as personal protective equipment and isolation and quarantine facilities. Key parameters and data sources The parameter values for the early versions of the NCEM were based on literature and data from other countries as well as local expert opinion regarding the types, duration and out- comes of hospital treatment. Parameter values and assumptions were regularly updated as the scientific knowledge base on COVID-19 expanded. As South African data became available, parameters were adjusted to reflect the local context. Parameter selection was guided by ongo- ing input from clinicians, virologists, intensive care specialists, immunologists and epidemiol- ogists on the SACMC. Table 2 contrasts the initial set of parameter values from late April 2020 and the updated set from September 2020; Table 3 provides an overview of the main data sources. The NCCM used three types of input data: 1) The type and required quantities of resources, such as the number of inpatient beds projected by the NCEM, human resources at all care lev- els, oxygen, oxygen delivery devices, SARS CoV-2 tests, infection control and prevention infra- structure; 2) the public-sector prices of these resources; and 3) the baseline volume of resources available for the COVID-19 health response for all items where the quantities required exceeded existing resource levels, such as in the case of hospital beds or ventilators. Costs were evaluated from the provider perspective, in this case the South African government. Despite the need to provide COVID-19 testing and care in both the public and the private sec- tors, this perspective is appropriate given that contracting arrangements were put in place to ensure private-sector services were offered at charges similar to public-sector prices. As a result, we used public-sector prices and salaries throughout, based on the most recent tariffs and public tenders (Table 4). PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 PLOS GLOBAL PUBLIC HEALTH The role of modelling and analytics in South African COVID-19 planning and budgeting S1 Appendix). With a need for planning support at a finer spatial granularity and lifting of restrictions on intra-country travel, a stochastic version of the NCEM (v3) was extended to cover the 52 districts of South Africa, linked through a connectivity matrix formulated by mobile data over time. NCEM v4 was necessary after the detection of the Beta variant in South Africa (Figure C in S1 Appendix). The model was further stratified to include 7 age groups across three subpopulations of interest: healthcare workers, the population with comorbidities and everyone else. Vaccination was included to incorporate a generic vaccine’s potential effec- tiveness against infection and severity. In anticipation of another variant and with the vaccina- tion programme underway for healthcare workers, NCEM v5 was developed to include a third Table 2. Key NCEM parameter values and their evolution between April and September 2020. Evolution of modelling purpose, structures and tools across the epidemic Projections and scenarios for short- and long-term COVID-19 burden. Government set three priorities for modelling and projections during the first wave of the COVID-19 epi- demic in South Africa: 1) to generate short- and long-term projections of COVID-19 cases, estimating the pace at which cases might increase and spread between provinces; 2) to project the expected number of severe and critical cases leading to hospital admission, as well as esti- mates of the corresponding resource requirements; and 3) to compute the cost of the health sector response to the epidemic at a provincial and national level in order to inform the adjust- ment of the health budget and the flow of resources around the country. To fulfil the first and second priority, we developed the NCEM, a compartmental transmis- sion model to estimate the total and reported incidence of COVID-19 in the nine provinces (and later, 52 districts) of South Africa. It was designed to simulate the impact of different behavioural scenarios, inform resource requirements and predict where gaps could arise based on the available resources within the South African health system. The model follows a gener- alised Susceptible-Exposed-Infectious-Removed (SEIR) structure accounting for disease sever- ity (asymptomatic, mild, severe and critical cases) and the treatment pathway as shown in Fig 2. Version 1 of the NCEM v1, stratified at the provincial level, accounted for the clinical pro- file of SARS-CoV-2 and the hospital care pathway (Figure A in S1 Appendix). With limitations on general ward and ICU capacity, the model was extended in v2 to account for limited access to hospital level care and restricted capacity in wards at the provincial level (Figure B in PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 5 / 18 PLOS GLOBAL PUBLIC HEALTH PLOS GLOBAL PUBLIC HEALTH Parameter April 2020 September 2020 Value* (range) Sources Value* (range) Sources Infection severity and transmission** Proportion of cases that are asymptomatic 75% [9–11] 75% (70%–80%) [8–11] Relative infectiousness of asymptomatic cases - - 80% (77.5%, 82.5%) [12–14] Estimated through calibration to admissions and fatalities count data (DATCOV) [15] Mild to moderate cases among the symptomatic (95.64%, 96.78%) adjusted based on [17] (94.55%–97.13%) Estimated through calibration to admissions and fatalities count data (DATCOV) [15,16] Severe cases among the symptomatic (2.46%–3.64%) (2.58%–5.00%) Critical cases among the symptomatic (1.16%–1.45%) (0.18%–0.55%) Proportion of cases that are fatal (0.30%, 0.412%) [17,18] Timeframes & treatment duration Time from infection to onset of infectiousness 4 days (2–9) [18,19–23], with additional input from analysis of NICD data 2 days (1–3) [24–34] with input from the National COVID-19 Modelling Consortium Time from onset of infectiousness to onset of symptoms 2 days (1–4) 4 days (3–5) Duration of infectiousness from onset of symptoms 5 days 5 days (4–6) [35,36] Time from onset of mild symptoms to testing 4 days (2–4) 4 days (3–5) [25,26,18–21,23] Time from onset of symptoms to hospitalisation 5 days (4–8) 5 days (4–6) Time from onset of symptoms to ICU admission 9 days (8–17) see below Duration of hospital stay 12 days (7–16) Duration from ICU admission to discharge 18 days (14–18) Duration from ICU admission to death 5 days (4–7) Time in non-ICU (never ICU) to death/ recovery 8 days (4–12) Lengths of stay: values and ranges sourced from NICD COVID-19 Hospital Sentinel Surveillance database (DATCOV) [15,4] Time in non-ICU for those destined for ICU 0 days (0–2) Time in ICU for those ventilated and destined to die 14 days (7–27) Time in ICU for those never ventilated and destined to die 11 days (7–18) Time in ICU for those ventilated and recovered 19 days (15–37) Time in ICU for those never ventilated and recovered 5 days (1–10) Time in non-ICU for those who were in ICU and recovered 0 days (0–6) https://doi.org/10.1371/journal.pgph.0001063.t002 S1 Appendix). With a need for planning support at a finer spatial granularity and lifting of restrictions on intra-country travel, a stochastic version of the NCEM (v3) was extended to cover the 52 districts of South Africa, linked through a connectivity matrix formulated by mobile data over time. NCEM v4 was necessary after the detection of the Beta variant in South Africa (Figure C in S1 Appendix). PLOS GLOBAL PUBLIC HEALTH The model was further stratified to include 7 age groups across three subpopulations of interest: healthcare workers, the population with comorbidities and everyone else. Vaccination was included to incorporate a generic vaccine’s potential effec- tiveness against infection and severity. In anticipation of another variant and with the vaccina- tion programme underway for healthcare workers, NCEM v5 was developed to include a third S1 Appendix). With a need for planning support at a finer spatial granularity and lifting of restrictions on intra-country travel, a stochastic version of the NCEM (v3) was extended to cover the 52 districts of South Africa, linked through a connectivity matrix formulated by mobile data over time. NCEM v4 was necessary after the detection of the Beta variant in South Africa (Figure C in S1 Appendix). The model was further stratified to include 7 age groups across three subpopulations of interest: healthcare workers, the population with comorbidities and everyone else. Vaccination was included to incorporate a generic vaccine’s potential effec- tiveness against infection and severity. In anticipation of another variant and with the vaccina- tion programme underway for healthcare workers, NCEM v5 was developed to include a third PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 6 / 18 PLOS GLOBAL PUBLIC HEALTH The role of modelling and analytics in South African COVID-19 planning and budgeting Table 3. Summary of NCEM data sources. National case and hospitalisation data from the South African National Institute for Communicable Diseases Statistics South Africa projected 2020 district population projections [37] Coronavirus COVID-19 (2019-nCoV) Data Repository for South Africa, Data Science for Social Impact Research Group @ University of Pretoria [38] Medical Research Council analyses of weekly excess deaths [39,40] Vaccine uptake and coverage from the National Department of Health’s Electronic Vaccination Data System Vodacom Mobile Event Database Google COVID-19 Community Mobility Reports Published and pre-print academic literature (cited in Table 2) Expert input from members of the SA COVID-19 Modelling Consortium and press releases and notices from the South African government’s COVID-19 Online Resource and News Portal (https://sacoronavirus.co.za/category/ press-releases-and-notices/) https://doi.org/10.1371/journal.pgph.0001063.t003 Table 3. Summary of NCEM data sources. PLOS GLOBAL PUBLIC HEALTH National case and hospitalisation data from the South African National Institute for Communicable Diseases Statistics South Africa projected 2020 district population projections [37] Coronavirus COVID-19 (2019-nCoV) Data Repository for South Africa, Data Science for Social Impact Research Group @ University of Pretoria [38] Medical Research Council analyses of weekly excess deaths [39,40] Vaccine uptake and coverage from the National Department of Health’s Electronic Vaccination Data System Vodacom Mobile Event Database Google COVID-19 Community Mobility Reports Published and pre-print academic literature (cited in Table 2) Expert input from members of the SA COVID-19 Modelling Consortium and press releases and notices from the South African government’s COVID-19 Online Resource and News Portal (https://sacoronavirus.co.za/category/ press-releases-and-notices/) https://doi.org/10.1371/journal.pgph.0001063.t003 Table 3. Summary of NCEM data sources. variant stratification for this hypothetical new variant. The model was updated at the begin- ning of the third wave to represent early data on the characteristics of the Delta variant (Figure D in S1 Appendix). With NCEM v6, vaccination was included in greater detail, with specific vaccination types based on those planned to be locally administered and additional dif- ferentiation between vaccine effectiveness in those with prior infection vs the immunologically naïve (Figure E in S1 Appendix). Lastly, just the NCEM updated to v7 just before the fourth wave with a fourth stratification for another hypothetical new variant. This produced various scenarios driven by different joint assumptions regarding its transmissibility and immune escape properties (Figure F in S1 Appendix). This version was updated to the characteristics of the Omicron variant within days of Omicron being named. Model versions v4-7 were updated to represent vaccine coverage and uptake across provinces and age groups at each time point based on vaccination data from the National Department of Health. The NCEM was originally developed as a deterministic compartmental model, where the characterisation of uncertainty changed over the course of the pandemic. Early on in the first wave, when uncertainty in parameter values was large and the least complex framework of the NCEM was implemented, uncertainty in parameter values was explored through sensitivity Table 4. Summary of NCCM data sources. Intervention/ ingredient Type of target population Data sources Target population Cost Baseline capacity PPE Healthcare workers at all levels DHIS Own analysis NDOH ICU beds, staff and linen Critically ill cases NCEM Own analysis NDOH Ventilators Critically ill cases (subset) NCEM Public tender NDOH Testing All cases who present for testing Own analysis NHLS NHLS Community health workers (CHW) 10,000 DHIS Public salaries - CHW supplies Thermometers for 10,000 CHW - Public tender - Isolation - Own analysis - O2 Severely + critically ill cases NCEM Own analysis NDOH Hospital beds, staff and linen Severely ill cases NCEM Own analysis - Drugs All cases (by level of severity) NCEM Own analysis - PHC staff 70% of symptomatic mild cases NCEM Own analysis - Fever clinics NDOH NDOH NDOH - 30-bed wards NDOH NDOH NDOH - Mortuary cupboards and cabinets Deaths by municipality NCEM NDOH NDOH https://doi.org/10.1371/journal.pgph.0001063.t004 Table 4. Summary of NCCM data sources. Table 3. Summary of NCEM data sources. PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 7 / 18 PLOS GLOBAL PUBLIC HEALTH The role of modelling and analytics in South African COVID-19 planning and budgeting Fig 2. Original NCEM model structure (v1). https://doi.org/10.1371/journal.pgph.0001063.g002 Fig 2. Original NCEM model structure (v1). https://doi.org/10.1371/journal.pgph.0001063.g002 Fig 2. Original NCEM model structure (v1). Fig 2. Original NCEM model structure (v1). https://doi.org/10.1371/journal.pgph.0001063.g002 https://doi.org/10.1371/journal.pgph.0001063.g002 analyses and uncertainty bounds estimated through random draws from specified parameter distributions. When the need arose to expand the model to a finer spatial granularity in v3, the NCEM was additionally implemented stochastically to account for greater uncertainty at small geographical levels. The inclusion of variants and vaccines into NCEM versions 4 onwards brought an additional layer of uncertainty in projection modelling, and scenario analyses were employed to depict uncertainty in the outcomes of unknown vaccine and variant characteristics. 8 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 PLOS GLOBAL PUBLIC HEALTH The role of modelling and analytics in South African COVID-19 planning and budgeting Resource planning. The National COVID-19 Cost Model (NCCM) takes inputs from the NCEM and cost inputs based on data from existing sources that were adapted to represent the type, number and prices of ingredients required in South Africa’s COVID-19 response. It cal- culates annual budgets for the NDOH’s response to COVID-19, allocating costs at the level of the provinces as well as NDOH, incremental to existing resources such as hospital beds and staff contingents. The NCCM was updated with new NCEM results when they became available and was changed to incorporate additional interventions when deemed relevant by policy makers and planners (for example, temporary inpatient infrastructure such as field hospitals and add-on clinic space). Additional updates included new clinical interventions once they were incorpo- rated into national COVID-19 management guidelines (such as dexamethasone treatment and high-flow nasal cannulae treatment), and prices and quantities as new tenders and data on actual resource use became available. For example, in July 2020 we adjusted our assumptions regarding inpatient length of stay downwards from the initial estimates (which were based on international literature) to results from our analysis the South African hospital sentinel surveil- lance database [15] (for more detail regarding the methods see [4]); and inpatient costs were updated based on a more detailed analysis of South African ingredients and prices [41]. Dashboard for disseminating model updates. PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 Table 3. Summary of NCEM data sources. Between early April and early September 2020, the NCEM was updated frequently and results made available to stakeholders within the South African government. Reports on a subset of these updates were additionally made pub- lic. To aid the timely dissemination of projections, we developed a web-based interactive appli- cation, the National COVID-19 Epi Model Dashboard (https://masha-app.shinyapps.io/ NCEMDashboard), which visualised the most important NCEM outputs on projected cases, hospitalisations and deaths. These included active cases and cumulative detected cases for symptomatic, severe and critical cases; hospital beds needed and cumulative admissions for non-ICU, ICU ventilated and ICU non-ventilated; and cumulative deaths. Resurgence monitoring. At the beginning of the second wave of COVID-19 cases in Sep- tember 2020, driven by the Beta variant which originated in South Africa, resurgence monitor- ing became a priority. The SACMC assisted in the development of a set of resurgence metrics to support planners and technical advisory teams to declare a resurgence or wave and respond according to the guidelines detailed in the Ministry of Health’s resurgence action plan [5]. To facilitate effective communication and dissemination of these metrics, we developed a second web-based dashboard called the SACMC Epidemic Explorer in two versions: a sub-district level version to guide planners at all government levels, and a district-level version (www. SACMCEpidemicExplorer.co.za) to inform the public. It allows users to explore the COVID- 19 epidemic in South Africa, analyses resurgence risk, monitor confirmed COVID-19 hospital admissions and deaths, and presents metrics to prepare for future outbreaks, including esti- mates of case levels, percentage change in cases and periods of consistent growth. We mea- sured the metrics’ performance against three main criteria: whether the resurgence metrics were consistent in their messaging; whether the metrics provided sufficient early warning; and whether the metrics relaxed the severity of their messaging at a suitable time and pace after the peak of the epidemic. Short-term forecasting. Towards the end of the second wave, short-term forecasts of COVID-19 cases, hospital admissions, and of the likelihood of when provinces would meet the third, then fourth wave criterion were developed. This enabled improved situational awareness and informed government resource planning. These two-week case and admission forecasts were updated weekly and three times weekly, respectively, and displayed on the SACMC Epi- demic Explorer for easy access. Table 3. Summary of NCEM data sources. PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 9 / 18 PLOS GLOBAL PUBLIC HEALTH The role of modelling and analytics in South African COVID-19 planning and budgeting Findings of the National COVID-19 Epi Model (First wave) In May 2020, using version 1, the model projected an estimated 8.01 and 8.62 million labora- tory-confirmed cases and 40,223 and 43,759 deaths in the optimistic and pessimistic scenarios, respectively, by 1 October. Active cases were estimated to peak in early July in the pessimistic, and mid-July in the optimistic scenario, with a maximum number of between 72,281 and 77,899 hospital beds and 31,656 and 24,150 ICU beds in use at peak. Total cumulative inci- dence was estimated to reach between 48.7 and 51.7 million cases (symptomatic or asymptom- atic) by 1 October (i.e., an attack rate of 82–88%), with between 8.01 and 8.62 million detected cases, assuming a detection factor of 1 in 6 cases. While these projections incorporated a cer- tain level of effectiveness of PHSM in their scenarios, they underestimated the severity and effect of government restrictions and population adherence to them and thus assumed that all exposure would happen during a single wave. A full set of projections for all nine provinces is available in our detailed reports [42,43]. The updates in version 2 took into account the variation in timing and level of peaks of the epi- demics between the provinces and between the districts in each province. This resulted in an esti- mated national peak in cases at a a similar time (i.e., mid-August 2020) to the optimistic scenario from version 1, but at a lower level. While the model projected a concomitant lower peak in the need for hospital (non-ICU) and ICU beds at a national level, bed capacity was still expected to be breached or overwhelmed in all provinces. We noted that increasing capacity to accommodate patients in hospital could allow the country to better leverage new therapeutic options, such as high-flow oxygen and dexamethasone, which had the potential to improve mortality outcomes. In version 3, we modelled the impact of four behavioural scenarios on the four provinces with the most advanced epidemics: Western Cape, Eastern Cape, Gauteng and KwaZulu- Natal. The application of each of these behavioural scenarios led to earlier and/or lower peak- ing of cases than our original projections. One scenario (in which the behavioural response threshold was assumed to be 110 deaths per day) peaked at roughly the same level but shifted the peak forward. The full analysis detailing the behavioural scenarios is available in [44]. Findings of the National COVID-19 Epi Model (First wave) p y p infections by September, equating to 25.5% (uncertainty range: 22.0%–28.6%) of the population —a much lower first-wave attack rate than estimated in our first version. Under the moderate testing scenario, cumulative detected cases were estimated to continue to grow until between 570,000 and 1.2 million cases by early November (and only marginally so thereafter), depending on testing rates. The peak number of general hospital (i.e., non-ICU) beds in use was estimated to have been reached in early-August, at around 8,000 beds (when we had projected that around 12,500 beds would have been needed). The peak number of ICU beds in use was estimated to have been reached around the same time, with around 1,100 beds—although more than 2,000 beds would have been needed. Total deaths are estimated to continue to increase until early November when the cumulative number of all deaths would reach 37,000 (of which 16,000 would have been in hospital); thereafter the growth rate was estimated to be very low [44]. PLOS GLOBAL PUBLIC HEALTH Budget item Description Scenario with 20,000 additional ICU beds Total cost [millions 2020 USD] % of total cost PPE Personal protective equipment for healthcare workers at all levels 360 18% Testing PCR tests only; no new laboratory instruments or extra staff 145 7% Central functions Port Health and surveillance 23 1% Intensive Care Unit beds incl. additional beds required, linen and staff costs 426 21% Ventilators Additional ventilation equipment required for inpatient care 74 4% Oxygen Oxygen cylinders for inpatient care, excludes oxygen equipment 179 9% Hospital beds incl. additional beds required, linen and staff costs 9 0% Drugs at ICU, general wards and primary healthcare clinics 211 10% PHC staff for screening, testing, clinical assessment, post-test follow-up 32 2% CHW supplies 1.1 thermometers per community healthcare worker 7 0.3% Isolation facilities Upgrading and repurposing of hotel and conference facilities to be used as facilities for isolation of mild cases 60 3% Fever clinics 1000 units to be added to primary health centres and community health centres 218 11% 30-bed COVID-19 wards Attached to existing hospital or to field hospital 299 15% TOTAL 2,043 https://doi.org/10.1371/journal.pgph.0001063.t005 PLOS GLOBAL PUBLIC HEALTH The role of modelling and analytics in South African COVID-19 planning and budgeting The role of modelling and analytics in South African COVID-19 planning and budgeting Table 5. The projected COVID-19 health budget for financial year 2020/21. Note that not all of these components were necessarily supported or included in the final allocations in the order of 1.5 billion USD. Budget item Description Scenario with 20,000 additional ICU beds Total cost [millions 2020 USD] % of total cost PPE Personal protective equipment for healthcare workers at all levels 360 18% Testing PCR tests only; no new laboratory instruments or extra staff 145 7% Central functions Port Health and surveillance 23 1% Intensive Care Unit beds incl. additional beds required, linen and staff costs 426 21% Ventilators Additional ventilation equipment required for inpatient care 74 4% Oxygen Oxygen cylinders for inpatient care, excludes oxygen equipment 179 9% Hospital beds incl. PLOS GLOBAL PUBLIC HEALTH additional beds required, linen and staff costs 9 0% Drugs at ICU, general wards and primary healthcare clinics 211 10% PHC staff for screening, testing, clinical assessment, post-test follow-up 32 2% CHW supplies 1.1 thermometers per community healthcare worker 7 0.3% Isolation facilities Upgrading and repurposing of hotel and conference facilities to be used as facilities for isolation of mild cases 60 3% Fever clinics 1000 units to be added to primary health centres and community health centres 218 11% 30-bed COVID-19 wards Attached to existing hospital or to field hospital 299 15% TOTAL 2,043 https://doi org/10 1371/journal pgph 0001063 t005 The NCCM estimated that the budget required for the COVID-19 health response for financial year 2020/21 would be around 2.1 and 2.7 billion USD under the NCEM’s optimistic and pessimistic scenarios, respectively (Table 5). Scenarios differed in the cost of those budget items that were directly linked to the number of projected cases, particularly for inpatient care (ICU and non-ICU beds, ventilators and oxygen) and drugs at all levels of the healthcare sys- tem. In both scenarios, the largest contributor to total cost were the procurement and staffing of additional ICU beds (21% and 26%, resp., in the optimistic and pessimistic scenario), per- sonal protective equipment (PPE) for healthcare workers at all levels (18% and 14%) and pre- fabricated facility infrastructure discussed at that time. This included additions to primary healthcare clinics for the management of non-severe patients (“fever clinics”) (11% and 9%) and 30-bed stand-alone COVID-19 wards as additions to hospitals (15% and 12%). Based on these results, about 1.5 billion USD was added under the COVID-19 Special Adjustment Budget in June 2020, funded through a combination of reprioritisation of funds from other departments and within the provincial health budgets and increased lending [7]. Most of this budget was used in expenditure on inpatient care and PPE, although over 12 months instead of the originally estimated 6 months, and allocated slightly differently. Few of the large infrastructure projects were implemented, except for the construction of three large field hospitals in the worst-hit provinces, in two cases through the repurposing of existing buildings, and with completion so delayed that very few patients were admitted. In total, fewer ICU beds were added due to severe constraints in the trained staff needed to staff them. Findings of the National COVID-19 Cost Model (First wave) Given the regular updates to the NCCM’s structure and inputs and the ongoing changes to the country’s COVID-19 management policies and clinical guidelines, model results changed almost weekly for the duration of the first wave. Amongst the many updates, we report on a version from the end of May 2020, which used input from the first NCEM version to inform an additional allocation to COVID-19 related healthcare for the 2020/21 financial year. PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 10 / 18 PLOS GLOBAL PUBLIC HEALTH PLOS GLOBAL PUBLIC HEALTH The NCCM estimated that the budget required for the COVID-19 health response for financial year 2020/21 would be around 2.1 and 2.7 billion USD under the NCEM’s optimistic and pessimistic scenarios, respectively (Table 5). Scenarios differed in the cost of those budget items that were directly linked to the number of projected cases, particularly for inpatient care (ICU and non-ICU beds, ventilators and oxygen) and drugs at all levels of the healthcare sys- tem. In both scenarios, the largest contributor to total cost were the procurement and staffing of additional ICU beds (21% and 26%, resp., in the optimistic and pessimistic scenario), per- sonal protective equipment (PPE) for healthcare workers at all levels (18% and 14%) and pre- fabricated facility infrastructure discussed at that time. This included additions to primary healthcare clinics for the management of non-severe patients (“fever clinics”) (11% and 9%) and 30-bed stand-alone COVID-19 wards as additions to hospitals (15% and 12%). Based on these results, about 1.5 billion USD was added under the COVID-19 Special Adjustment Budget in June 2020, funded through a combination of reprioritisation of funds from other departments and within the provincial health budgets and increased lending [7]. Most of this budget was used in expenditure on inpatient care and PPE, although over 12 months instead of the originally estimated 6 months, and allocated slightly differently. Few of the large infrastructure projects were implemented, except for the construction of three large field hospitals in the worst-hit provinces, in two cases through the repurposing of existing buildings, and with completion so delayed that very few patients were admitted. In total, fewer ICU beds were added due to severe constraints in the trained staff needed to staff them. Findings from resurgence modelling, forecasts and scenario modelling (Wave 2 onwards) The resurgence monitoring dashboard provided continuous updates three times a week on the state and growth of the epidemic across provinces, districts and sub-districts from December 2020 onwards. The metrics on the dashboard classified all areas as being in a state of control, Table 5. The projected COVID-19 health budget for financial year 2020/21. Note that not all of these components were necessarily supported or included in the final allocations in the order of 1.5 billion USD. Findings from resurgence modelling, forecasts and scenario modelling (Wave 2 onwards) The resurgence monitoring dashboard provided continuous updates three times a week on the state and growth of the epidemic across provinces, districts and sub-districts from December 2020 onwards. The metrics on the dashboard classified all areas as being in a state of control, 11 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 PLOS GLOBAL PUBLIC HEALTH The role of modelling and analytics in South African COVID-19 planning and budgeting alert or response, which triggered a series of actions laid out in a Department of Health work- plan [5]. The short-term forecasts provided two-week forecasts on the trajectory of cases and admissions at the provincial level from May 2021 onwards. These projections were placed within the context of the relationship between weekly admissions and hospital-based case fatal- ity rates that was seen in prior waves, with one notable exception: At the beginning of the fourth wave (Omicron), we did not provide admissions forecasts due to uncertainty regarding the relationship between cases and admissions under this novel variant. The scenario modelling for the third wave accounted for province-level data on seropreva- lence and the dominance of new variants. The modelling estimated that, across most provinces and behavioural scenarios, the peak admissions of the third wave would be lower than that of the second wave. However, we saw that a slow, weak behavioural response would increase admissions for severe or critical COVID-19 cases across most age groups. Younger age groups were expected to have fewer admissions than in the second wave in most scenarios. Our analy- sis by province showed substantial variation in the size of the third wave between provinces, reflecting different age distributions, seroprevalence and prevalence of comorbidities. The third wave was highest in more urban provinces due to the higher concentration of working- age adults and people with co-morbidities. Across provinces, the time from an initial increase in transmission to the peak was estimated to be 2–3 months on average. A full report detailing scenario projections for each of the nine provinces is available in [45,46]. Lastly, our scenarios for the fourth wave projected that hospital admissions during the fourth wave would be less than during the third wave. This scenario considered a large range of assumptions regarding immune escape properties and transmissibility, a hypothetical new variant, loss of protection against severe infection and increase in contacts. Policy impact While we are confident that our projections have provided important data to the evidence on which decisions and actions of the South African government were based, we are equally aware that they were merely one aspect that comes to bear on how these decisions were derived and which actions were implemented.—Other factors with a bearing on government decision making were clinical evidence, economic and social considerations, and aspects of political and practical feasibility. We can, therefore, claim no direct causal link between our data and any of the actions of the South African government, including decisions regarding what public health and social measures to implement at what stage. We do know, however, that staff within the national and provincial departments of health used our projections throughout the pan- demic to plan for and reallocate required resources. Moreover, since December 2020our resur- gence monitoring application was used to plan for local additional waves. Equally, SACMC members were part of the Ministerial Advisory Committee on COVID-19 and of a working group of the Ministerial Advisory Committee on COVID-19 Vaccines, and contributed to some of these advisories. Findings from resurgence modelling, forecasts and scenario modelling (Wave 2 onwards) The projected reduction in hospital admissions was in large part due to high population levels of combined immunity from previous infection and, to a lesser extent, vaccination (owing to low vaccine coverage at the time). This remained true in our updated sets of scenarios incorporating the higher transmissibility and immune evasion properties of Omicron compared to previous variants. Discussion The process of providing policy-relevant research on the evolution and impact of an aggressive novel pathogen required a relevant, timely and responsive modelling approach; ongoing re- 12 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 PLOS GLOBAL PUBLIC HEALTH The role of modelling and analytics in South African COVID-19 planning and budgeting assessment of the availability of local and international data and evidence; and effective, trans- parent navigation of uncertainty. Partly based on above, this section gives an overview of the lessons we learned through our experience providing modelling and analytics support throughout the COVID-19 epidemic in South Africa, and expands these lessons to hold value for future pandemic preparedness efforts in South Africa and other low- or middle-income countries. The modelling approach needs to be relevant and responsive to evolving policy priorities Over the course of an epidemic, the type of questions that are most important to decision mak- ers changes. Modellers need to work closely with model users so that the outputs developed are relevant and timely, serving the dynamic needs of the user. Further, establishing strong relationships and embedding continuous engagement with stakeholders throughout the modelling process is fundamental to accelerating the use of modelling output into decision making. In South Africa, policy needs changed over time. At first, urgent long-term projec- tions of the shape and peak of the first wave were required. Then, the need shifted to modelling the impact of behaviour to explain how the first wave had diverted from expectation. Subse- quently, there was a need for real-time resurgence monitoring during the ensuing waves (each driven by a new variant). This new approach also needed to incorporate all previous learnings into third wave projections (alongside two-week forecasts of hospital-relevant data). Further- more, just ahead of the fourth wave, scenarios were provided that included a potential novel variant with immune escape properties. Finally, we updated our fourth wave scenarios with the results of our own rapid-fire analysis of Omicron properties within days of the emergence of this variant. The approach discussed in this paper demonstrates the value of the adaptive nature of our policy-driven modelling work. PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 Models should take local context into account There are several challenges to modelling in low- and middle-income countries in general. Data systems and surveillance infrastructure are often underdeveloped. Constrained resources, especially during health emergencies, lead to overwhelmed hospital staff who are unable to feed data systems in real time. This can cause delays and errors in reporting. Heterogeneity in population characteristics and access to health infrastructure are vital elements in the disease ecosystem and essential inputs to disease models. Infectious disease models should account for the local context, such as data availability, health systems dynamics, demography, contact pat- terns, acceptability of interventions and cultural influences. In countries where data availability is scarce, it is important to understand what adaptations are needed and how models may per- form under different data constraints, and to communicate the resulting uncertainty effec- tively. As such, the adaptation of plug-and-play models from resource-richer countries remains an inferior option, as these are usually ill-equipped to provide reliable, ongoing, real- time decision-making support tailored to local needs, data and epidemic specifics. One exam- ple of this is our mis-specification of mortality rates in v1 of our model which was a result of a misinterpretation of international data on case fatality rates, which were the only available data in March/April 2020 [42]. Incidentally, updating the model with locally derived mortality rates after the first South African wave resulted in very similar, albeit more correct, estimates of the number of overall deaths [44]. Another is our decision to not incorporate the impact of indi- vidual PHSM, despite clear and regular requests from policy makers to help with decisions regarding which individual restrictions would still be necessary. This decision was taken because, despite regular reviews, we did not find a robust enough dataset applicable to a low- or middle-income setting or South Africa specifically regarding the impact of these measures. Modellers need to be flexible in the tools they apply At different stages of the epidemic, the varying availability of data and most pressing policy questions should inform the selection of an appropriate modelling or analytics approach. Short-term modelling can account for rapid and frequent changes, quickly incorporating new data and enabling a short lead time between updates and communication of findings. This is particularly relevant for important but rapidly changing inputs such as available hospital capacity [42–44]. Additionally, our initial projections had intentionally projected the number of beds needed without taking capacity and cost constraints into account, the actual capacity to accommodate severe and critical cases was much lower. As such, we added outputs (including budgets) based on actual bed use. The ability to project both bed need and bed use during the first wave, predict the extent of the health burden across provinces during the third and fourth waves, and monitor resurgence patterns and bed occupancy at the sub-district level, were pos- sibly amongst our most useful contributions. However, even these outputs’ usefulness was lim- ited by the non-fungibility of healthcare resources in many instances. The strongest constraint on inpatient bed availability was human resources, which could not be easily shifted and much less created over a short time span. Additionally, more than two thirds of deaths happened out- side of hospitals, potentially pointing to COVID-19 patients being disheartened by reports of locally overwhelmed hospitals [47–49], being turned away by overburdened emergency rooms, becoming too sick too quickly to seek care in time, or dying in transit where emergency transport was scarce. PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 13 / 18 PLOS GLOBAL PUBLIC HEALTH The role of modelling and analytics in South African COVID-19 planning and budgeting Modellers need to make judgement calls about when to produce new projections- and if at all We learned that it is in the hands of the modeller to judge if sufficient data are available and/or modelling can responsibly support the decisions to be made. Ahead of the second wave in South Africa in October 2020, model predictions of the shape and timing of the peak of this second wave were urgently requested. The SACMC decided to first assess the driving forces behind the resurgence, considering factors such as increased mobility, PHSM fatigue, lower seroprevalence or new variants. In the absence of additional information at the time (with the Beta variant only discovered in December 2020), the SACMC made the difficult decision not to produce model-based projections. Instead, we choose to develop a set of metrics that could detect and monitor the second wave. Further updates to the NCEM were only resumed in anticipation of the third wave and the vaccine roll-out programme, once more information was available. At the beginning of the fourth wave, our use of epidemiological tools such as reinfection analysis [50] allowed us to quickly produce bounding estimates for Omicron’s combined transmissibility and immune escape properties [51], which in turn provided reliable estimates of bed needs for this wave. Acknowledgments The authors acknowledge the support of the National Department of Health and all planners, stakeholders and decision-makers who made use of these data systems and analyses to respond to the COVID-19 epidemic in South Africa. A large number of colleagues within the National Institute of Communicable Diseases, the Ministerial Advisory Committee on COVID-19, the Clinton Health Access Initiative, provincial and national Departments of Health and National Treasury alerted us to policy-relevant questions, assisted the development of our models by providing data, discussing details of model structure and reviewing assumptions and results. We are especially grateful to decision makers and planners at all levels of the health system who continuously used our outputs in the management of COVID-19 in South Africa. Communicating uncertainty clearly and transparently is vital when reporting model findings Managing expectations regarding the limitations of models, the quality of data and the assumptions used, improves the likelihood of model outputs being used responsibly and appropriately by decision makers. We used several widely accepted approaches for doing this, including using scenario and sensitivity analyses and clearly marked uncertainty ranges for 14 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0001063 July 3, 2023 PLOS GLOBAL PUBLIC HEALTH The role of modelling and analytics in South African COVID-19 planning and budgeting input parameters and results. We found that uncertainty, while central to our understanding of our role as modellers, was not always useful to our audience of health planners. Especially where additional health resources had to be made available, users reverted to simply using our median estimates to inform budgets and order exact quantities. By the same token, we realised that communicating our methods and the sources of our assumptions to the general public was equally fundamental to building their trust in our results—though it was somewhat harder. Conclusion In developing the NCEM, NCCM and a number of dashboards and additional outputs such as reports and briefing materials, the SACMC supported national and provincial government to plan several months ahead, expanding hospital facilities where needed, and procuring addi- tional resources. As the country is continuing its path towards endemicity, the SACMC con- tinues to serve the planning needs of the government, tracking the development of cases and admissions and developing models to further support the national vaccine rollout. Disease modelling was a source of regularly updated scientific evidence for decision making in the South African epidemic. Much progress was made in developing models rapidly in an emergency setting. However, many challenges remain and need to be overcome to incorporate local context, needs of policymakers and sub-optimal data systems, and to build disease model- ling capacity to better prepare for future health emergencies. References 1. 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Screening for potential prophylactics targeting sporozoite motility through the skin
Malaria journal
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Douglas et al. Malar J (2018) 17:319 https://doi.org/10.1186/s12936-018-2469-0 Douglas et al. Malar J (2018) 17:319 https://doi.org/10.1186/s12936-018-2469-0 Malaria Journal Open Access © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background:  Anti-malarial compounds have not yet been identified that target the first obligatory step of infection in humans: the migration of Plasmodium sporozoites in the host dermis. This movement is essential to find and invade a blood vessel in order to be passively transported to the liver. Here, an imaging screening pipeline was established to screen for compounds capable of inhibiting extracellular sporozoites. Methods:  Sporozoites expressing the green fluorescent protein were isolated from infected Anopheles mosquitoes, incubated with compounds from two libraries (MMV Malaria Box and a FDA-approved library) and imaged. Effects on in vitro motility or morphology were scored. In vivo efficacy of a candidate drug was investigated by treating mice ears with a gel prior to infectious mosquito bites. Motility was analysed by in vivo imaging and the progress of infec- tion was monitored by daily blood smears. Results:  Several compounds had a pronounced effect on in vitro sporozoite gliding or morphology. Notably, monen- sin sodium potently affected sporozoite movement while gramicidin S resulted in rounding up of sporozoites. How- ever, pre-treatment of mice with a topical gel containing gramicidin did not reduce sporozoite motility and infection. Conclusions:  This approach shows that it is possible to screen libraries for inhibitors of sporozoite motility and highlighted the paucity of compounds in currently available libraries that inhibit this initial step of a malaria infec- tion. Screening of diverse libraries is suggested to identify more compounds that could serve as leads in developing ‘skin-based’ malaria prophylactics. Further, strategies need to be developed that will allow compounds to effectively penetrate the dermis and thereby prevent exit of sporozoites from the skin. Keywords:  Plasmodium, Sporozoite, Gliding motility, MMV Malaria Box, Gramicidin, Monensin While often overlooked, the sporozoite stage of the life cycle presents a possible opportunity for prophylaxis [28–32]. Sporozoites form in oocysts within the mosquito vector and need their motility first to be released into the haemocoel of the insect [33], where they passively drift before actively invading salivary glands [34–36]. Dur- ing mosquito probing for a blood meal, sporozoites flow out with the saliva and are deposited in the skin of the mammalian host [30, 37–40]. Sporozoites, powered by an actomyosin system, move rapidly through the dermis using a form of locomotion referred to as gliding motility [30, 41, 42]. Sporozoites then associate with blood vessels and enter the blood stream whereby they passively drift before invading hepatocytes [29, 30, 43–45]. Ross G. Douglas*, Miriam Reinig, Matthew Neale and Friedrich Frischknecht* Ross G. Douglas*, Miriam Reinig, Matthew Neale and Friedrich Frischknecht* Backgroundh Thirdly, sporozoites might possibly be targeted by com- pounds directly applied to the skin, perhaps administered in the form of a daily body lotion or soap, thus avoiding the difficult pharmacological parameters of toxicity and bioavailability that many orally administered candidates encounter. Lastly, inhibitors of sporozoite motility could display broader inhibition of other stages and thus might also inhibit the active invasion of merozoites (needed for red blood cell invasion) and motility of midgut penetrat- ing ookinetes. chosen as any hits identified from this screen would possess desired drug-like properties, thereby accelerat- ing the discovery to application process and thus save time, money and licensing complications. A screen of the entire library against Plasmodium berghei liver stage invasion and/or development has already been per- formed [8]. Ninety-seven relevant hits from this screen (which could be targeting motility, invasion and/or intra- cellular development) were acquired as a potential drug short-list and assessed for direct effects on sporozoites. In vitro screening assay for sporozoite motility inhibition Rodents infected with P. berghei (NK65 strain express- ing GFP under the CSP promoter) were anesthetized, and starved mosquitoes allowed to feed. Salivary glands of infected mosquitoes (days 17–24 post-infection) were isolated by dissection, parasites placed into RPMI-1640 P/S buffer (supplemented with 50,000 units ­l−1 penicil- lin and 50  mg  l−1 streptomycin), released by mechani- cal crushing and briefly centrifuged. Sporozoites were resuspended in activation medium (RPMI-1640 P/S supplemented with 6% bovine serum albumin) and ali- quoted into a 384-well plate to a final amount of approxi- mately 2000 sporozoites per well. An equal volume of inhibitor (in RPMI-1640 P/S) was added promptly to the sporozoites to give a final concentration of either 1 µM or 10  µM and mixed by gentle pipetting. Some com- pounds of the Malaria Box were excluded due to high background fluorescence and thus affected the visualisa- tion of sporozoites: MMV006309 and MMV009127. The sporozoite-inhibitor mixture was centrifuged for 3 min at 1000 rpm to maximise sporozoite numbers adhering to the glass, incubated for 30 min and each well imaged at 1 Hz for 30 s. Backgroundh The increasing emergence of resistance to front-line anti-malarial drug artemisinin emphasizes the need for identification and development of novel drug candidates [1–3]. To reduce the occurrence of drug resistance, the malaria parasite Plasmodium should be blocked at mul- tiple stages of the life cycle [3–7]. In line with this, many studies have attempted to screen for compounds that are potent inhibitors of liver stage development, blood stage growth, gametocyte integrity, or transmission into the mosquito (or a combined potency of all of these) [8–27]. *Correspondence: ross.douglas@med.uni‑heidelberg.de; freddy.frischknecht@med.uni‑heidelberg.de Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany *Correspondence: ross.douglas@med.uni‑heidelberg.de; freddy.frischknecht@med.uni‑heidelberg.de Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany Sporozoites are a viable target for malaria prophylaxis for several reasons. Firstly, sporozoite deposition into the © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Douglas et al. Malar J (2018) 17:319 Page 2 of 10 Douglas et al. Malar J (2018) 17:319 skin presents a population bottleneck. Approximately 1–100 sporozoites are introduced into the skin during probing and thus only a small number of parasites need to be inhibited and/or cleared by the immune system [30, 38, 46]. Secondly, the skin step is the longest extracellu- lar stage of the life cycle in the human host (estimated to be more than 10 min) [37] and thus, due to this long exposure outside of a host cell, might be possibly more vulnerable to appropriate drugs or immune responses than merozoites. Stalling sporozoites in the skin could allow for sufficient time for the phagocytic cells of the immune system to clear them [47]. Indeed, inhibiting sporozoite migration can be achieved by antibodies tar- geting the circumsporozoite protein CSP [42, 48, 49]. Backgroundh To identify a maximum number of com- pounds, and due to the large number of compounds screened, the limitations of the number of sporozoites that one needs for screening and the nature of the assay itself (live imaging on parasites that only move for a short time), all initial hit identification in the pilot screen was done with a single assay per compound. All compounds from both libraries were first assessed with automated tracking software ToAST [50] and subsequently visually for changes in motility (by maximum intensity z-pro- jections) and sporozoite morphology. Compounds that showed potent inhibition in the pilot screen were further assessed for inhibition reproducibility with at least two additional biological replicates. Sporozoites were classed as moving if they moved more than 1 parasite length dur- ing the 30-s acquisition. The percentage residual motile population was then calculated and compared to unin- hibited controls (buffer solution containing an equiva- lent amount of DMSO). Compounds displaying > 75% To date, there have been no compound library screens performed on whole sporozoites to identify direct inhibi- tors of extracellular sporozoite motility. Here, the results of screens using two available drug libraries against motile sporozoites are presented. Using this approach, three compounds from the MMV Malaria Box (out of six initial hits) and antimicrobial ionophores monensin and gramicidin were identified as possible lead candidates for the potential use in malaria skin phase prophylaxis should an appropriate delivery method become available. These data also show that only a few compounds show inhibitory effects, suggesting that compounds identified from screens against a multiplying parasite might not be suited for repurposing to inhibit motile extracellular stages. MMV Malaria Box screen identifies potent inhibitors of sporozoite motilityh The Malaria Box has been used as a screening toolbox for the various stages of the Plasmodium life cycle [9] (Fig. 1a). Here, it was investigated whether compounds from this library were capable of directly inhibiting in vitro sporozoite motility. To this end, sporozoites were isolated from freshly dissected mosquito salivary glands and incubated with library compounds for 30 min. The effects of these compounds were then assessed by direct visualization (Fig. 1b). This identified six compounds that reproducibly displayed > 50% inhibition of sporozoite motility (Fig. 1c, Additional file  1: Table  S1, Additional file 2: Figure S1). As a positive inhibitor control, sporozo- ites were treated with 1 µM cytochalasin D (Cyto D), an actin toxin previously shown to be a potent inhibitor for sporozoite movement [52]. Treatment with Cyto D com- pletely abrogated sporozoite motility and resulted in more Compound libraries Two compound libraries were tested for effects on iso- lated sporozoites: the MMV Malaria Box and a short- listed version of a library containing FDA-approved drugs [8]. The Malaria Box is a library of approximately 400 compounds that were initially identified as hits of Plasmodium falciparum asexual blood stage develop- ment [10] and later screened for effects at other life cycle stages as well as different pathogens [9]. The FDA- approved drug library was made up of 1037 drugs that have received approval for human or animal use in the treatment of a spectrum of diseases. This library was Page 3 of 10 Douglas et al. Malar J (2018) 17:319 inhibition at these conditions were considered for in vivo characterization. adherent sporozoites consistent with previous observa- tions (Fig. 2a) [52, 53]. Interestingly, of the hits identi- fied, there were two pairs that shared the same starting scaffold (MMV665953 and MMV665852; MMV665794 and MMV007224). MMV665953 and MMV665852 are part of a family of four related compounds in the Malaria Box with the other two compounds (MMV000911 and MMV001318) not showing any noticeable activity against sporozoites in the pilot screen. In terms of potency, three compounds (MMV665953, MMV665852 and MMV007224) displayed approximately > 75% inhibition (Fig. 1a). However, due to reported toxic effects on hepat- ocytes [9] and a lack of a currently available topical treat- ment, these compounds were not further tested in vivo. A sub‑set of compounds from an FDA‑approved drug library are micromolar inhibitors of sporozoites library are micromolar inhibitors of sporozoites A previous study made use of an FDA-approved drug library to screen for liver stage drug candidates, resulting in the identification of decoquinate as a promising mul- tistage anti-malarial [8]. In this study, sporozoites were incubated with the compounds and added to liver cells. As a read-out, the development of liver stage parasites was used. This could be affected by either inhibiting liver stage growth or by liver cell invasion or gliding motility of sporozoites. A pilot screen was performed on the 97 drugs that were anticipated to have an effect on extracel- lular sporozoite motility and possibly not on liver devel- opment (Additional file 1: Table S1). From these only a small set of compounds showed prominent inhibition at the screening concentration of 1  µM (> 75% inhibition compared to DMSO control, Fig. 2b, c). These included zinc pyrithione, niclosamide and ionophores monensin sodium and gramicidin. Interestingly, monensin sodium completely blocked sporozoite motility by affecting proper sporozoite adhesion to the glass surface while not affecting shape (Fig. 2b). Gramicidin S treatment resulted in a pronounced rounding up of sporozoites presumably through membrane destabilization (Fig. 2c). Curiously tyrothricin, a mixture of tyrocidines and gramicidins, had a lesser effect on sporozoite morphology than grami- cidin alone. Nonetheless at 10 µM concentration a simi- lar effect on sporozoites could be observed as for 1 µM gramicidin. Given this observation, and that an inex- pensive topical gel formulation containing tyrothricin (and hence gramicidin) was available, the antibacterial ­Tyrosur® gel was selected for further in vivo analysis. In vivo validation of ­Tyrosur® gel y g To fully characterize the in vivo efficacy of one of the hits, a rodent model of infection was employed. All animal experiments were performed according to FELASA B and GV-SOLAS standard guidelines. Animal experiments were approved by the German authorities (Regierung- spräsidium Karlsruhe, Germany). Approximately 100 mg of ­Tyrosur® gel formulation (Engelhard Arzneimittel) was applied to the ear 4  h before the bite experiment. Mice were then anesthetized with a ketamine/xylazine mixture (87.5 mg kg ­ketamine−1, 12.5 mg kg−1 xylazine, administered intraperitoneally) and infected mosqui- toes allowed to bite on a single mouse ear for 10–20 min. For in vivo imaging on the ears of living mice, mice were anesthetized as above and the hair removed by treatment with hair removal cream (Veet) for 5 min 24 h prior to mosquito bite. The next day, mice were again anesthe- tized and infected mosquitoes allowed to bite the treated ear. Upon observation of a mosquito bite, the anesthe- tized mouse was immediately moved onto a heated chamber wide field microscope (Zeiss Axiovert TM200), the bite site identified and sporozoites imaged at 1 frame every 3 s for 5 min as described previously [51]. To moni- tor infection progression post-mosquito bite, mice were given standard drinking water and monitored daily from day 3 after biting by Giemsa staining to measure the pre- patent period and subsequent blood stage growth and compared to control mice, which were treated by a con- trol cream (Vaseline). Mann–Whitney statistical tests were conducted for non-parametric datasets. Pre‑treatment with ­Tyrosur® gel does not significantly affect sporozoite infection ability in mice ® Tyrosur® gel is used to treat and prevent infections of the skin [54, 55]. In order to assess whether pre-treatment of mouse skin with ­Tyrosur® gel was able to affect the Douglas et al. Malar J (2018) 17:319 Page 4 of 10 Fig. 1  Overview of previous and current screening strategies. a Previously, researchers have screened for compounds active at multiple stages (indicated in red text) yet there are no studies directly attempting to stop skin phase sporozoites (green text). b Summary of the current screening approach. Plasmodium berghei sporozoites are isolated from freshly dissected Anopheles stephensi mosquitoes and incubated with compounds for 30 min. Sporozoites are imaged and the effects on motility and morphology noted. Scale bar: 10 μm. c The Malaria Box is a condensed hit library from a large set of blood stage positive hits. The motility screen identified 6 compounds that displayed noticeable inhibition of extracellular sporozoites Fig. 1  Overview of previous and current screening strategies. a Previously, researchers have screened for compounds active at multiple stages (indicated in red text) yet there are no studies directly attempting to stop skin phase sporozoites (green text). b Summary of the current screening approach. Plasmodium berghei sporozoites are isolated from freshly dissected Anopheles stephensi mosquitoes and incubated with compounds for 30 min. Sporozoites are imaged and the effects on motility and morphology noted. Scale bar: 10 μm. c The Malaria Box is a condensed hit library from a large set of blood stage positive hits. The motility screen identified 6 compounds that displayed noticeable inhibition of extracellular sporozoites Fig. 1  Overview of previous and current screening strategies. a Previously, researchers have screened for compounds active at multiple stages (indicated in red text) yet there are no studies directly attempting to stop skin phase sporozoites (green text). b Summary of the current screening approach. Plasmodium berghei sporozoites are isolated from freshly dissected Anopheles stephensi mosquitoes and incubated with compounds for 30 min. Sporozoites are imaged and the effects on motility and morphology noted. Scale bar: 10 μm. c The Malaria Box is a condensed hit library from a large set of blood stage positive hits. The motility screen identified 6 compounds that displayed noticeable inhibition of extracellular sporozoites ability of sporozoites to infect new hosts, a mouse model was employed (Fig. 3a). Pre‑treatment with ­Tyrosur® gel does not significantly affect sporozoite infection ability in mice ® Mice were treated with ­Tyrosur® or control gels 4 h prior to exposure to infected mosqui- toes that were allowed to probe and feed on the treated (or control) region. Mice were subsequently placed on a heated stage of a microscope and fluorescent sporozo- ites filmed as they migrated in the skin (Fig. 3a). Image analysis revealed no difference between the two groups of mice in terms of sporozoite speed and migration pattern (Fig. 3b, c). Thus, pre-treatment of mice ears with a topi- cal application of gel did not affect sporozoite motility in the host skin. Consistent with these observations, mice exposed to infected mosquito bites showed no signifi- cant difference in prepatent period compared to controls, suggesting that this treatment also does not significantly affect sporozoite viability after skin exit and entry into the liver (Fig. 2d). Discussionh The skin phase of Plasmodium is surprisingly often over- looked in reviews describing drug discovery or vaccina- tion strategies in the malaria field [28]. Not only is this unfortunate in terms of omission of an important part of the parasite biology, but it also misses the important con- sideration of the skin being an additional area for infec- tion prevention. The possibility of skin phase prophylaxis has been proposed in previous studies [29–32]. Indeed, antibodies to the major surface protein of sporozoites have been shown to affect sporozoite movement in the skin [42] and could therefore contribute to the effects of the RTS,S vaccine [56, 57]. Many groups have per- formed screens assessing for antimalarial candidates act- ing at various or combined stages of the parasite life cycle [8–27]. However, to date only one paper has specifically screened and analysed inhibitors of whole sporozoites Page 5 of 10 Douglas et al. Malar J (2018) 17:319 Fig. 2  Screening against freshly isolated salivary gland sporozoites. a 3 Malaria Box compounds (at 10 µM) displayed potent inhibition of sporozoite motility (> 75% inhibition). b Selected drugs from the FDA approved library also displayed potent inhibition at 1 µM, with monensin sodium treatment pronouncedly inhibiting proper sporozoite attachment and motility. In both a and b, Cytochalasin D was used as a positive inhibitor control. c Gramicidin (1 µM) was potent in causing sporozoites to round up, while tyrothricin needed to be at a tenfold higher concentration for a similar effect. Data represented as mean ± SD. Scale bars: 10 µm Fig. 2  Screening against freshly isolated salivary gland sporozoites. a 3 Malaria Box compounds (at 10 µM) displayed potent inhibition of sporozoite motility (> 75% inhibition). b Selected drugs from the FDA approved library also displayed potent inhibition at 1 µM, with monensin sodium treatment pronouncedly inhibiting proper sporozoite attachment and motility. In both a and b, Cytochalasin D was used as a positive inhibitor control. c Gramicidin (1 µM) was potent in causing sporozoites to round up, while tyrothricin needed to be at a tenfold higher concentration for a similar effect. Data represented as mean ± SD. Scale bars: 10 µm compounds published previously [9] are probably acting either during invasion of the hepatocyte and/or during subsequent intracellular development, but not before. Discussionh Time lapse recording enabled analysis of migration as shown exemplary by a projection of the sporozoite path over 450 s (right). Scale bar: 10 μm. b In vivo imaging of sporozoites deposited in the skin by mosquito bites moved at similar speeds (Mann–Whitney test, red line indicates median speed); and, c with similar mean square displacement (MSD) when compared to controls. d Mice were monitored after mosquito bite to assess any ‘post-skin’ effects of Tyrosur treatment. Parasite emergence in the blood was similar between groups indicating no prominent effect of Tyrosur after skin exit. Data represented as mean ± SEM Fig. 3  Pre-treatment with a tyrothricin and gramicidin containing topical gel ­(Tyrosur®) did not inhibit in vivo parasite progression. a Mice ear hair was removed 24 h before the experiment and mosquito bites 4 h post-topical treatment. Anesthetized mice were transferred to a heated chamber and the bite site imaged to reveals sporozoites (left image). Time lapse recording enabled analysis of migration as shown exemplary by a projection of the sporozoite path over 450 s (right). Scale bar: 10 μm. b In vivo imaging of sporozoites deposited in the skin by mosquito bites moved at similar speeds (Mann–Whitney test, red line indicates median speed); and, c with similar mean square displacement (MSD) when compared to controls. d Mice were monitored after mosquito bite to assess any ‘post-skin’ effects of Tyrosur treatment. Parasite emergence in the blood was similar between groups indicating no prominent effect of Tyrosur after skin exit. Data represented as mean ± SEM leads to generate selective agents that stop sporozoite motility prior to invading the liver. gliding machinery components and is important for both merozoite red blood cell invasion and ookinete motility [61–63]. It is thus reasonable that inhibition of this kinase would affect sporozoite motility as well, although inducible knockout of PKG did not appear to affect liver cell invasion of sporozoites suggesting addi- tional targets of these compounds and/or compensa- tion by other kinases after deletion [64]. In the second screen, 97 compounds were used from a study that screened for liver stage inhibitors [8]. In that study, sporozoites were added together with the com- pounds to hepatocytes and the read out was liver stage growth. Thus, this assay setup could not distinguish between a compound affecting parasite motility, inva- sion, viability or growth. Four potent molecules inhib- ited sporozoite motility at 1 µM concentration. Discussionh The small number of hits that were identified as sporo- zoite inhibitors from the blood stage inhibiting Malaria Box library also suggest that the sporozoites show few overlapping targets with other stages. Indeed there is strong evidence for global downregulation of translation in sporozoites [59, 60] and thus fewer targets could be available. that could in principle directly act on motile parasites in the skin itself, although it investigated sporozoite-liver cell interactions [32]. A second paper made use of a com- putational screen for a particular complex of proteins and evaluated the hits on sporozoites yet did not screen libraries on whole sporozoites [58]. In this study, two compound libraries were screened to test for in  vitro sporozoite inhibition. The MMV Malaria Box contained three molecules that fulfilled the criteria of potent inhibition of motility (MMV665953, MMV665852, MMV007224). These were part of the list also identified in two separate liver stage screens (which identified 43 candidates) but it is important to note that the compounds identified in the current study were all previously identified as toxic for hepatocytes [9]. None- theless, the shorter list of identified compounds might still serve as useful leads in stopping sporozoites that now require further optimization for delivery. Further, these hits also assist the understanding of the liver stage hits identified previously. These data strongly suggest that the other liver stage hits observed for Malaria Box MMV665953 and MMV665852 were potent mol- ecules while structurally similar compounds of the N,N′-Diarylurea backbone family (MMV000911 and MMV001318) were not. The consistent feature of the two hits is the preservation of the meta/para halides and thus these functional groups should be considered in any subsequent derivative design. The current sug- gested target for this group of compounds is possibly cGMP-dependent protein kinase (PKG) [9], a key regu- lator of multiple cellular processes. Interestingly, PKG has been shown to be involved in phosphorylation of Douglas et al. Malar J (2018) 17:319 Page 6 of 10 Fig. 3  Pre-treatment with a tyrothricin and gramicidin containing topical gel ­(Tyrosur®) did not inhibit in vivo parasite progression. a Mice ear hair was removed 24 h before the experiment and mosquito bites 4 h post-topical treatment. Anesthetized mice were transferred to a heated chamber and the bite site imaged to reveals sporozoites (left image). Discussionh Further, orally administered drugs should not be excluded as it is also possible that, if able to permeate the dermal layer via the blood, one could achieve the same effect of sporozoite motility in the skin. Such dermal penetration has been suggested previously in the case of passive intravenous transfer of CSP antibodies [42]. Our platform could thus also be used to quantitatively evalu- ate other antibodies against sporozoite proteins. with 1 µM monensin before applying to the hepatocytes only resulted in a 70% reduction [70]. This suggested the primary potency was mediated through host cell medi- ated effects. At the same concentration in the assays used in this study, there was a reduction in sporozoite motility. This difference is probably best explained by the differ- ent environments of sporozoite motility. It is reasonable to suggest that effects in a simple 2D environment (our assay) has more striking effects on adhesion while the three-dimensional environment during cell traversal [70] could require more compound for the same effect. Indeed a similar effect between environments has been observed with mutated parasite lines, which were largely unable to move in 2D but showed no defect in 3D [73, 74]. Surrogate systems that mimic the three dimensional nature of the skin are currently being developed that could be used to test any future potential drug candidates in a 3D environment prior to in vivo testing [75]. Tyrothricin is isolated from Bacillus brevis and con- sists of a mixture of cyclic decapeptides, gramicidin S and tyrocidine A. Similar to monensin, gramicidin is a poly- peptide that produces membrane pores and affects cation gradients [76]. Given the general targeting of membranes, it is not surprising that ionophores have been shown to be effective in inhibiting Plasmodium blood stage growth [71, 77–79]. It is interesting that tyrothricin, a mixture of ionophoric peptides, was not as potent in sporozo- ite rounding as gramicidin. This suggests that the active ingredient against sporozoites is more specifically grami- cidin (which is only a fraction of tyrothricin). Tyrocidine peptides appear to be the primarily potent molecules against blood stage parasites with an IC50 value in the low micromolar range [79]. The difference between potencies of tyrothricin and gramicidin against sporozo- ites might provide a subtle hint that membrane suscepti- bilities are different across the life cycle. Discussionh Of these four compounds the two most potent candidates (mon- ensin and gramicidin) belonged to the ionophore class of compounds. This category of antibiotic is particularly attractive since it targets membranes and therefore has reduced likelihood of generation of resistant strains [69]. Monensin is a polyether antibiotic that, when inserting into membranes, results in sodium and potassium fluxes that negatively affects ion homeostasis within cells [70]. It has been employed in ruminant cattle, primarily for the treatment of coccidiosis. Monensin has been shown to be active against Plasmodium at different stages including asexual blood stages, gametocytes and oocyst formation [71, 72] although long incubation times were required. Interestingly, pretreatment of hepatocytes with monen- sin had a potent effect on sporozoite and Toxoplasma tachyzoite invasion; while pre-treatment of sporozoites Thrombospondin-related anonymous protein (TRAP) is a prominent parasite adhesin involved in parasite gliding and organ penetration [34, 65, 66]. The other potent hit from the MMV library, MMV007224, has been recently identified as a molecule that might immo- bilize an aldolase–TRAP interaction thereby affect- ing sporozoite motility [58]. While aldolase does not have a direct role in motility [67, 68], affecting TRAP dynamics on the parasite plasma membrane by stabiliz- ing a non-specific interaction could have consequences for efficient motility. The aldolase–TRAP study also identified other compounds from the library that were not notably active in the assay used here, presumably because these displayed weaker activities compared to MMV007224. Taken together, this shortlist of MMV malaria box compounds are useful in understanding contributors to parasite motility and could be used as Douglas et al. Malar J (2018) 17:319 Page 7 of 10 inhibition even with large amounts of gel applied, it does suggest that the drug is not able to sufficiently permeate the dermal layer where sporozoites are predominantly located after  the bite. Thus, reduced drug accessibility to the essential skin layer could be the major cause of treatment failure. Recent publications indicate good pro- gress on developing and modelling dermal penetration of exogenous molecules [86–89] but there still remains a long way to go before this could be used effectively in the field. Since this is a critical point, future work should thus focus on applying enhanced delivery methods that might allow targeting of the parasite and prevention of infec- tion. Conclusion In this study, a screening pipeline has been established and utilised to directly assess the potential effects of dif- ferent drug candidates on extracellular sporozoite motil- ity. Through this approach, a small set of molecules have been identified, including three MMV Malaria Box com- pounds and two ionophores, that have potent effects on sporozoites viability in  vitro. However, much further development is needed such that compounds can be effectively delivered to an intact dermis. Given the small numbers of hits on sporozoites, screening of larger librar- ies is needed. Authors’ contributions FF conceived the study. FF and RD designed experiments. RD, MR and MN conducted experiments and analysed the data. All authors contributed to data interpretation. RD and FF wrote the manuscript (with contributions from all authors). All authors read and approved the final manuscript. Discussionh Different effects between other ionophores were also noticed: monensin affected proper sporozoite adhesion while gramicidin led to rounding up of sporozoites. While the molecu- lar details are currently not clear regarding the altered response, it is reasonable to speculate that differences in pore sizes formed by these agents could affect dynamics of both the plasma and organellar membranes [80–84].f Additional files Additional file 1: Table S1. Initial pilot screen of MMV Malaria Box and FDA approved compounds. Additional file 1: Table S1. Initial pilot screen of MMV Malaria Box and FDA approved compounds. Additional file 2: Figure S1. Structures of inhibitors showing >50% inhibition. Ionophores have been used effectively in both the topi- cal and systemic treatment of gram-positive bacterial infections [85]. Given the micromolar in vitro inhibition of the tyrothricin mixture, with the presence of gramici- din in its overall composition and the availability of a gel formulation for topical application, we decided to fur- ther analyse ­Tyrosur® gel for possible in vivo effects on deposited sporozoites. However, treatment of mice ears with ­Tyrosur® gel did not significantly affect sporozoite infection ability after mosquito bite. Given the lack of References 1. Cowman AF, Healer J, Marapana D, Marsh K. Malaria: biology and disease. Cell. 2016;167:610–24. 24. Hovlid ML, Winzeler EA. Phenotypic screens in antimalarial drug discov- ery. Trends Parasitol. 2016;32:697–707. 2. Tilley L, Straimer J, Gnadig NF, Ralph SA, Fidock DA. Artemisinin action and resistance in Plasmodium falciparum. Trends Parasitol. 2016;32:682–96. 2. Tilley L, Straimer J, Gnadig NF, Ralph SA, Fidock DA. Artemisinin action and resistance in Plasmodium falciparum. Trends Parasitol. 2016;32:682–96. 25. 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Consent for publication All authors approved the manuscript for submission. 15. Guiguemde WA, Shelat AA, Bouck D, Duffy S, Crowther GJ, Davis PH, et al. Chemical genetics of Plasmodium falciparum. Nature. 2010;465:311–5. Availability of data and materials The datasets supporting the conclusions of this article are included within the article (and its additional files). 14. Gamo FJ, Sanz LM, Vidal J, de Cozar C, Alvarez E, Lavandera JL, et al. Thousands of chemical starting points for antimalarial lead identification. Nature. 2010;465:305–10. Ethics approval and consent to participate 16. Ekland EH, Schneider J, Fidock DA. Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches. FASEB J. 2011;25:3583–93. All animal experiments were performed according to FELASA B and GV-SOLAS standard guidelines. Animal experiments were approved by the German authorities (Regierungspräsidium Karlsruhe, Germany). 17. 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Light microscopic autoradiography and biochemical analysis. J Neurosci. 1984;4:1086–92. 83. Stein BS, Bensch KG, Sussman HH. Complete inhibition of transferrin recycling by monensin in K562 cells. J Biol Chem. 1984;259:14762–72. 84. Matheke ML, Fliesler SJ, Basinger SF, Holtzman E. The effects of monensin on transport of membrane components in the frog retinal photorecep- tor. I. Light microscopic autoradiography and biochemical analysis. J Neurosci. 1984;4:1086–92. 85. Banerjee S, Argaez C. In: Topical antibiotics for infection prevention: a review of the clinical effectiveness and guidelines. Ottawa (ON); 2017. 86. Kuchler S, Radowski MR, Blaschke T, Dathe M, Plendl J, Haag R, Schafer- Korting M, Kramer KD. Nanoparticles for skin penetration enhance- ment—a comparison of a dendritic core-multishell-nanotransporter and solid lipid nanoparticles. Eur J Pharm Biopharm. 2009;71:243–50. 87. Contri RV, Fiel LA, Alnasif N, Pohlmann AR, Guterres SS, Schafer-Korting M. 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Comparison and evaluation of digital signature schemes employed in ndn network
arXiv (Cornell University)
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International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 Al Imem Ali1 Al Imem Ali1 1 PRINCE ISITC, H. Sousse University of Sousse, 4011 Hammam Sousse, Tunisia ABSTRACT It is well known that Named Data networking ensure data integrity so that every important data has to be signed by its owner in order to send it safely inside the network. Similarly, in NDN we have to assure that none could open the data except authorized users. Since only the endpoints have the right to sign the data or check its validity during the verification process , we have considered that the data could be requested from various types of devices used by different people, these devices could be anything like a smartphone, PC, sensor node etc.b, with a different CPU descriptions, parameters, and memory sizes, however their ability to check the high traffic of a data during the key generation and/or verification period is definitely a hard task and it could exhaust the systems with low computational resources. RSA and ECDSA as digital signature algorithms have proven their efficiency against cyber-attacks, they are characterized by their speed to encrypt and decrypt data, in addition to their competence at checking the data integrity. The main purpose of our research was to find the optimal algorithm that avoids the system’s overhead and offers the best time during the signature scheme .KEYWORDS RSA, ECDSA, NDN, MSS, Digital signature algorithms, Security,Encryption, Decryption, Merkle scheme, Eliptic Curve International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 This new concept will bring back user’s trust. There are numerous digital signatures algorithms used in NDN such as RSA and ECDSA characterized by their high level of security and their speed to encrypt and decrypt data according to [1], moreover their efficiency to generate signatures and verify the data integrity with reduced key sizes. Besides RSA and ECDSA, there is also MSS (Merkle signature scheme) defined by [10] which is an interesting alternative for well- established signature schemes such as RSA, and ECDSA proved their eligibility against cyber- attacks e.g. timing attacks. The remaining part of the paper is organized as follows: Section 1 involves the NDN security concept with details and enumerates the advantages and disadvantages of RSA and ECDSA algorithms besides their limitation. Section 2 present a modular reduction used for accelerating one of those protocols RSA or ECDSA. Section 3 describes the simulation process used to clarify and illustrate the differences between RSA and ECDSA. The paper is concluded in section 4. 2. DIGITAL SIGNATURE The digital signature provides a means of integrity checking [11]. This is done to provide assurance for the receiver that the data was in fact sent by the assumed party. The integrity plays a critical role in virtual society and it’s important to protect it from coming out to the public ensure data integrity so that every important data has to be signed by its owner in order to send it safely inside the network. Similarly, NDN assure that none could open the data except authorized users. The digital signatures in use today can be classified according to the high underlying mathematical problem, which provides the basis for their security: • Integer Factorization (IF) problem: RSA signature schemes can be seen as an example under this classification[6]. • Integer Factorization (IF) problem: RSA signature schemes can be seen as an example under this classification[6]. • Elliptic Curve (EC) discrete logarithm problem represented in the Elliptic curve digital signature algorithm[4]. • Elliptic Curve (EC) discrete logarithm problem represented in the Elliptic curve digital signature algorithm[4]. RSA and ECDSA as digital signature algorithms have proven their efficiency against cyber- attacks, they are characterized by their speed to encrypt and decrypt data, in addition to their competence at checking the data integrity RSA and ECDSA as digital signature algorithms have proven their efficiency against cyber- attacks, they are characterized by their speed to encrypt and decrypt data, in addition to their competence at checking the data integrity 2.1. RSA “The RSA algorithm was developed at Massachusetts Institute of Technology (MIT) in 1977 by Ron Rivest, Adi Shamir and Leonard Adelman[6] “. The RSA concept is based on the factorization of big numbers which means the larger sequence of numbers you have, the more you are protected. The RSA provides a strong security; therefore an adversary should not be able to break RSA by factoring due to its complexity and large keys. RSA is used to encrypt/decrypt data and also has the ability to sign and/or verify the data packets. RSA does not mandate the use of a particular hash function, so the security of the signature and encryption are partly dependent on the choice of hash function used to compute the signature. 1.INTRODUCTION The digital signature algorithms uses some sort of complex operations aims to prevent the data from being accessible only for the authorized users, and computing such operations could exhaust the systems with limited computational resources; the problem statement affects the integrity of the data directly and minimizes the security level to facilitate the process of penetration, then the algorithms must be selected depending on the degree to maintain the integrity of the information regardless of the type of device. The NDN project team [13] considered security as an important factor; the researchers designed a new security model and implemented it not only into the architecture (e.g. communication channel) but also inside the packet that carry the information itself. Security are implemented directly by using a digital signature in each NDN data packets, and then it’s important for us to check the efficiency of these implemented protocols. The data packet could carry any type of information: credit cards number, personal information, and passwords, top secret information etc., then the role of the digital signature is to keep the data packet away from the cyber-attacks. DOI : 10.5121/ijesa.2015.5202 15 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 2.2. ECDSA “The Elliptic Curve Digital Signature Algorithm (ECDSA) is the elliptic curve analogue of the Digital Signature Algorithm (DSA). It was accepted in 1999 as an ANSI standard, and was accepted in 2000 as IEEE and NIST standards. It was also accepted in 1998 as an ISO standard, and is under consideration for inclusion in some other ISO standards, the range of parameters 16 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 offered by the standard can provide security for a number of years, provided the lowest figures are gradually discarded, taking into account the progress of computing power” [14]. offered by the standard can provide security for a number of years, provided the lowest figures are gradually discarded, taking into account the progress of computing power” [14]. Unlike the ordinary discrete logarithm problem and the integer factorization problem, no sub exponential-time algorithm is known for the elliptic curve discrete logarithm problem. Elliptic curve digital signature (ECDSA) was developed in 1985 by Neal Koblitz and Victor Miller. “ECDSA schemes provide the same functionality as RSA schemes including sign and/or verify signed packets There are some environments where 1024-bit RSA cannot be implemented while Unlike the ordinary discrete logarithm problem and the integer factorization problem, no sub exponential-time algorithm is known for the elliptic curve discrete logarithm problem. Elliptic curve digital signature (ECDSA) was developed in 1985 by Neal Koblitz and Victor Miller. Unlike the ordinary discrete logarithm problem and the integer factorization problem, no sub exponential-time algorithm is known for the elliptic curve discrete logarithm problem. Elliptic curve digital signature (ECDSA) was developed in 1985 by Neal Koblitz and Victor Miller. “ECDSA schemes provide the same functionality as RSA schemes including sign and/or verify signed packets. There are some environments where 1024-bit RSA cannot be implemented, while 192-bit ECDSA can. For this reason, the strength-per-key-bit is substantially greater in an algorithm that uses elliptic curves” [4]. g g ( ) p y “ECDSA schemes provide the same functionality as RSA schemes including sign and/or verify signed packets. There are some environments where 1024-bit RSA cannot be implemented, while 192-bit ECDSA can. For this reason, the strength-per-key-bit is substantially greater in an algorithm that uses elliptic curves” [4]. The Elliptic curve cryptography (ECC) is a very efficient technology to realize public key cryptosystems and public key infrastructures (PKI). 2.3. RSA and ECDSA digital signature schemes The essential elements of the digital signature schemes for RSA and ECDSA which defined by three computational procedures or algorithms: 2.2. ECDSA The security of a public key system using elliptic curves is based on the difficulty of computing discrete logarithms in the group of points on an elliptic curve defined over a finite field according to the [7]. The claim is that a 192 bit ECDSA key is similar to a 1024 bit RSA key in terms of the security that it offers. The performance tests therefore make comparisons according to these claims and attempt to provide more insight into the most suitable public key cryptography algorithm for a mobile framework given its limitations. The following table from the article wrote by [12], represents the recommended comparative key lengths for RSA and ECDSA used in software implementations. RSA needs larger key length but instead ECDSA requires significantly smaller key size with same level of security which offers faster computations and less storage space. ECDSA ideal for constrained environments: Tablets, Smart phones, RFID systems, Sensors etc. Table 1. Equivalent key lengths for RSA and ECDSA RSA key length (bits) ECDSA key length (bits) 1024 192 2048 256 2.3. RSA and ECDSA digital signature schemes International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 Here are some remarkable differences between RSA and ECDSA investigated by Khalique in[2]: • ECDSA offers same level of security with smaller key sizes. • Data size for RSA is smaller than ECDSA. • Encrypted message is a function of key size and data size for both RSA and ECDSA. ECDSA key size is relatively smaller than RSA key size, thus encrypted message in ECDSA is smaller. • Computational power is smaller for ECDSA. • ECDSA provides faster computations and less storage space • ECDSA key sizes are so much shorter than comparable RSA keys • The length of the public and private keys is much shorter in ECDSA. This results in faster processing times, and lower demands on memory and bandwidth. Note: “Some researchers have found that ECDSA is faster than RSA for signing and decryption process, however ECDSA is a bit slower for signature verification and encryption”[8]. Note: “Some researchers have found that ECDSA is faster than RSA for signing and decryption process, however ECDSA is a bit slower for signature verification and encryption”[8]. 2.3.2. Signing procedure The producer generates a signing process to transform/change the data from its original format to a new protected form. Each time it is used the procedure takes as input a signature key generated using the key generation procedure and data from some pre-determined data space. The signing procedure transforms the data and produces a signature as an output for the producer or the legal owner. 2.3.3. Verifying procedure Consumers who receive desired data packet in reverse need to be able to check that the signature appended to the message is correct, in the sense that it is a value which would be produced if the signing procedure was applied to the received data packet using the Producer's signing key. The verifying procedure takes as input the data and signature together with the public key of the purported consumer and then either accepts or rejects the signature. If the verifying procedure outputs ‘Accept,’ then the message are accepted as valid; otherwise it is rejected as invalid and the consumer sends a new interest packet. 2.4. Comparison of ECDSA with RSA Here are some remarkable differences between RSA and ECDSA investigated by Khalique in[2]: 2.3.1. Key generation procedure The key generation procedure is used to generate the keys that are used by the signing procedure and the verifying procedure. Each time it is used the procedure generates a key pair consisting of a Private/signature key and the corresponding Public/verification key. It is important to note that the key generation procedure uses a random number generator and will generate a different pair each time it is used. SK is always known as the secret key because in applications the signing key is kept secret. PK is always known as the public key, the verification key is distributed to all users who want to verify signatures. 17 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 Here are the most RSA and ECDSA remarkable limitations stated in [2]: Here are the most RSA and ECDSA remarkable limitations stated in [2]: • Key generation is very slow. • Key generation is very slow. • Speed of encrypting of data is slow. • Message length should be less than the bit length otherwise algorithm will fail. • Message length should be less than the bit length otherwise algorithm will fail. • RSA is factorization based algorithm so that every time RSA initialization takes two large prime number p and q. • RSA is factorization based algorithm so that every time RSA initialization takes t large prime number p and q. 3.THE MONTGOMERY REDUCTION “Peter Montgomery has devised a way to speed up arithmetic in a context in which a single modulus is used for a long-running computation”[5]. Montgomery multiplication algorithm reduces the computation time taken by a computer when there are a large number of multiplications to be [9]. The Montgomery reduction algorithm above uses the shift and/or adds operation to change the mode of calculation and makes it easier. The method can be used to reduce memory consumption, the execution time. The Montgomery method uses modular reduction to minimize the cycles taken by the signature schemes. 4.EXPERIMENTAL RESULTS Our objective was to compare two signature algorithms RSA and ECDSA used in NDN, using an (Intel core i5-2450 M CPU 2.50GHz and 4GB -RAM) machine. We combined NDNx simulator with Open SSl library in order to measure: • The required time to sign the data packet. • The required time to verify the data packet. • The required time to generate the private and/or public keys. Moreover, we used the Montgomery simulator to compare the ECDSA computation tasks by calculating the necessary operations and/or cycles between the ordinary and the Montgomery multiplication methods in order to resample the algorithm and speed up ECDSA. During the simulation process we observed that RSA performs better with shorter keys while ECDSA showed their slowness during the verification process. This weakness point needed to be solved, thus we implemented the montgomery multiplication technique in order to speed up ECDSA and upgrade its performance. 2.5. Advantages of ECDSA The ECDSA offered remarkable advantages over other cryptographic system mentioned by [2] • It provides greater security with smaller key sizes. • It provides effective and compact implementations for cryptographic operations requiring smaller chips. • Due to smaller chips less heat generation and less power consumption. • It is mostly suitable for machines having low bandwidth, low computing power, less memory. • It has easier hardware implementations. • It has easier hardware implementations. 18 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 4.2. RSA key generation The following table represents how much time taken by the algorithm to generate the Public/Private keys using different key lengths. The mean time generated by RSA-512 and RSA- 1024 recorded the fastest time due to its small key sizes compared with RSA-2048 and RSA- 4096. The RSA-4096 key generation was out of control and it exhausted the machine performance during the simulation process and increased the system’s overhead, and registered the slowest time during the signature process, wich could exhaust the battery lifetime and energy on wireless devices; Generating the RSA keys to encrypt the data by the producers especially if some of them are using a wireless device will lockup the system for some time and may completley drain the battery. 4.1. RSA speed Performance The obtained results showed that the verification process do more faster than the signing process, moreover we noticed that the more we increase the key size, there were no big difference during the verification process, the obtained results were too close to each other for the different key sizes which means that even if the RSA key size got doubled RSA still fast compared with the signing process. The RSA-512 and RSA-1024 recorded the best time during the verification and/or signing process compared with RSA-2048 and RSA-4096. We've got unreasonable that won’t suit the specifications of embedded systems. 19 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 4.3. ECDSA speed performance During the ECDSA speed performance, we observed that the signature generation and/or verification time does not differ until the larger key sizes: ECDSA-256ECDSA-521. The following table represents the generated time for each key size. We observed that ECDSA overpass RSA during the signature generation process. However RSA defeat ECDSA in performance during the verification process. ECDSA-192 and ECDSA-160 registered the best time during the sign/verification process. The ECDSA keys keeps the same level of security as RSA and provides quicker computation, lower power consumption, memory and bandwidth savings as an addition. Table 4. ECDSA speed performance time statics Key length(Bits) ECDSA- 160 ECDSA- 192 ECDSA- 224 ECDSA- 265 ECDSA- 384 ECDSA- 521 Sign per key size(sec) 0.0003 0.0003 0.0007 0.0009 0.0016 0.0033 Verify per key size(sec) 0.0015 0.002 0.0024 0.0039 0.0082 0.018 Table 4. ECDSA speed performance time statics 20 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 4.4. ECDSA key generation ECDSA can create keys in superior speed to RSA comparable key lengths. The overall comparison showed that generating a 2048 bit key for RSA takes significantly longer than generating a 1024 bit key. Table 5. ECDSA key generation time statics Key length(Bits) ECDSA- 160 ECDSA- 192 ECDSA- 224 ECDSA- 265 ECDSA- 384 ECDSA- 521 Key generation(sec) 0.161 0.165 0.229 0.305 0.799 1.584 Table 5. ECDSA key generation time statics However, This is not the case for ECDSA where there is small increase in the execution time. This is already expected since there is a 1024 bit difference between the two RSA keys and only small difference between the two ECDSA keys. 1. M = Mont (m, R2 mod n), result = R mod n where Mont (u, v) is uvR-1 mod n 2. For i from t down to 0 do the following: 2.1 result = Mont (result, result) 2.2 If ea. = 1 then A = Mont (result, M) 3. result = Mont(result,1) 4. Return(result). 4. Return(result). The Montgomery reduction algorithm above uses the shift and/or adds operation to change the mode of calculation and makes it easier. The method can be used to reduce memory consumption, the execution time. The Montgomery method uses modular reduction to minimize the cycles used by each process: signing/verification process. We’ve made four scenarios and for each scenario we’ve used a different bit lengths sequence. Since it was hard to calculate that huge number of exponents our research were only limited to 64 bit lengths. We’ve been through calculating the operations needed per each method, the Montgomery method uses an extra module called Add/shift multiplier used to optimize the big charge of operations by the standard multiplication method. International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 1. M = Mont (m, R2 mod n), result = R mod n where Mont (u, v) is uvR-1 mod 4.5.1. Scenario 1: Standard method vs. Montgomery method statics: 16 bits length The first scenario showed that the Montgomery method took fewer cycles than the standard method, the Add and/or shift module optimized the modular reduction to only 2 % (see figure 1) from the global operations. Figure1. Standard /Montgomery Method calculation Percent: 16 bits 4.5. The Montgomery reduction The Montgomery reduction algorithm are applied to speed up modular exponentiation and use shift operations in the place of modular reductions. One such algorithm is the square and multiply algorithm from [3]: 1. Set result = 1. If e = 0 then return (result). 2. Set A = m.wa 3. If k0 = 1 then set result = m 4. For i from 1 to t do the following 4.1 Set A = A² mod n 4.2 If ei = 1 then set result = A. result mod n. 5. Return  result 1. Set result = 1. If e = 0 then return (result). 2. Set A = m.wa 3. If k0 = 1 then set result = m 4. For i from 1 to t do the following 4.1 Set A = A² mod n 4.2 If ei = 1 then set result = A. result mod n. The standard multiplication method is a completely hard task to accomplish its work, however the Montgomery method takes shorter path to get the intended results as the same as the standard method. The algorithm above are up to the same results from each side. Our interests were in replacing the current ECDSA algorithm with a modified algorithm which we called “modified ECDSA” and to show the differences between. We suspended RSA algorithm from upgrading its performance for the reason that installing such algorithm on light-weight devices will adversely affect their performance and delay the decryption process. ECDSA in counterpart could be a replacement for RSA system, their comptability to be installed in any system with different memory sizes and CPU description and parameters, ECDSA provide the same level of security as RSA but with shorter keys: The smaller key sizes of ECDSA potentially allow for less computationally able light-weight devices and wireless systems to use cryptography for secure data transmissions, data verification and offers less heat generation and less power consumption, less storage space and offers an optimized memory and bandwidth and faster signature generation. To reduce the cycles used by the ECDSA we used this algorithm from [3]: 21 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 Figure1. Standard /Montgomery Method calculation Percent: 16 bits In addition it saved 42 cycles against the standard method. The number of cycles saved, aims to reduce the CPU execution time and to speed up the digital signatures procedure such as verification or generation time. 22 22 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 Figure 2. Standard method vs. Montgomery method statics: 16 bits length Figure 2. Standard method vs. Montgomery method statics: 16 bits length 4.5.2. Scenario 2: Standard method vs. Montgomery method statics: 32 bits length The second scenario showed a reduction by 121 cycles, the Add and/or shift module gain another 79 compared with the last Montgomery results. The number of the modular reductions used by the standard method are greater than the Montgomery’s, then it delays the computation tasks. Figure 3. Standard /Montgomery Method calculation Percent: 32 bits Figure 3. Standard /Montgomery Method calculation Percent: 32 bits The second scenario showed a reduction by 121 cycles, the Add and/or shift module gain another 79 compared with the last Montgomery results. The number of the modular reductions used by the standard method are greater than the Montgomery’s, then it delays the computation tasks. The second scenario showed a reduction by 121 cycles, the Add and/or shift module gain another 79 compared with the last Montgomery results. The number of the modular reductions used by the standard method are greater than the Montgomery’s, then it delays the computation tasks. The second scenario showed a reduction by 121 cycles, the Add and/or shift module gain another 79 compared with the last Montgomery results. The number of the modular reductions used by the standard method are greater than the Montgomery’s, then it delays the computation tasks. 23 23 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 Figure 4. Standard method vs. Montgomery method statics: 32 bits length Figure 4. Standard method vs. Montgomery method statics: 32 bits length 4.5.3.Scenario 3: Standard method vs. Montgomery method statics: 48 bits length The third scenario is a complementary for the other two scenarios; we kept getting good results since the Montgomery method succeeded to reduce the number of cycles. This time we got rid of 188 cycles. Moreover the modular reduction used by the standard Method is greater than the Montgomery’s, and then it delays the computation tasks. Figure 5. Standard /Montgomery Method calculation Percent: 48 bits Figure 5. Standard /Montgomery Method calculation Percent: 48 bits The shift and/or add operations might be greater, but it helps to shorten the numbers of modular reduction. The numbers of modular reduction used in the standard method are extremely hard task. The shift and/or add method aims to reduce the numbers of operations used in modular reduction. 24 24 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 Figure 6. Standard method vs. Montgomery method statics: 48 bits length 4.5.4.Scenario 4: Standard method vs. Montgomery method statics: 64 bits length Figure 7. Standard /Montgomery Method calculation Percent: 64 bits The last scenario showed big differences from the previous scenarios we got rid of 297 cycles which means that we took the right road to prove that the Montgomery could be a replacement of the standard multiplication method. Figure 6. Standard method vs. Montgomery method statics: 48 bits length Figure 6. Standard method vs. Montgomery method statics: 48 bits length 4.5.4.Scenario 4: Standard method vs. Montgomery method statics: 64 bits length Figure 7. Standard /Montgomery Method calculation Percent: 64 bits Figure 7. Standard /Montgomery Method calculation Percent: 64 bits Figure 7. Standard /Montgomery Method calculation Percent: 64 bits The last scenario showed big differences from the previous scenarios we got rid of 297 cycles which means that we took the right road to prove that the Montgomery could be a replacement of the standard multiplication method. Figure 8. Standard method vs. Montgomery method statics: 64 bits length Figure 8. Standard method vs. Montgomery method statics: 64 bits length 25 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 One of our objective were to equilibrate ECDSA and RSA and improve ECDSA recorded time. The Montgomery method offers an optimized multiplication sequences which aims to speed up the regular ECDSA algorithm process. the next part shows that the Montgomery method is an effective way to accelerate the regular ECDSA. 4.6 Modified ECDSA speed Performance The results are shown in Table 6 represents the Modified ECDSA speed performance during the verification and signing process. We’ve got reasonable results for both sides as we can see 0.0001 ms for signing and between 0.0003 ms and 0.0002 ms for verification. It is safe to assume that embedded systems with low computational ressources will be more than capable of verifying ECDSA digital signatures. In terms of speed our verification routine outperforms RSA and ECDSA for all common security parameters. Table 6. Modified ECDSA speed performance time statics Key length(Bits) ECDSA- 160 ECDSA- 192 ECDSA- 224 ECDSA- 265 ECDSA- 384 ECDSA- 521 Sign per key size(sec) 0.0001 0.0001 0.0001 0.0001 0.0002 0.0005 Verify per key size(sec) 0.0002 0.0003 0.0003 0.0005 0.0007 0.001 Table 6. Modified ECDSA speed performance time statics 5.1 Performance Comparison: Standard Method, Montgomery Method The Montgomery multiplication method showed interesting results and succeeded to reduce the number of operations with the Add/shift method. The number of the modular reduction used by the standard method is greater than the Montgomery’s, and then it delays the computation tasks. The shift and/or add operations might be greater, but it helps to shorten the numbers of modular reduction. The numbers of modular reduction used in the standard method are extremely hard task then the shift and/or add method aims to reduce the numbers of operations used in modular reduction and so it could accelerate the digital signature process such as ECDSA and improve its performance and release the system from additional tasks. The first table summarizes the Montgomery vs. Standard method performance: Table 7. Performance Comparison: Standard Method, Montgomery method Method/Factors Number of operations Cycles Standard Method Less Many Montgomery Method Greater Optimized Table 7. Performance Comparison: Standard Method, Montgomery method Method/Factors Number of operations Cycles Standard Method Less Many Montgomery Method Greater Optimized Table 7. Performance Comparison: Standard Method, Montgomery method 26 26 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 5.2 Performance Comparison: RSA, ECDSA, MODIFIED ECDSA ECDSA in counterpart could be a replacement for RSA system, their comptability to be installed in any system with different memory sizes and CPU description and parameters, ECDSA provide the same level of security as RSA but with shorter keys: The smaller key sizes of ECDSA potentially allow for less computationally able light-weight devices and wireless systems to use cryptography for secure data transmissions, data verification and offers less heat generation and less power consumption, less storage space and offers an optimized memory and bandwidth and faster signature generation. One of our objective were to equilibrate ECDSA and RSA and improve their times. The Montgomery method we employed offers an optimized multiplication sequences which aims to speed up the regular ECDSA algorithm process. Our method changed the state of the regular algorithm’s verification time from slow to fast and the algorithm’s sigining time from fast to faster. The following discussion profiles the above comparison according to the steps generated by each algorithm. 5.2 Performance Comparison: RSA, ECDSA, MODIFIED ECDSA The results that were obtained for all the performance measurements have been categorised according to the dependent variables. The goal with this round of tests was to provide recommendations regarding the chosen algorithms with respect to their performance and compared to the level of security provided. The second table summarizes the digital signatures algorithms including the recommended domains: Table 8. Performance Comparison: RSA, ECDSA, Modified ECDSA We suspended RSA algorithm from upgrading its performance for the reason that installing such algorithm on light-weight devices will adversely affect their performance and delay the decryption process. ECDSA in counterpart could be a replacement for RSA system, their comptability to be installed in any system with different memory sizes and CPU description and parameters, ECDSA provide the same level of security as RSA but with shorter keys: The smaller key sizes of ECDSA potentially allow for less computationally able light-weight devices and wireless systems to use cryptography for secure data transmissions, data verification and offers less heat generation and less power consumption, less storage space and offers an optimized memory and bandwidth and faster signature generation. One of our objective were to equilibrate ECDSA and RSA and improve their times. The Montgomery method we employed offers an optimized multiplication sequences which aims to speed up the regular ECDSA algorithm process. Our method changed the state of the regular algorithm’s verification time from slow to fast and the algorithm’s sigining time from fast to faster. Security Complexity Domain Key Creator Execution Time Verify/s Sign/s RSA High Integer Factorization PC, laptops, Super computers Fast Slow Fast Fast ECDSA High Discrete logarithm Light- weight devices Faster Fast Slow Fast Modified ECDSA (Our) High Discrete logarithm Light- weigh devices Faster Fast Fast Faster Table 8. Performance Comparison: RSA, ECDSA, Modified ECDSA Table 8. Performance Comparison: RSA, ECDSA, Modified ECDSA Table 8. Performance Comparison: RSA, ECDSA, Modified ECDSA Security Complexity Domain Key Creator Execution Time Verify/s Sign/s RSA High Integer Factorization PC, laptops, Super computers Fast Slow Fast Fast ECDSA High Discrete logarithm Light- weight devices Faster Fast Slow Fast Modified ECDSA (Our) High Discrete logarithm Light- weigh devices Faster Fast Fast Faster We suspended RSA algorithm from upgrading its performance for the reason that installing such algorithm on light-weight devices will adversely affect their performance and delay the decryption process. 5.2.4 Signature Time The tests that were conducted for both RSA and ECDSA have shown that the RSA time signature performs poorly compared with ECDSA and modified ECDSA ones. The time required for RSA operations to generate signatures quickly rises due to its larger keys which is not acceptable and could delay the packets from being transfered. The time required for ECDSA times also rise, but at a much slower rate due to the convergence between the keys size. 5.2.3. Verification Time In terms of verification time we found that the difference between regular ECDSA and RSA was significant to. This is most likely due to the fact that the regular ECDSA uses a complex operations rather than RSA. For RSA the verification period is very fast since it is simpler in terms of cryptographic computations as only a minimum of modular multiplications is necessary. The work on resampling the ECDSA operations would speed up the signatures algorithm while maintaining the same security level, the Montgomery method we implemented succeeded to to accelerate the signature scheme and gave us better performance by minimizing the time required to verify the number of packets of each data and succeeded to reduce the number of operations during the computation tasks and made it more simple. Our algorithm( Modified ECDSA) has shorten the diffrences between ECDSA and RSA in terms of signing and verification time which could lead us to a new level where we can group security, speed, stability and comptabilty together. 5.2.2. Execution Time In terms of Execution time, we found that the difference between RSA and ECDSA was significant the resulting graph in Table 3 and Table 5 showing the execution time with standard deviation that RSA takes significantly longer compared to ECDSA to generate its key pair. This result is expected since the RSA keys are significantly larger than the ECDSA keys. 5.2.1. Key generation The key creation for ECDSA was significantly faster than RSA due to the difference in the key lengths. The comparison shows very small ratios whatever the security level. This means the key generation with ECDSA is always faster than the key generation with RSA. The RSA keys are generated using large prime numbers thus take significantly longer than the smaller ECDSA keys that are generated. 27 5.CONCLUSION The Montgomery method uses fewer cycles in order to speed up the execution time. Then the Montgomery method we’ve integrated reduced the number of operations during the computation task and succeeded to accelerate the ECDSA sign and/or verification process. As future works, we considered that the NDN network will be fully loaded by signed packets coming from different sources and/or destinations. The verification of each signature is definitely a hard task; we considered that verifying a group of signed packets together aims to reduce the verification time. As a solution we propose ‘batch verification’ technique in order to put together multiple signatures in the same queue for global verification and time saving. However the batch size is limited to a specific size and a specific number of signatures; in order to solve that we propose to compress each signature in order to add more inside the batch. The batch verification can be integrated inside one of the digital signature protocols such as: ECDSA. Then our idea summarized in verifying the maximum number of signatures instantly. REFERENCES [1] Kaur, Goyal V. “A Comparative Analysis of ECDSA VS. RSA Algorithm”, International journal of computer science and informatics, ISSSN (Print):2231-5292, volume-3, Issue-1, 2003. [1] Kaur, Goyal V. “A Comparative Analysis of ECDSA VS. RSA Algorithm”, International journal of computer science and informatics, ISSSN (Print):2231-5292, volume-3, Issue-1, 2003. [1] Kaur, Goyal V. “A Comparative Analysis of ECDSA VS. RSA Algorithm”, International journal of computer science and informatics, ISSSN (Print):2231-5292, volume-3, Issue-1, 2003. [2] Khalique, Singh k., Sood S.”Implementation Of Elliptic Curve Digital Signature Algorithm”, I i l j l f li i (0975 8887) V l 2 N 2 2010 [2] Khalique, Singh k., Sood S.”Implementation Of Elliptic Curve Digital Signature Algorith International journal of computer applications (0975-8887), Volume 2–No.2, 2010. [3] Menezes, Oorschot P., and Vanstone S, "Handbook of Applied Cryptography”, CRC Press, 1996. [4] Jhonson, Menezes A., Vanstone S, “ The Elliptic Curve Digital Signature Algorithm (ECDSA)” , Certicom research, Canada, 2001. [5] Buell. “Montgomery Multiplication”, 2005 [6] Milanov, “The RSA Algorithm”, 2009. [7] Federal Office for Information Security, “Technical Guideline TR-03111 Elliptic Curve Cryptography Version 2.0”, Germany, 2012. [8] Kessler G. C, “An Overview of Cryptography”, 2014. 8] Kessler G. C, “An Overview of Cryptography”, [9] Warren, Jr. “Montgomery Multiplication”, 2012. g y p [10] Buchman, Garcia L. C. C., Dahmen E., Doring M., and Klintsevich E. “CMSS- An Improved Merkle Signature Scheme”, Lecture Notes in Computer Science Volume 4329,pp 349-363. 2006. [11] Limor Elbaz, VP. Research, “Using Public Key Cryptography in Mobile Phones”, Discretix technologies Ltd, 2002. [12] Katiyar , Dutta K. , Gupta S. “A Survey On Elliptic Curve Cryptography For Pervasive Computing Environments”, In international journal of computing applications, 11(10);41-46, 2010. [13] NDN project team, “Named Data Networking (NDN) Project 2012-2013 Annual Report”, 2013. [14] Rao, Gan Q, “ The Performance Analysis of Two Digital Signatures Schemes Based on Secure Charging Protocol”, 2005. 5.CONCLUSION RSA and ECDSA used to protect the data packet inside the NDN network and to recommend the preferred one depending on the results we’ve got. In addition, we considered that the time is an important factor that a user wouldn’t wait the whole day waiting for encrypting and decrypting the data, the work on resampling the operations would speed up the signatures algorithm while maintaining the same security level, we present the Montgomery method aims to accelerate the signature scheme for better performance and the reduction of the wasted time. To compare the evaluation performance of the RSA and ECDSA digital signatures we used Open-SSL for comparison, and as results we found that: The key generation time for ECDSA was significantly faster than RSA due to the difference in the key lengths. The RSA keys that are generated using large prime numbers thus take significantly longer than the smaller ECDSA keys that are generated. The execution time between RSA and ECDSA was significant. This result is expected since the RSA keys are significantly larger than the ECDSA keys. 28 The verification time between regular ECDSA and RSA was significant to. This is most likely due to the fact that the regular ECDSA uses a complex operations rather than RSA. Our algorithm( Modified ECDSA) has shorten the diffrences between ECDSA and RSA in terms of 28 28 International Journal of Embedded systems and Applications(IJESA) Vol.5, No.2, June 2015 signing and verification time which could lead us to a new level where we can group security, speed, stability and comptabilty together. signing and verification time which could lead us to a new level where we can group security, speed, stability and comptabilty together. The time required for RSA operations to generate signatures quickly rises due to its larger keys which is not acceptable and could delay the packets from being transfered. The time required for ECDSA times also rise, but at a much slower rate due to the convergence between the keys size. The Montgomery method uses fewer cycles in order to speed up the execution time. Then the Montgomery method we’ve integrated reduced the number of operations during the computation task and succeeded to accelerate the ECDSA sign and/or verification process. ECDSA times also rise, but at a much slower rate due to the convergence between the keys size. Author: Ali Al Imem is currently a Researcher at Higher Institute of Computer Sciences and Communication Techniques, Hammam Sousse , Tunisia. Mr. Ali received his MSc. in Networks from ISITCOM, Tunisia. Mr Ali’s areas of interest include Embedded systems, Cryptography and Cloud Computing. Ali Al Imem is currently a Researcher at Higher Institute of Computer Sciences and Communication Techniques, Hammam Sousse , Tunisia. Mr. Ali received his MSc. in Networks from ISITCOM, Tunisia. Mr Ali’s areas of interest include Embedded systems, Cryptography and Cloud Computing. Ali Al Imem is currently a Researcher at Higher Institute of Computer Sciences and Communication Techniques, Hammam Sousse , Tunisia. Mr. Ali received his MSc. in Networks from ISITCOM, Tunisia. Mr Ali’s areas of interest include Embedded systems, Cryptography and Cloud Computing. 29 29
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A Missing Piece of Mutual Learning Model of March (1991)
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a) Graduate School of Economics, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan, moon-wealth@nifty.com b) Graduate School of Economics, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan, nobuta@e.u-tokyo.ac.jp A part of this paper was originally published as Mitomi and Takahashi (2014) in Japanese. Annals of Business Administrative Science 14 (2015) 35–51 Available at www.gbrc.jp http://dx.doi.org/10.7880/abas.14.35 Online ISSN 1347-4456 Print ISSN 1347-4464 ©2015 Global Business Research Center Annals of Business Administrative Science 14 (2015) 35–51 Available at www.gbrc.jp http://dx.doi.org/10.7880/abas.14.35 Online ISSN 1347-4456 Print ISSN 1347-4464 ©2015 Global Business Research Center Annals of Business Administrative Science 14 (2015) 35–51 Available at www.gbrc.jp http://dx.doi.org/10.7880/abas.14.35 Online ISSN 1347-4456 Print ISSN 1347-4464 ©2015 Global Business Research Center 1 Computer simulations are not only used in organizational learning but also have various uses in the garbage can model (Cohen, March, & Olsen, 1972; Inamizu, 2012; Takahashi, 1997), the communication competition model (Kuwashima, Takahashi, & Tamada, 2005; Takahashi, Kuwashima, & Tamada, 2006), or theoretical shaping of learning curves (Takahashi, 2013). A Missing Piece of Mutual Learning Model of March (1991) Yuki MITOMIa) and Nobuo TAKAHASHIb) Yuki MITOMIa) and Nobuo TAKAHASHIb) Abstract: The mutual learning model described in “Exploration and Exploitation in Organizational Learning” (March, 1991) concludes that “slow learning on the part of individuals maintains diversity longer, thereby providing the exploration,” based on the results of computer simulations. However, the simulations of March (1991) excluded both ends of the socialization rate domain. When compensating for those missing portions, there is an optimal socialization rate that actually maximizes the average knowledge level. This is because low learning on the part of individuals actually causes frequent lock-ins and impedes learning. This optimal socialization rate may be a common rate for socialization, and we cannot deny this possibility by using only computer simulations. Moreover, this high knowledge level is achieved in a non-equilibrium state. Keywords: exploration and exploitation, organizational learning, mutual learning model, computer simulation, socialization rate 35 Mitomi and Takahashi Introduction When multiple learners (including the organization) learning simultaneously affect each other, March referred to the learning process as the ecology of learning (Takase, 1991, p. 60). The ecology model of learning of March featured a selection process of organizational routines under the influence of other learners. A learner acquires other learners’ experiences codified as technology, codes, procedures, and routines (Levitt & March, 1988). Furthermore, the diffusion (Rogers, 1962) of learners’ experiences and routines in the organization makes this model even more complex. Mathematical analysis is typically abandoned and replaced by computer simulation analysis due to the complexity of the model. (e.g., Levinthal & March, 1981; Lounamaa & March, 1987). Throughout the 1980s, March was the primary proponent of this type of research (Huber, 1991).1 Levinthal and March (1993) called the phenomenon of preferring exploitation to exploration a “myopia of learning” (Sato, 2012). March (1991) entitled “Exploration and Exploitation in Organizational Learning” primarily developed and analyzed two computer simulation models: (A) a mutual learning model and (B) a competitive ecology model. However, Takahashi (1998) mathematically analyzes model (B) and does not require a computer simulation. In model (B), a reference organization having the normal performance distribution with mean m and variance v2 competes with N organizations having a standard normal performance distribution with mean 0 and variance 1. Let u be a value with an upper probability of 1(N  1) in the standard 36 A missing piece of mutual learning model of March (1991) normal distribution table (u  0.44 for N  2, u  1.34 for N  10, u  2.33 for N  100), Takahashi (1998) obtains the following equation: m  u(1  v) (1) Let P* be the probability of the reference organization having the best performance within the group. March (1991) constructs the “competitive equality lines” by conducting 5,000 simulations for each value v2 from 0 to 2 in steps of 0.05 and plotting points (m, v2) where P*  1(n  1). The three lines of N  2, N  10, and N  100 illustrated in Figure 6 in March (1991, p. 82) cross the vertical axis of mean m at approximately 0.2, 0.8, and 1.7, respectively. Reality There is an external reality, which is called the “state of nature” in statistical decision theory. A reality is an m-dimensional vector, with each component having a value of 1 or −1. r  (r1, r2, …, rm) Assumption 1: Each component ri of a reality is given an initial value of 1 or −1 with a probability of 1/2. Introduction However, using Equation (1) the mean must be, respectively, 0.44, 1.34, and 2.33 37 Variance of the performance distribution Mean of the performance distribution N = 2 N = 10 N = 100 2.0 1.5 1.0 0.5 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 Figure 1. Competitive equality lines Source: Takahashi (1998, Figure 6) Variance of the performance distribution Mean of the performance distribution N = 2 N = 10 N = 100 2.0 1.5 1.0 0.5 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 Variance of the performance distribution Variance of the performance distribution 37 Mitomi and Takahashi when variance is 0. More specifically, Figure 1 illustrates the parabolas that touch the vertical axis at these points. Takahashi (1998) is suspicious regarding the validity of March’s (1991) simulation program at least at the end point 0 of domain of variance. As March’s (1991) programs have not been published, this paper creates a computer simulation program for model (A) (Appendix A) in free software language R for statistical computing, and checks the validity of the program at end points of the domain wherein we can analyze mathematically without simulation. We then compensate for the missing piece of March’s (1991) mutual learning model (A) to identify the true conclusion. Mutual Learning Model March’s (1991) model is vaguely formulated because of less use of mathematical notation. This paper fully uses Takahashi’s (1998) notation and reformulates March’s (1991) model mathematically. The beliefs of n members and an organizational code Each of n members has a belief for each component of the reality at each time period. This belief is not a probability, whereas the belief of statistical decision theory represents a probability. For each 38 A missing piece of mutual learning model of March (1991) component in an m-dimensional vector of the reality, each belief has a value of 1, 0, or −1. For member j, an m-dimensional vector of belief can be expressed as bj  (bj1, bj2, …, bjm) bj  (bj1, bj2, …, bjm) bj  (bj1, bj2, …, bjm) Similarly, the belief of the organizational code is expressed as c  (c1, c2, …, cm) c  (c1, c2, …, cm) with each component having a value of 1, 0, or −1. Assumption 2: Each component of an m-dimensional vector bj that expresses the belief of each organizational member is given an initial value of 1, 0, or −1, each having an equal probability of 1/3. Assumption 3: The initial value of each component of an m-dimensional vector c that expresses the belief of the organizational code is 0. Learning from the superior group For each component of a belief, we examine whether it matches the component of the known reality. Thereafter, we define knowledge level L as the number of matching components divided by m. The group of individuals whose knowledge level is higher than that of the organizational code is called a “superior group.” Assumption 4: 1) If the superior group is empty or there is no majority opinion regarding the ith component of the superior group, the ith component ci of an organizational code’s belief does not change. 2) If the majority opinion regarding the ith component of the superior group is the same as the ith component ci of an organizational code’s belief, ci does not change. 3) If the majority opinion regarding the ith component of the superior group differs 39 Mitomi and Takahashi from the ith component ci of an organizational code’s belief, ci changes with probability q  1 – (1 − p2)ki in accordance with the majority opinion, where ki (ki  0) is the number of members whose ith component of belief differs from ith component of organizational code’s belief minus the number of members who do not (within the superior group). Changes in components are independent of each other. Probability p2 is expressed as the learning rate of the organizational code. 2 However, some vagueness remains; according to March (1991, p. 75), these do not necessarily have to be entirely the same. As can be seen later in this paper, in an 80 period simulation, an equilibrium is often not attained, and March’s statement may be in reference to that fact. Learning from the organizational code As a result of socialization, it is possible for the value of each component of a member’s belief to change to either 1, 0, or −1 due to influence from the organizational code (for example, changing from 1 to −1, or 0 to 1). Assumption 5: 1) If the ith component of the organizational code ci  0, the ith component bji of organizational member j’s belief continues to have the same value. 2) If ci  bji , bji continues to have the same value. 3) If ci  bji, bji takes on the same value of ci with probability p1. Probability p1 is called the socialization rate, and is common to all members. Probability p1 is called the socialization rate, and is common to all members. In our simulation program in R, the number m of dimensions of reality is set at 30, the number n of members is set at 50, and simulations were conducted in 80 time periods as same setting as March’s (1991) original simulation. However, we set seeds of the random number generator to 501, 502,…,600 for a total of 100 different random number settings, and then we average the results of 100 simulations. 40 A missing piece of mutual learning model of March (1991) Equilibrium and Lock-in March (1991) defines the state of sharing2 same belief among all organizational members and the organizational code as an equilibrium, and thus states “this equilibrium is stable” (March, 1991, p. 75). As a backdrop to this definition, March assumed that “the beliefs of individuals and the code converge over time” (March, 1991, p. 75). Based on the definition of an equilibrium, in such a state the beliefs of organizational members and an organizational code are identical, and the equilibrium is certainly stable. On the other hand, although the state may be stable, it is not an equilibrium; rather, this is called “lock-in.” Lock-in at the learning rate p2  0 of the organizational code Lock-in at the learning rate p2  0 of the organizational code A typical case of lock-in occurs when an organizational code’s learning rate p2  0, and then when q  0. In this case, the organizational code is unchanged according to Assumption 4. From Assumption 3, the initial value of each component of an organizational code is 0, and thus it is unchanged throughout. In actuality, from Assumption 5 (1), if each component of an organizational code is 0, then the beliefs of organizational members will not be affected by an organizational code at all. In other words, the beliefs of organizational members are unchanged from the initial state. Indeed, this is not an equilibrium, though it is a lock-in from the outset. In this case, reality component ri and belief component bji of member j are the same when the initial values (ri, bji)  (1, 1) or (−1, −1). Thus, from Assumptions 1 and 2, the probability of reality and 41 Mitomi and Takahashi Table 1. Average knowledge levels at the learning rate p2  0 Table 1. Average knowledge levels at the learning rate p2  0 p2 Socialization rate p1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 0.334 0.334 0.334 0.334 0.334 0.334 0.334 0.334 0.334 0.334 0.334 Note: m = 30, n = 50, 80 periods, 100 simulations belief being the same is as follows: (1/2)(1/3)  (1/2)(1/3)  1/3. In other words, if learning rate p2  0, then the average knowledge level of members should be 1/3 regardless of socialization rate p1. On the other hand, for the organizational code, each component remains unchanged from the initial value 0 and thus the knowledge level remains unchanged from 0. In the same setting as March (1991), reality dimensions m  30, organizational members n  50, and 80 periods, we conduct 100 simulations for each socialization rate p1 in steps of 0.1, and the average knowledge level of 100 simulations are indicated in Table 1. As with the theoretical value, regardless of the value of the socialization rate p1, the average knowledge level is 0.334, or approximately 1/3. Indeed, lock-in occurs from the outset, and this average knowledge level is a reflection of the probability for initial values of (ri, bji)  (1, 1) or (−1, −1) being 1/3. Table 2. Average knowledge levels at the learning rate p1  0 On the other hand, from Assumption 3, the initial value for each component of an organizational code’s belief is 0 and the initial knowledge level is 0; however, from Assumption 4, when the ith component of the majority opinion of the superior group is either 1 or On the other hand, from Assumption 3, the initial value for each component of an organizational code’s belief is 0 and the initial knowledge level is 0; however, from Assumption 4, when the ith component of the majority opinion of the superior group is either 1 or 1, the ith component ci of an organizational code’s belief changes with probability q  1 – (1 – p2)ki in accordance with the majority opinion. This change continues until the ith component of the organizational code’s belief matches with the (unchanging) ith component of the majority opinion of the superior group’s belief. However, even upon stabilization, the belief of the organizational code will certainly not be the same as the minority opinion of the superior group. In any case, this is not an equilibrium but a lock-in. component of the majority opinion of the superior group is either 1 or 1, the ith component ci of an organizational code’s belief changes with probability q  1 – (1 – p2)ki in accordance with the majority opinion. This change continues until the ith component of the organizational code’s belief matches with the (unchanging) ith component of the majority opinion of the superior group’s belief. However, even upon stabilization, the belief of the organizational code will certainly not be the same as the minority opinion of the superior group. In any case, this is not an equilibrium but a lock-in. In the same setting as March (1991), reality dimensions m  30, organizational members n  50, and 80 periods, we conduct 100 simulations for each learning rate p2 in steps of 0.1, and the average knowledge level of 100 simulations are indicated in Table 2. As with the theoretical value, regardless of the value of the learning rate p2, the average knowledge level is 0.334, or approximately 1/3. Lock-in at the socialization rate p1  0 Another typical case of lock-in occurs when the socialization rate p1 is 0. In this case, the belief of organizational members is not affected by the organizational code from Assumption 5. Accordingly, from Assumption 2, each component of each organizational member’s beliefs is unchanged, with an initial value of 1, 0, or 1, each with an equal probability of 1/3. The average knowledge level in this case is 1/3. 42 A missing piece of mutual learning model of March (1991) Table 2. Average knowledge levels at the learning rate p1  0 p1 Learning rate p2 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 0.334 0.334 0.334 0.334 0.334 0.334 0.334 0.334 0.334 0.334 0.334 Note: m = 30, n = 50, 80 periods, 100 simulations Table 2. Average knowledge levels at the learning rate p1  0 3 Figure 1 of March (1991) has an overall average knowledge level that is higher than Figure 2 by approximately 0.1, with greater variance and no convergence of average knowledge level as the socialization rate p1 approaches 1. Takahashi (1998) proposes modifications to more closely resemble Figure 1 of March (1991); see Appendix B for more detail. Follow-up Simulations and True Conclusion Based on the above considerations, our program created in R reproduced our theoretical predictions, we therefore conduct simulations in the same setting as March (1991): reality dimensions 43 Mitomi and Takahashi p2 = 0.1 p2 = 0.5 p2 = 0.9 Socialization rate p1 1.0 0.9 0.8 0.7 0.6 0.5 0.1 0.3 0.5 0.7 0.9 Average knowledge level Figure 2. Average knowledge levels of March’s (1991) model. Note: m  30, n  50, 80 periods, 100 simulations p2 = 0.1 p2 = 0.5 p2 = 0.9 Socialization rate p1 1.0 0.9 0.8 0.7 0.6 0.5 0.1 0.3 0.5 0.7 0.9 Average knowledge level Figure 2. Average knowledge levels of March’s (1991) model. Figure 2. Average knowledge levels of March’s (1991) model. Note: m  30, n  50, 80 periods, 100 simulations m  30, organizational members n  50, 80 periods, and for each socialization rate p1 from 0.1 to 0.9 in steps of 0.1. m  30, organizational members n  50, 80 periods, and for each socialization rate p1 from 0.1 to 0.9 in steps of 0.1. However, unlike March (1991), the simulation was conducted 100 times for each setting. These are follow-up simulations to find whether we could reproduce the results illustrated in Figure 1 of March (1991); the average knowledge level of 100 simulations are illustrated in Figure 2.3 As with Figure 2, Figure 1 of March (1991) appears to be a graph of a monotonically decreasing function, causing March to conclude that “slower socialization (lower p1) leads to greater knowledge at 44 A missing piece of mutual learning model of March (1991) equilibrium than does faster socialization, particularly when the code learns rapidly (high p2)” (March, 1991, p. 75). Although a closer examination indicates that the socialization rate is a probability in definition, and should take on a value of between 0 and 1, Figure 1 of March (1991) is limited to a range of 0.1 to 0.9. Thus, either end of the domain, particularly the graph with a range of 0 to 0.1, which has particularly large changes, is missing. When we compensate for that missing graph, we arrive at an entirely different conclusion. In fact, we know the theoretical results when socialization rate p1 is 0, we experience lock-in, and the theoretical knowledge level is 1/3, as has already been demonstrated. Follow-up Simulations and True Conclusion In the results of our simulations indicated in Table 2, the average knowledge level, 0.334, is right about at the theoretical value. In other words, there should theoretically be no monotonically decreasing function. Thus, we use the same settings of Figure 2: reality dimensions m  30, organizational members n  50, and 80 periods. However, this time we conduct 100 simulations for each socialization rate p1 from 0 to 0.1 in steps of 0.01. When we tie this to Figure 2, we obtain Figure 3 (we have also compensated for p1  1). As is clearly illustrated in Figure 3, there is a peak where socialization rate p1 is between 0 and 0.1. When the learning rate p2 is 0.1, the highest average knowledge level is 0.756 at p1  0.06. When the learning rate p2 is 0.5, the highest average knowledge level is 0.868 at p1  0.07. When learning rate p2 is 0.9, the highest average knowledge level is 0.901 at p1  0.07. In other words, the statement that “slower socialization (lower p1) leads to greater knowledge at equilibrium than does faster socialization” (March, 1991, p. 75) is clearly a mistake, as can be seen from the shape of the graph. The conclusion that “slow learning on the part of individuals maintains diversity longer, thereby providing the exploration that allows the knowledge found in the 45 Mitomi and Takahashi Mitomi and Takahashi p2 = 0.1 p2 = 0.5 p2 = 0.9 1.0 p2 = 0.1 p2 = 0.5 p2 = 0.9 The range of Figure 2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.1 Socialization rate p1 Average knowledge level Figure 3. Average knowledge levels of March’s (1991) model with a wide range of 0 to 1 Note: m  30, n  50, 80 periods, 100 simulations p2 = 0.1 p2 = 0.5 p2 = 0.9 1.0 p2 = 0.1 p2 = 0.5 p2 = 0.9 The range of Figure 2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.1 Socialization rate p1 Average knowledge level p2 = 0.1 p2 = 0.5 p2 = 0.9 p2 = 0.1 p2 = 0.5 p2 = 0.9 The range of Figure 2 0.6 0.8 1.0 owledge level The range of Figure 2 organizational code to improve” (March, 1991, p. Follow-up Simulations and True Conclusion 76) cannot be arrived at using this model. Actually, there is an optimal socialization rate that maximizes the average knowledge level. This is because low learning on the part of individuals causes lock-ins frequently, and impedes learning. The optimal socialization rate of 0.06 to 0.07 may be a common rate for socialization, and we cannot deny this possibility by using only computer simulation method. organizational code to improve” (March, 1991, p. 76) cannot be arrived at using this model. Actually, there is an optimal socialization rate that maximizes the average knowledge level. This is because low learning on the part of individuals causes lock-ins frequently, and impedes learning. The optimal socialization rate of 0.06 to 0.07 may be a common rate for socialization, and we cannot deny this possibility by using only computer simulation method. 46 A missing piece of mutual learning model of March (1991) Equilibrium rate Socialization rate p1 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 p2 = 0.1 p2 = 0.5 p2 = 0.9 Figure 4. Equilibrium rate of March’s (1991) original model. Note: m  30, n  50, 80 periods, 100 simulations Equilibrium rate Socialization rate p1 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 p2 = 0.1 p2 = 0.5 p2 = 0.9 Figure 4. Equilibrium rate of March’s (1991) original model. 1.0 Figure 4. Equilibrium rate of March’s (1991) original model. N t 30 50 80 i d 100 i l ti Figure 4. Equilibrium rate of March’s (1991) original model. Moreover, maximization of the knowledge level does not require an equilibrium. Figure 4 illustrates the equilibrium rate, which is the number of equilibriums at the end of the 80th period divided by 100. As we have already stated, when socialization rate p1 is 0, all simulations are lock-in’s and the equilibrium rate should be 0. Even when the socialization rate p1 is 0.1, the equilibrium rate is low, at between 0.20 and 0.31. In other words, when the average knowledge level is highest in Figure 3, the equilibrium rate is only around 20%. The vertical axis of Figure 1 in March (1991) was intentionally labeled “average equilibrium knowledge.” However, a high knowledge level was actually obtained without equilibria. Acknowledgements This work was supported by JSPS KAKENHI Grant Number 26380454 for FY 2014–2018. Follow-up Simulations and True Conclusion In actuality, the fact that an equilibrium is not a desirable state is one important finding from our simulations, and an important conclusion of Takahashi et al. (2006) who conducted simulations using a communication competition model. Moreover, maximization of the knowledge level does not require an equilibrium. Figure 4 illustrates the equilibrium rate, which is the number of equilibriums at the end of the 80th period divided by 100. As we have already stated, when socialization rate p1 is 0, all simulations are lock-in’s and the equilibrium rate should be 0. Even when the socialization rate p1 is 0.1, the equilibrium rate is low, at between 0.20 and 0.31. In other words, when the average knowledge level is highest in Figure 3, the equilibrium rate is only around 20%. The vertical axis of Figure 1 in March (1991) was intentionally labeled “average equilibrium knowledge.” However, a high knowledge level was actually obtained without equilibria. In actuality, the fact that an equilibrium is not a desirable state is one important finding from our simulations, and an important conclusion of Takahashi et al. (2006) who conducted simulations using a communication competition model. 47 Mitomi and Takahashi Mitomi and Takahashi Rogers, E. M. (1962). Diffusion of innovations. 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Takahashi, N. (1997). A single garbage can model and the degree of anarchy in Japanese firms. Human Relations, 50, 91–108. Takahashi, N. (1998). Soshikiruchin to soshikinai ekoroji [Organizational routine and intraorganizational ecology]. Soshikikagaku [Organizational Science], 32(2), 54–77. Takahashi, N., Kuwashima, K., & Tamada, M. (2006). Komyunikeishon kyoso moderu to gorisei [Communication competition model and rationality]. Keizaigakuronshu [Journal of Economics], 72(3), 2–20. Society of Economics, University of Tokyo. Takahashi, N. (2013). Jumping to hasty experience curves: The learning curve revisited. Annals of Business Administrative Science, 12, 71–87. doi: 10.7880/abas.12.71 Received September 29, 2014; accepted October 24, 2014 49 Mitomi and Takahashi (supplementary materials, http://dx.doi.org/10.7880/abas.14.5) (supplementary materials, http://dx.doi.org/10.7880/abas.14.5) 50 (supplementary materials, http://dx.doi.org/10.7880/abas.14.5) # A Model of Mutual Learning in accordance with March (1991) n<-50 # The number of individuals. m<-30 # The number of dimensions. p1<-0.1 # The socialization rate. p2<-0.1 # The organization learning rate. set.seed(501) # A random seed is 501. # Reality has m dimensions. # Each dimension has a value of -1 or 1 with independent equal probability 0.5. r<-matrix(runif(m),1,m) for(i in 1:m){ if(r[i] >= 0.5){ r[i]<- 1 }else{ r[i]<- -1} } # Each of n individuals holds prior beliefs about reality. # For each dimension of reality, each prior belief has a value of -1, 0, or 1 with equal probability 1/3. b<-matrix(runif(n*m),n,m) for(i in 1:n){ bP<-b[i,] for(j in 1:m){if(bP[j] <= 0.3333333){ bP[j]<- -1 }else if(( bP[j]> 0.3333333) && (bP[j] <= 0.6666667)){ bP[j]<- 0 }else{ bP[j]<- 1 } b[i,j]<-bP[j] }} # CKL is the knowledge level of the organization code. C<-matrix(rep(0),1,m) CKLC<-function(C,r){ TrC<-0 for(i in 1:m){ if(C[,i] == r[,i]){ TrC<- TrC+1 } } CKL<- TrC/m ; return(CKL) } CKL<-CKLC(C,r) # MKL is the knowledge level of individuals. MKL<-matrix(rep(0),n,1) MKLC<-function(b,r){ for(j in 1:n){ TrC<-0 ; B<-b[j,] for(i in 1:m){ if(B[i] == r[,i]){ TrC<- TrC+1 } } MKL[j,]<- TrC/m } ; return(MKL)} MKL<-MKLC(b,r) MKLA<-matrix(rep(0),1,80) # The average of individual knowledge levels of all 80 periods. EQAR<-matrix(rep(0),1,80) # The equilibrium rate of all 80 periods. # EQWI is 1 (equilibrium) if all the beliefs of the organization code and individuals are equal. EQWI<-function(b,C){ TrEN<-0 ; TrE<-0 for(j in 1:n){ B<-b[j,] for(i in 1:m){ if(B[i] == C[,i]){ TrE<- TrE+1 } } } nm<-n*m ; if(TrE == nm){ TrEN<-1 } ; return(TrEN)} SG<-matrix(rep(0),1,n) # The ith dimension is 1 if the ith member belongs to the superior group. SGB<- matrix(rep(2),1,m) # The initial value is 2, which means no dominant belief within the superior group. # Individuals modify their beliefs as a consequence of socialization into the organization code. B<-matrix(rep(0),n,m) # Individual beliefs, code belief, and superior group belief at each period. BT<-NULL CT<-NULL SGBT<-NULL # The simulation starts. t<-1 # The number of periods. t <= 80. while(t <= 80){ ## Individuals learn from the organization code. # If the code is 0 on a particular dimension, then individual belief is not affected. # If the code differs on any particular dimension from the individual belief, # then individual belief changes to that of the code with probability p1 (when A is below p1). (supplementary materials, http://dx.doi.org/10.7880/abas.14.5) for(j in 1:n){ for(i in 1:m){ if((C[i] != 0) && (C[i] != b[j,i])){ A<-runif(1) }else{ A<- 1.1 } ; if(A <= p1){ b[j,i] <- C[i] } } } # The superior group is composed of individuals whose knowledge levels are superior to that of the code. for(j in 1:n){ MKLB<-MKL[j,] ; if(MKLB > CKL){ SG[,j] <- 1 }else{ SG[,j] <- 0 } } # Individuals are selected from the superior group. SGb<-cbind(b,t(SG)) ; SGb<-subset(SGb,SGb[,m+1] == 1) ; SGb<-SGb[,1:m] ; SGN<-sum(SG) if(SGN != 0){ SGbN <- matrix(rep(0),m,3) # The number of individual beliefs within superior group. if(SGN != 1){ for(j in 1:SGN){ SGb2<-SGb[j,] for(i in 1:m){ if(SGb2[i] == -1){ SGbN[i,1]<-SGbN[i,1]+1 }else if(SGb2[i] == 0){ SGbN[i,2]<-SGbN[i,2]+1 }else{ SGbN[i,3]<-SGbN[i,3]+1 } } } }else{SGb2<-SGb ; for(i in 1:m){ if(SGb2[i] == -1){ SGbN[i,1]<-SGbN[i,1]+1 }else if(SGb2[i] == 0){ SGbN[i,2]<-SGbN[i,2]+1 }else{ SGbN[i,3]<-SGbN[i,3]+1 } } } # SGN is dominant belief within the superior group. for(i in 1:m){ if((SGbN[i,1] <= SGbN[i,2]) && (SGbN[i,2] < SGbN[i,3])){ SGB[1,i] <- 1 }else if((SGbN[i,2] <= SGbN[i,1]) && (SGbN[i,1] < SGbN[i,3])){ SGB[1,i] <- 1 }else if((SGbN[i,1] <= SGbN[i,3]) && (SGbN[i,3] < SGbN[i,2])){ SGB[1,i] <- 0 }else if((SGbN[i,3] <= SGbN[i,1]) && (SGbN[i,1] < SGbN[i,2])){ SGB[1,i] <- 0 }else if((SGbN[i,2] <= SGbN[i,3]) && (SGbN[i,3] < SGbN[i,1])){ SGB[1,i] <- -1 }else if((SGbN[i,3] <= SGbN[i,2]) && (SGbN[i,2] < SGbN[i,1])){ SGB[1,i] <- -1 }else{ SGB[1,i] <- 2 #if there is no dominant belief } } # Calculation of k. k<-matrix(rep(0),1,m) for(i in 1:m){ if(C[i] == 1){ NM<-SGbN[i,2]+SGbN[i,1] ; MM<-SGbN[i,3] ; k[i] <- NM - MM }else if(C[i] == 0){ NM<-SGbN[i,1]+SGbN[i,3] ; MM<-SGbN[i,2] ; k[i] <- NM - MM }else if(C[i] == -1){ NM<-SGbN[i,2]+SGbN[i,3] ; MM<-SGbN[i,1] ; k[i] <- NM - MM } } ## The organization code adapts to the individual beliefs within the superior group. for(i in 1:m){if((SGB[,i] != 2) && (C[,i] != SGB[,i]) && (k[,i] > 0)){ A<-runif(1) ; q<- 1- (1-p2)^k[,i] ; if(A <= q){ C[,i] <- SGB[,i] } } } } } CKL<-CKLC(C,r) #Knowledge level of the code after adaptation. MKL<-MKLC(b,r) #Knowledge level of individuals after socialization. MKLA[1,t]<-sum(MKL)/n #Average knowledge level of individuals after socialization. # Calculation of equilibrium rate. eqm<-0 for(j in 1:n){ b2<-b[j,] for(i in 1:m){ if(b2[i] == C[,i]){ eqm<-eqm+1 } } } EQAR[1,t]<-eqm/(n*m) BT[[t]]<-b CT[[t]]<-C SGBT[[t]]<-SGB t<-t+1 } # Print MKLA EQAR EQWI(b,C) # Individual beliefs, code belief, and superior group belief at each period. # The number of periods. t <= 80. } # Print MKLA EQAR EQWI(b,C) } # Print MKLA EQAR EQWI(b,C) 50 A missing piece of mutual learning model of March (1991) Appendix B: Revised Model by Takahashi (1998) Takahashi (1998) attempted revisions to the model used by March (1991) to reproduce March’s Figure 1. Takahashi (1998) corrected the majority opinion of the superior group in Assumption 4 to the majority opinion of the superior group “except for 0” by removing 0 from the belief components to avoid lock-in as much as possible from the outset. In addition, he proposed that Assumption 4 (3), “ki (ki  0) is the number of members whose ith component of belief differs from ith component of organizational code’s belief minus the number of members who do not (within the superior group)” be modified to “ki is the number of the majority minus the number of the minority (within the superior group)”. When conducting simulations using this modification as in Figure 2, the result is Figure 5, and the shape is quite similar to Figure 1 of March (1991). 1.0 0.9 0.8 0.7 0.6 0.5 0.1 0.3 0.5 0.7 0.9 Average knowledge level Socialization rate p1 p2 = 0.1 p2 = 0.5 p2 = 0.9 Figure A1. Average knowledge levels of Takahashi’s (1998) revised model 1.0 0.9 0.8 0.7 0.6 0.5 0.1 0.3 0.5 0.7 0.9 Average knowledge level Socialization rate p1 p2 = 0.1 p2 = 0.5 p2 = 0.9 0.9 Figure A1. Average knowledge levels of Takahashi’s (1998) revised model Note: m  30, n  50, 80 periods, 100 simulations Note: m  30, n  50, 80 periods, 100 simulations Note: m  30, n  50, 80 periods, 100 simulations 51
https://openalex.org/W4389886019
https://journals.uni-lj.si/aas/article/download/15568/13502
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Endosperm protein polymorphism of common and tartary buckwheat
Acta agriculturae Slovenica
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1 Selo pri Žirovnici 31/B, 4274 Žirovnica, Slovenia 2 Research Institute of Crop Production, Prague-Ruzyně, Drnovská 507, Prague 6 Ruzyně 161 06, Czech Republic 3 Czech University of Agriculture Prague, Institute of Tropical and Subtropical Agriculture, Kamýcká 129, Prague 6 – Suchdol, 165 21, Czech Republic Agris category code: F30 COBISS code 1.01 Marija LIČEN1, Vaclav DVOŘÁČEK2, Petra ČEPKOVÁ3, Anna MICHALOVÁ2 Marija LIČEN1, Vaclav DVOŘÁČEK2, Petra ČEPKOVÁ3, Anna MICHALOVÁ Received: February 16; accepted: May 31, 2004 Prispelo 16. februarja 2004; sprejeto 31. maja 2004. Received: February 16; accepted: May 31, 2004 Prispelo 16. februarja 2004; sprejeto 31. maja 2004. Acta agriculturae slovenica, 83 - 1, junij 2004 Acta agriculturae slovenica, 83 - 1, junij 2004 str. 171 - 179 Agrovoc deskriptors: Fagopyrum esculentum; Fagopyrum tataricum; varieties, analytical methods, identification, electrophoresis, endosperm, proteins, polymorphism ABSTRACT The endosperm protein polymorphism of seven accessions of common buckwheat (Fagopyrum esculentum Moench) and two accessions of tartary buckwheat (Fagopyrum tataricum Gaertn.) were studied by SDS-PAGE electrophoresis. The position and the intensity of the main four electrophoretic protein bands (marked as “a”, “b”, “c” and “d”) of cultivar Siva (common buckwheat) were estimated and the polymorphism was studied using seeds from open pollinated heterozygous plants. Two high significant correlations (correlation coefficient 0.559 for “a” and “c” bands, and 0.264 for “a” and “d” bands) were established among the intensities of electrophoretic protein bands of common buckwheat, while other four possible relations were not significant. The results indicate significantly higher protein polymorphism of endosperm within individual accessions than among accessions of common buckwheat. In contrary to common buckwheat, there was in material studied found no variability in endosperm protein bands between or within two accessions of tartary buckwheat. Key words: Fagopyrum esculentum; Fagopyrum tataricum; SDS-PAGE electrophoresis; endosperm proteins; polymorphism Key words: Fagopyrum esculentum; Fagopyrum tataricum; SDS-PAGE electrophoresis endosperm proteins; polymorphism 1 INTRODUCTION Buckwheat (Fagopyrum esculentum Moench) proteins are nutritionally important because of their excellent amino acid composition (Kreft I., 1994; Kreft I., 1995; Bonafaccia et al., 2003a) and thus very high biological value of buckwheat proteins (BV = 86) which is much higher than that of cereal proteins (Skrabanja et al., 2000). The major (13S globulin) and minor classes of storage protein represent about 33 and 6.5% of total seed proteins, respectively (Radović et al., 1996). Buckwheat proteins may prevent gallstone formation more strongly than soy protein isolates, retard mammary carcinogenesis by lowering serum estradiol, and they suppress colon carcinogenesis by reducing cell proliferation (Kayashita et al., 1999; Tomotake et al., 2000; Liu et al., 2001). This is maybe connected with limited digestibility of buckwheat protein (Ikeda et al., 1986; Ikeda et al., 1993; Skrabanja et al., 2000). The high content of protein in some buckwheat grain milling fractions suggests a potential application of these materials for special dietary products. Tannin-protein complexes are in the gastrointestinal tract potent radical cation scavengers and may act as a radical sink (Luthar, 1992; Riedel and Hagerman, 2001). Buckwheat endosperm proteins may contain some selenium ( Bonafaccia et al., 2003b), a trace element, essential in human nutrition. As buckwheat does not contain gluten proteins, it is a common supplement for patients with celiac disease (Skerritt, 1986; Wieslander and Norbäck, 2001). Proteins are along with starch among the main endogenous factors responsible for the textural characteristics of buckwheat products (Ikeda et al., 1997). Correlations between protein content and hardness, cohesiveness, adhesiveness, springiness and chewiness evaluated on buckwheat dough prepared from the endosperm fractions were reported (Ikeda et al., 1997; Ikeda et al., 1999). All textural characteristics, except for adhesiveness, were in significant negative correlation to protein content. Buckwheat is an undemanding low-input plant (Kreft I., 1989; Petr, 1995; Kalinova and Moudry, 2003). Polymorphism of electrophoretic protein pattern in buckwheat was first reported by Kreft I. et al. (1978). The genetic polymorphism related to buckwheat nutritive value was studied earlier (Michalová, 1998; Michalová, et al. 1998; Baburkova et al., 2000; Kalinova et al., 2002). High polymorphisms of the cotyledon storage proteins, but high endosperm protein homogenity were reported by Rogl and Javornik (1996). However till now there was no research report on polymorphism in endosperm proteins of common buckwheat. IZVLEČEK POLIMORFIZEM BELJAKOVIN ENDOSPERMA PRI NAVADNI IN TATARSKI AJDI S pomočjo SDS-PAGE elektroforeze smo analizirali sedem vzorcev navadne (Fagopyrum esculentum Moench) in dva vzorca tatarske ajde (Fagopyrum tataricum Gaertn.). Pri cv. Siva smo analizirali semena s tujeprašnih rastlin in ocenili pojavljanje in intenzivnost glavnih štirih elektroforeznih črt (označene z “a”, “b”, “c” in “d”). Pri ovrednotenju intenzivnosti pojavljanja elektroforeznih črt, smo ugotovili dve visoko statistično značilni korelaciji (korelacijski koeficient 0,559 pri “a” in “c” elektroforeznih črtah ter 0,264 pri “a” in “d” elektroforeznih črtah), medtem ko ostale štiri možne korelacije intenzivnosti elektroforeznih črt niso bile statistično značilne. Rezultati nakazujejo na visoko statistično značilni polimorfizem beljakovin Acta agriculturae slovenica, 83 - 1, junij 2004 172 Acta agriculturae slovenica, 83 - 1, junij 2004 172 endosperma med analiziranimi semeni cv. Siva, med tem ko med posameznimi vzorci ni razlik v pojavljanju elektroforeznih črt. Med dvema vzorcema in znotraj vzorca tatarske ajde nismo našli razlik v pojavljanju elektroforeznih črt. endosperma med analiziranimi semeni cv. Siva, med tem ko med posameznimi vzorci ni razlik v pojavljanju elektroforeznih črt. Med dvema vzorcema in znotraj vzorca tatarske ajde nismo našli razlik v pojavljanju elektroforeznih črt. Ključne besede: Fagopyrum esculentum; Fagopyrum tataricum; SDS-PAGE elektroforeza; beljakovine endosperma; polimorfizem 2.2 Dissection of buckwheat seeds Prior to dissection the unhusked buckwheat seeds were soaked for 1 hour in distilled water. Under magnifying glass the husk and testa were removed from each analyzed seed and the endosperm was carefully separated from cotyledons (embryo) and placed each endosperm separately into a 1,5 ml micro centrifuge tube. Each dissected endosperm was smashed in 300 µm of extraction buffer (0.5 M Tris- HCl pH 6.8, 20 % glycerol, 4 % SDS, 3 % 2-merkaptoetanol, Bromphenol blue) with a glass stick. After 2 hours of extraction, the samples were incubated in water bath at 95 ºC for 5 minutes, centrifuged for 15 min at 10 000 rp/min and 15 µm of supernatant was applied for electrophoresis. 1 INTRODUCTION The aim of this investigation was to describe the polymorphism of buckwheat endosperm proteins, as a basis for breeding selection towards the designed endosperm protein constitution. 173 LIČEN, M., et al: Endosperm protein polymorphism of common and tartary... LIČEN, M., et al: Endosperm protein polymorphism of common and tartary... 2.1 Plant material The material that we used in this study was obtained from the Gene Bank at the Research Institute of Crop Production, Prague-Ruzyně, Czech Republic, and from the gene bank maintained at the Center for Plant Biotechnology, Breeding and Genetics, Biotechnical Faculty, University of Ljubljana. Samples are listed in table 1. able 1: List of accessions used for analysis No. of sample Accession 1. Grey buckwheat cv. Siva (SVN) 2. Z50-0064 La Harpe (FRA) 3. Z50-0063 Pyra (CZE) 4. Z50-0062 Bolshevik 4 (RUS) 5. Z50-0051 Botansoba (JAP) 6. Z51-0014 Tartary buckwheat (USA) 7. Z51-0012 Tartary buckwheat (CZE) 8. Z50-0070 NS-SP-LXH (SVK) 9. Z50-0111 Emka (POL) Table 1: List of accessions used for analysis Sample number 1 was common buckwheat cv. Siva, 105 seeds from open pollinated heterozygous plants were analyzed. Samples number 2 – 9 were accessions collected and provided by the Research Institute of Crop Production, Prague-Ruzyně, Czech Republic; approximately 10 seeds from open pollinated heterozygous plants respectively self-pollinated homozygous plants of tartary buckwheat (number 6 and 7) were analyzed. Acta agriculturae slovenica, 83 - 1, junij 2004 174 Acta agriculturae slovenica, 83 - 1, junij 2004 174 Acta agriculturae slovenica, 83 - 1, junij 2004 174 2.3 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method For one run of SDS-PAGE electrophoresis two parts of gels were prepared; separation (10% T) (1.5 M Tris- HCl pH 8.8, 10 % T acrylamide stock, 1% SDS, 1% ammonium persulfate, TEMED) and concentration gel (1.5 M Tris- HCl pH 6.8, 4 % T 1.3 % C acrylamide stock, 1% SDS, 1% ammonium persulfate, TEMED) (Laemmli, 1970). For one run of electrophoresis, two 1.5 mm thick gels were prepared. The apparatus (for vertical electrophoresis) carried out the electrophoresis at 40 mA for 1 hour, and about 3 hours at 60 mA. The proteins were stained for 24 hours with 0.05% Commasie Brilliant Blue R- 250, 5% ethanol, and 12% TCA, and then destained in deionised water. Intensity of the electrophoretic protein bands was estimated by the scale from 0 (there is no band) to 5 (very thick band). Statistical analysis was performed using Image analysis Excel (Microsoft Co., USA). 3.1 Endosperm proteins of buckwheat cv. Siva The endosperm protein cv. Siva was characterized 3 – 4 strong protein with different intensity and high number of weak bands with molecular weight extent of 20 – 100 kDa. Our population study of cv. Siva endosperm protein variability was focused on clearly visible main 4 bands with molecular weight 95 kDa respectively 50 – 60 kDa. The results of endosperm protein polymorphism of cv. Siva are shown in figure 1. After the estimation of the intensities of the bands by the scale from 0 (no band) to 5 (very thick band), the correlation between patterns of 105 seeds was established. Correlation coefficients between pairs of the appearances of “a”, “b”, “c”, and “d” bands are presented in table 2. Figure 1: Electropherograms of cv. Siva buckwheat endosperm SDS-PAGE. Among all bands the 4 bands (“a”, “b”, “c”, “d”) are the most intense. Band “b” does not appear in all samples a ~ 95 kDa b ~ 63 kDa c ~ 59 kDa d ~ 54 kDa Figure 1: Electropherograms of cv. Siva buckwheat endosperm SDS-PAGE. Among all bands the 4 bands (“a”, “b”, “c”, “d”) are the most intense. Band “b” does not appear in all samples Electrophoretic band “b” (~ 63 kDa) appears just in some seeds and the intensity is quite variable. Band “a” (~ 95 kDa) appears in endosperm extracts of all studied seeds and here the band intensity is almost in all samples the same. The intensity of “c” (~ 59 kDa) and “d” (~54 kDa) bands was different from seed to seed, but these bands appear always in the combination with the occurrence of band “a”. There was no significant correlation between “a” and “b” or “b” and “c” bands; there was negative, but nont significant relation between “b” and “d” or “c” and “d” bands. Significant correlation appeared between “a” and “c”, and “a” and “d” bands (table 2). LIČEN, M., et al: Endosperm protein polymorphism of common and tartary... 175 Table 2: Correlation coefficients between pairs of the appearance of a, b, c an bands in cv. *Valuation: / (no band, 0); + (intensity 1); ++ (intensity 2); +++ (intensity 3); ++++ (intensity 4); +++++ (intensity 5) 3.2 Endosperm protein bands of other accessions of common buckwheat No essential differences of endosperm proteins appeared among other samples 2, 3, 4, 5, 8 and 9 (common buckwheat). In all samples, bands “a”, “c”, and “d” occurred in the same position and with the similar intensity as in cv. Siva buckwheat, however band “b” does not appear in any of samples 2-5, 8 or 9. This is in agreement with the results of Rogl and Javornik (1996), who find no essential variability in the electrophoretic protein band in common buckwheat. They obtained higher polymorphism of the cotyledon storage proteins. We can also agree with the statemen of Rogl and Javornik (1996), the commonest differentiating feature in the electrophoretic profile of out-breeding crops is quantitative differences in band intensities rather than the complete presence / absence of bands. It can also notice that strictly heterogamy of common buckwheat causes significantly higher intra varietal variability than the variability which we can find among cultivars. It’s possible to expect (on the basis of endosperm protein polymorphism) that higher genetic gain (improvement of agronomical or qualitative traits) will be obtained with the selection work inside of individual current cultivars (degrease of genetic basis) than prompt mutual crossing among different cultivars. 3.1 Endosperm proteins of buckwheat cv. Siva On the basis of band positions we only found out 2 genotypes which differed in the presence (ratio 38%) / absence (ratio 62%) of “b” protein band. When we implicated the factor of the band intensity in our estimation, we obtained 19 genotypes with percentage ratio of individual genotypes (electrophoreotypes) from 2% to 17%. That corresponds with biological character of open pollinated heterozygous plants and gives the possibility for selection breeding towards the designed combination of endosperm proteins (Kreft I., 1995). 3.1 Endosperm proteins of buckwheat cv. Siva Siva buckwheat endosperm extracts from 105 seeds band / band a b c d a - 0.1039 0.5591* 0.2636* b - 0.0347 -0.1770 c - -0.0600 d - * Correlation significant at p<0.01 * Correlation significant at p<0.01 Buckwheat endosperm proteins are important factors of the final nutritional and functional value and influence value of other constituents with the importance for human health, as rutin (Michalova et al., 1998; Kreft S. et al., 1999; Petr et al., 1999; Park et al., 2000; Fabjan et al., 2003), resistant starch (Skrabanja and Kreft I., 1998; Skrabanja et al., 1998; Skrabanja et al., 2001; Kreft I. and Skrabanja, 2002) and mineral elements (Bonafaccia et al., 2003b). The found correlations among intensity of specific storage protein molecules, respectively presence / absence of “b” band, can have important roll for future finding their relation to quantity or quality grain parameters. Table 3: Number of different electroforetic phenotypes combinations and percent ratio in cv. Siva population Electrophoretic band Combinations of electroforetic phenotypes a b c d Percentage ratio of evaluated phenotypes 1. ++++ / ++++ ++++ 13.3% 2. +++ / ++++ +++++ 17.1% 3. +++ / +++ + 2.8% 4. ++ / ++++ +++++ 6.6% 5. ++ / ++++ +++ 3.8% 6. ++ / ++ ++++ 3.8% 7. ++ / + +++++ 4.8% 8. + / ++++ ++++ 0.9% 9. + / + +++++ 5.7% 10. + / + ++ 2.8% 11. ++++ ++++ ++++ +++++ 3.8% 12. +++ + ++++ ++++ 3.8% 13. +++ + +++ ++++ 1.5% 14. +++ ++ ++++ ++++ 6.6% 15. +++ ++ ++++ +++ 2.8% 16. +++ ++ ++++ +++++ 0.9% 17. ++ +++ ++++ ++ 5.7% 18. ++ + ++++ ++++ 4.8% 19. ++ + ++++ ++ 1.9% *Valuation: / (no band, 0); + (intensity 1); ++ (intensity 2); +++ (intensity 3); ++++ (intensity 4); +++++ (intensity 5) Table 3: Number of different electroforetic phenotypes combinations and their percent ratio in cv. Siva population Table 3: Number of different electroforetic phenotypes combinations and their percent ratio in cv. Siva population Table 3: Number of different electroforetic phenotypes combinations and their percent ratio in cv. Siva population Acta agriculturae slovenica, 83 - 1, junij 2004 176 Different genotypes combinations of cv. Siva endosperm and their percent ratio are presented in table 3. 4 CONCLUSIONS The electrophoretical protein evaluation of cv. Siva indicated high polymorphism mainly in band intensity of endosperm proteins. We confirmed significant positive correlation among protein bands “a” - “c”, and “a” - “d” No pronounced difference among endosperm proteins of different accessions of common buckwheat was found. The significantly higher intra varietal protein variability was found in contrast to protein variability among cultivars. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of common buckwheat endosperm proteins seems to be less suitable tool for the identification of the standard buckwheat cultivars. Clear protein difference was only noticed between both different species common and tartary buckwheat. 3.3 Endosperm proteins of tartary buckwheat The variation of endosperm proteins in tartary buckwheat was studied and compared to endosperm proteins of common buckwheat cv. Siva (Figure 2). There is no essential difference in the endosperm proteins bands between two accessions of tartary buckwheat. However the important difference was found between common and tartary buckwheat samples. On the electropherogram of tartary buckwheat the band “d” (weight range around 59 kDa) is less intensive than on electropherogram of any common buckwheat and at the tartary buckwheat at the position of “b” there was no band; however at the adjacent position of “c” an intense double band always appears in tartary buckwheat. LIČEN, M., et al: Endosperm protein polymorphism of common and tartary... 177 Fiure 2: Two different electropherograms of cv. Siva buckwheat endosperm (samples 1 and 1a) compared to electropherogram of tartary buckwheat (sample 7) 1 1a 7 Fiure 2: Two different electropherograms of cv. Siva buckwheat endosperm (samples 1 and 1a) compared to electropherogram of tartary buckwheat (sample 7) 6 REFERENCES Baburkova M., Juza J., Moudry J., Pejcha J. 2000. The effect of genotype and agronomical practices on the structure of yield factors of buckwheat. Rostl. Výr., 46, 5: 225- 230. Bonafaccia G., Marocchini M., Kreft I. 2003a. Composition and technological properties of the flour and bran from common and tartary buckwheat. Food Chem., 80: 9-15. Bonafaccia G., Gambelli L., Fabjan N., Kreft I. 2003b. Trace elements in flour and bran fromcommon and tartary buckwheat. Food Chem., 83: 1-15. Fabjan N., Rode J., Košir I. J., Wang Z., Zhang Z., Kreft I. 2003. Tartary buckwheat (Fagopyrum tataricum Gaertn.) as a source of dietary rutin and quercetin. J. Agric. Food Chem., 51: 6452-6455. Ikeda, K., Fujiwara, J., Asami, Y., Arai, R., Bonafaccia, G., Kreft I., I., Yasumoto, K. 1999. Relationship of protein to the textural characteristics of buckwheat products: analysis with various buckwheat flour fractions. Fagopyrum, 16: 79-83. Ikeda, K., Oku, M., Kusano, T., Yasumoto, K. 1986. Inhibitory potency of plant antinutrients towards the in vitro digestibility of buckwheat protein. J. Food Sci., 51: 1527-1530. Ikeda, K., Kishida, M. 1993. Digestibility of proteins in buckwheat seed. Fagopyrum, 13: 21- 24. Ikeda, K., Kishida, M., Kreft I., I., Yasumoto, K. 1997. Endogenous factors responsible for the textural characteristics of buckwheat products. J. Nutr. Sci. Vitaminol., 43: 101- 111. Kalinova J., Moudry J. 2003. Evaluation of frost resistance in varieties of common buckwheat (Fagopyrum esculentum Moench). Plant, Soil and Environment, 49 (9): 410-413. Kalinova J., Moudry J., Curn V. 2002. Technological quality of common buckwheat (Fagopyrum esculentum Moench.). Rostl. Výr., 48 (6): 279-284 Kayashita J., Shimaoka I., Nakajoh M., Kishida N., Kato N. 1999. Consumption of a buckwheat protein extract retards 7,12-dimethylbenz[alpha]anthracene-induced mammary carcinogenesis in rats. Bioscience, Biotechnology, and Biochem., 63: 1837-1839. Kreft. I. 1989. Breeding of determinate buckwheat. Fagopyrum, 9: 57-59. Kreft I. 1994. Traditional buckwheat food in Europe. Bull. Res. Inst. Food Sci., 57: 1-8. Kreft I. 1995. Ajda. CZD, Kmečki glas, Slovenia, 1 -112. Kreft I. 1995. Ajda. CZD, Kmečki glas, Slovenia, 1 -112. Kreft I., Javornik B., Strel B. 1978. Polymorphism of electrophoretic protein patterns in buckwheat. Research Reports, 31: 67-69. Kreft I., Škrabanja V. 2002. Nutritional properties of starch in buckwheat noodles. J. Nutr. Sci. Vitaminol., 48: 47-50. Kreft S., Knapp M., Kreft I. 1999. Extraction of rutin from buckwheat (Fagopyrum esculentum Moench) seeds and determination by capillary electrophoresis. J. Agric. Food Chem., 47: 4649-4652. Laemmli U. 1970. 5 ACKNOWLEDGEMENT This investigation was performed in the frame of scientific cooperation Slovenia - Czech Republic, financed by the Ministry of Education, Science and Sports of Slovenia and the Ministry of Science of the Czech Republic: Project Contact. We would like to thank Mrs. Branka Juvančič for invaluable technical assistance, Prof. Branka Javornik for making possible the utilization of the electrophoretic equipment and Prof. Ivan Kreft for useful suggestions in this research. Acta agriculturae slovenica, 83 - 1, junij 2004 178 Acta agriculturae slovenica, 83 - 1, junij 2004 178 6 REFERENCES Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227: 680-685. Liu Z., Ishikawa W., Huang X., Tomotake H., Kayashita J., Watanabe H., Nakajoh M.,Kato N. A. 2001. Buckwheat protein product suppresses 1,2-dimethylhydrazine-induced colon carcinogenesis in rats by reducing cell proliferation. J. Nutr., 131: 1850-1853. LIČEN, M., et al: Endosperm protein polymorphism of common and tartary... LIČEN, M., et al: Endosperm protein polymorphism of common and tartary... 179 Luthar Z. 1992. The content and the distribution of tannin in the buckwheat seeds (Fagopyrum esculentum Moench). Ph.D. Thesis, University of Ljubljana, Ljubljana, Slovenia. Michalová A., 1998. Variability of selected characteristics in sets of buckwheat, millet and amaranth; selection of perspective genotypes and comparison of their nutritive value. Research Reports, 71: 115 -125. Michalová A., Dotlačil L., Čejka L. 1998. Evaluation of common buckwheat cultivars. Rostl. Výr., 44 (8): 361-368. Park C.H., Kim Y. B., Choi Y. S., Heo K., Kim S. L., Lee K. C., Chang K. J., Lee H. B. 2000. Rutin content in food products processed from groats, leaves and flowers of buckwheat. Fagopyrum, 17: 63-66. Petr J. 1995. Buckwheat and proso millet production. Metodiky pro Zavadeni Vysledku Vyzkumu do Zemedelske Praxe, No. 7, 35 pp. Petr J., Skerik J., Dlouhy J. 1999. Cadmium, lead and mercury contents in ecologically grown crops. Scientia Agric. Bohemica, 30: (4) 285-299. Radović S. R., Maksimović V. R., Varkonji-Gašić E. I., 1996. Characterization of buckwheat seed storage proteins. J. Agric. Food Chem., 44: 972- 974. Riedl K.M., Hagerman A.E., 2001. Tannin-protein complex as radical scavengers and radical sinks. Journal of agricultural and food chemistry, 49,10: 4917-4923. Rogl S., Javornik B, 1996. Seed protein variation for identification of common buckwheat (Fagopyrum esculentum Moench) cultivars. Euphytica, 87: 111-117. Skerritt J. H. 1986. Molecular comparison of alcohol-soluble wheat and buckwheat proteins. Cereal Chem., 63: 365-369. Skrabanja V., Kreft I. 1998. Resistant starch formation following autoclaving of buckwheat (Fagopyrum esculentum Moench) groats. An in vitro study. J. Agric. Food Chem., 46: 2020-2023. Skrabanja V., Laerke H. N., Kreft I. 1998. Effects of hydrothermal processing of buckwheat (Fagopyrum esculentum Moench) groats on starch enzymatic availability in vitro and in vivo in rats. J. Cereal Sci., 28: 209-214. Skrabanja V., Laerke H. N., Kreft I. 2000. Protein-polyphenol interactions and in vivo digestibility of buckwheat groat proteins. Pflügers Archiv - Eur. J. Physiol., 440: R129-R131. Skrabanja V., Liljeberg Elmståhl H. G. Wieslander G., Norbäck D. 2001. Buckwheat allergy. Allergy, 56: 703-704. 6 REFERENCES M., Kreft I., Björck I. M. E. 2001. Nutritional properties of starch in buckwheat products: studies in vitro and in vivo. J. Agric. Food Chem., 49: 490-496. Tomotake H., Shimaoka I., Kayashita J., Yokoyama F., Nakajoh M., Kato N. 2000. A buckwheat protein product suppresses gallstone formation and plasma cholesterol more strongly than soy protein isolate in hamsters. J. Nutr., 130: 1670-1674. Wieslander G., Norbäck D. 2001. Buckwheat allergy. Allergy, 56: 703-704.
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https://www.degruyter.com/document/doi/10.1515/abitech-2022-0040/pdf
de
Warum wird das DINI-Zertifikat immer wieder angepasst?
ABI-Technik
2,022
cc-by
472
ABI Technik 2022; 42(3): 226 ABI-Technik-Frage Daniel Beucke, Isabella Meinecke und Thomas Severiens Warum wird das DINI-Zertifikat immer wieder angepasst? https://doi.org/10.1515/abitech-2022-0040 Das DINI-Zertifikat ist das wesentliche Instrument zur Standardisierung für Open-Access-Publikationsdienste. Das Zertifikat wurde von der DINI-Arbeitsgruppe Elektronisches Publizieren (DINI-AG E-Pub) entwickelt. Es wird turnusmäßig aktualisiert. Daniel Beucke, Isabella Meinecke und Thomas Severiens sind Sprecher*innen der DINI-AG E-Pub. Für ABI Technik beantworten sie diesmal die Technikfrage: Warum wird das DINI-Zertifikat immer wieder angepasst? Das DINI-Zertifikat für Open-Access-Publikationsdienste1 ebnet den Weg für eine offene Wissenschaftskommunikation durch die freie Bereitstellung wissenschaftlicher Materialien als wesentlicher Baustein zukunftsorientierter forschungsnaher Dienste. Das DINI-Zertifikat gibt es bereits seit 2004; es wird von der DINI-AG E-Pub angeboten. Das Zertifikat besteht aus einem Katalog von Kriterien, die für Einrichtung und Betrieb eines entsprechenden Open-Access-Publikationsdienstes zu berücksichtigen sind. Für die zertifizierten Einrichtungen bietet es damit quasi eine Garantie, dass der eigene Service den Anforderungen offener Wissenschaft gerecht wird und bleibt. Die DINI-AG E-Pub erarbeitet alle drei Jahre einen angepassten Kriterienkatalog, der auf den aktuellen Stand der technischen und redaktionellen Anforderungen eingeht. Technische Austauschprotokolle, Standards der Wissensbeschreibung und rechtliche Anforderungen an Services entwickeln sich stetig weiter und erfordern eine regelmäßige Anpassung des Zertifikatstexts. Seit den letzten Versionen wird in einem Request-for-Comments-Verfahren zudem das Feedback der interessierten Fachgemeinschaft eingeholt und bei der Überarbeitung berücksichtigt, so dass das Zertifikat zum Standard für entsprechende Publikationsdienste geworden ist. In der 2022er-Version wird auf die internationale Vernetzung, die Unterstützung der Nutzenden als Autor:innen und Rezipient:innen sowie die Nachhaltigkeit der Infrastruktur des Services fokussiert. Dazu wurde das Zertifikat um die spezifischen Anforderungen für eine Zertifizierung österreichischer Publikationsdienste erweitert. Aufgrund von Anmerkungen aus der Fachgemeinschaft zur besseren Benutzbarkeit und Verständlichkeit werden im 2022er-Zertifikat die bisherigen Anhänge zur OAI-Schnittstelle in den Kriterienkatalog integriert, einige Kriterien werden im Inte1 https://dini.de/dienste-projekte/dini-zertifikat/ (08.07.2022). resse der Nutzenden-Freundlichkeit zusammengefasst. Das seit 2021 überarbeitete Gemeinsame Vokabular für Publikations- und Dokumententypen, ein De-facto-Standard für die Beschreibung von Publikationstypen, wird bindend für die Zertifizierung nach der neuen Version. DataCite wird neben Dublin Core als Metadatenformat empfohlen, um z. B. Autor:innen mit Namen und ORCID identifizieren zu können. Nach Abschluss der Arbeiten an der aktuellen Zertifikatsversion werden neue Entwicklungen in den Blick genommen, die für die kommende 2025er-Version als bedeutsam erachtet werden. So werden bereits weitere Schritte der Internationalisierung angedacht, etwa durch Vernetzung mit europäischen Projekten, die ebenfalls an internationalen Standards für Open-Access-Publikationsdiensten wirken. Darüber hinaus erfordert der Trend zu integrierten heterogenen Repositorien, die unterschiedliche Formate, Metadaten und Zugangsformen beinhalten, eine Reaktion. Auch hieran zeigt sich: Das DINI-Zertifikat ist als überzeugender Standard immer am Puls der Zeit. Autoreninformationen Daniel Beucke Niedersächsische Staats- und Universitätsbi­blio­thek Göttingen beucke@sub.uni-goettingen.de orcid.org/0000-0003-4905-1936 Isabella Meinecke Staats- und Universitätsbi­blio­thek Hamburg isabella.meinecke@sub.uni-hamburg.de orcid.org/0000-0001-8337-3619 Thomas Severiens Institut für Wissenschaftliche Information, Osnabrück thomas.severiens@jade-hs.de orcid.org/0000-0001-6303-5073 Open Access. © 2022 bei den Autorinnen und Autoren, publiziert von De Gruyter. Namensnennung 4.0 International Lizenz. Dieses Werk ist lizensiert unter der Creative Commons
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https://pure.au.dk/ws/files/274021624/1_s2.0_S0264410X21011506_main.pdf
English
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Coverage and factors associated with receiving campaign polio vaccines in an urban population in Guinea-Bissau
Vaccine
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cc-by
5,646
a b s t r a c t Article history: Received 1 February 2021 Received in revised form 13 August 2021 Accepted 25 August 2021 Available online 12 October 2021 Keywords: Oral polio vaccine Supplementary immunization activities Immunization coverage Background: Polio eradication campaigns are intended to complement routine immunization. Studies addressing factors associated with campaign coverage are warranted to identify children missed by cam- paigns. Article history: Received 1 February 2021 Received in revised form 13 August 2021 Accepted 25 August 2021 Available online 12 October 2021 Article history: Received 1 February 2021 Received in revised form 13 August 2021 Accepted 25 August 2021 Available online 12 October 2021 Methods: Bandim Health Project runs demographic surveillance with registration of routine immuniza- tion and campaign participation data in urban Guinea-Bissau. We assessed coverage and factors associ- ated with receiving campaign polio vaccines in children aged 0–35 months in two polio eradication campaigns conducted in 2017 and 2018 using univariate and multivariate regression models. tion and campaign participation data in urban Guinea-Bissau. We assessed coverage and factors associ- ated with receiving campaign polio vaccines in children aged 0–35 months in two polio eradication campaigns conducted in 2017 and 2018 using univariate and multivariate regression models. Results: Campaign coverage reached 84% in 2017 and 88% in 2018. We found lower coverage among chil- dren of young and not formally educated mothers in univariate analyses; Children <9 months and Fula children had lower campaign coverage in both univariate and multivariate analyses. Keywords: Oral polio vaccine Supplementary immunization activities Immunization coverage Results: Campaign coverage reached 84% in 2017 and 88% in 2018. We found lower coverage among chil- dren of young and not formally educated mothers in univariate analyses; Children <9 months and Fula children had lower campaign coverage in both univariate and multivariate analyses. Conclusions: To increase campaign coverage in urban Guinea-Bissau attention may be directed at inform- ing young mothers, mothers of young children, mothers without formal education, and the Fula ethnic group about campaigns.  2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). Vaccine 39 (2021) 6720–6726 Vaccine 39 (2021) 6720–6726 journal homepage: www.elsevier.com/locate/vaccine journal homepage: www.elsevier.com/locate/vaccine M. Buus a,b,c, I. da Silva b, S. Nielsen a,b, S.M. Thysen a,b, A.B. Fisker a,b,⇑ Department of Clinical Research, Odense University Hospital/University of Southern Denmark, Denmark H Network Bissau Guinea Bissau a Bandim Health Project, OPEN, Department of Clinical Research, Odense University Hospital/University of Southern Denmark, Denmark b Bandim Health Project, INDEPTH Network, Bissau, Guinea-Bissau c Department of Public Health, Aarhus University, Aarhus, Denmark 1. Background suggest that campaigns are important in combatting poverty- related inequity[7–10]. Global polio eradication campaigns are carried out with the goal of eradicating polio by 2023 and ceasing all oral polio vaccination (OPV) by 2024[1]. Over the past 30 years, cases of wild poliovirus infection have been reduced from >350,000 in 1988[2] to 174 in 2019[3] due to vaccination. Guinea-Bissau, a sub-Saharan low- income country has provided OPV through the Expanded Pro- gramme on Immunization (EPI) since the early 1980s[4]. Studies addressing determinants of campaign coverage are war- ranted to characterize children who are missed in campaigns, thereby enabling targeted efforts. Recent studies on vaccination coverage in urban populations show that children from less wealthy households are often under immunized[11,12]. Major actors provide urban immunization toolkits to combat these inequities[13,14], and Gavi, the Vaccine Alliance, encourages coun- tries to identify and target urban disadvantaged populations[14]. Supplementary immunization activities including polio eradica- tion campaigns are intended to complement routine immunization by vaccinating hitherto unvaccinated children and boosting immu- nity in children already immunized. Though campaigns target chil- dren of wider age groups, regardless of prior vaccination history, some studies suggest that the same children are missed by both strategies and that the effect of campaigns on progress may there- fore be less than anticipated[5,6]. On the other hand, other studies We describe coverage and factors associated with coverage in two successive polio eradication campaigns in an urban population in Guinea-Bissau in 2017 and 2018 to elucidate which children are missed in campaigns. https://doi.org/10.1016/j.vaccine.2021.08.090 0264-410X/ 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ⇑Corresponding author at: Bandim Health Project, OPEN, Department of Clinical Research, Odense University Hospital/University of Southern Denmark, Stud- iestræde 6, 1455 Copenhagen K, Denmark. E-mail address: afisker@health.sdu.dk (A.B. Fisker). 2.1. Study population Bandim Health Project (BHP) has maintained a health and demographic surveillance system (HDSS) in suburbs of Bissau, the capital of Guinea-Bissau, since 1978. BHP’s HDSS covers a pop- M. Buus, I. da Silva, S. Nielsen et al. Vaccine 39 (2021) 6720–6726 and multivariate binomial regression models and described as rel- ative risks (RR) with 95% confidence interval (CI) for receipt of campaign OPV. ulation of approximately 100,000 residents in six suburbs of Bis- sau. All individuals are assigned unique ID numbers. Households are visited bimonthly by field assistants registering new pregnan- cies and births. Children <36 months are visited at home every 3– 4 months to collect information on survival, health and vaccination status. Children aged 0–35 months who were registered in the HDSS at the time of either of the campaigns were eligible for the present study. In a multivariate regression model, we excluded variables with >10% missing information and selected between highly correlated variables. To this model, we added timely receipt of routine OPV, as this variable had special interest. Due to convergence problems, we estimated the RR from multivariate logistic regression models and used the exponentiated coefficients from the margins of the predictors to derive the RR. 3.3.1. Child factors While sex and number of siblings were not associated with campaign participation in either campaign, data from both cam- paigns indicated that the oldest children (27–35 months) were 23% (95% CI 18–28%) more likely to have participated than the youngest children (0–8 months) (Table 1). 2.2. Assessed background factors Finally, we assessed whether 2017-campaign participation was associated with participation in the 2018-campaign among chil- dren eligible for both campaigns. Through HDSS routine data collection, at the first visit after birth, socio-economic factors (maternal education, type of sanitary facilities, household electricity and ethnicity) are registered. At the home visits to children <36 months, mid-upper arm circumference (MUAC) is measured and vaccination status recorded from vaccina- tion cards. Data were analyzed using Stata 16.0 (StataCorp, College Station, TX). 3. Results We assessed whether scheduled doses of polio vaccine had been received timely. OPV doses were scheduled at 6, 10 and 14 weeks of age. Furthermore, inactivated polio vaccine (IPV) scheduled at 14 weeks was introduced in 2016. However, due to IPV shortage, vaccination with IPV was paused in mid-2017. We classified chil- dren’s vaccination status based on vaccination cards inspected before the campaign and least 4 weeks after the scheduled age for a particular dose of polio vaccine. We defined a timely vaccina- tion as a vaccine administered within the interval 4 days prior to and 4 weeks after the scheduled age[15–18]. Children who had received OPV/IPV outside the timely interval or had no record of receiving the vaccine, were classified as not timely. In addition to the categorical variable for each dose of OPV1-3, we combined the measures into one indicator identifying children who had received one or more scheduled OPV1-3 as not timely. Though not counted in the common assessment of vaccination coverage [19], OPV is also given at birth (OPV0), but not commonly after 2 weeks of age. Therefore, we also assessed OPV0 coverage by 2 weeks of age among children with a seen vaccination card after 2 weeks of age and before the campaign. 3.1. Study population 6368 children aged 0–35 months were resident in the urban study area at the time of the 2017-campaign, and 6029 children at the time of the 2018-campaign. Data on campaign participation were attempted collected for 6158 (97%) children after the 2017- campaign and 5698 (95%) children after the 2018-campaign (Fig. 1). The distribution of socio-economic factors among children for whom no participation data were obtained indicated that they were slightly worse off and had younger mothers than the groups that had participation data collected. Differences were less pro- nounced when compared to non-participants than to participants (Supplementary table 1). 3.2. Campaign coverage Participation data were obtained for 4199 children after the 2017-campaign, 3544 participants and 655 non-participants, retrieving a coverage of 84%. In 2018, the coverage was 88% (3377/3836). Intervals between campaigns and the date of data collection were approximately 2 months for both participants and non-participants (Supplementary table 1). One of the field assistants, working in the Bandim area, identified most of the non-participants after both the 2017-campaign (37%) and the 2018-campaign (26%) (Supplementary table 1). Mothers reported lower coverage (84%) than other caretakers (87%) in the 2017- campaign but not in the 2018-campaign (mothers: 89%, other care- takers: 87%) (Supplementary table 1). MUAC-for-age z-scores and birthweight z-scores were calcu- lated using the WHO Child Growth Standards igrowup_standard. ado package. As MUAC-for-age z-scores are only defined for chil- dren >3 months, we created a composite variable for describing nutritional status, where birthweight z-scores were used in place of missing MUAC-for-age z-scores. 2.3. Campaign coverage The polio campaigns targeted children aged 0–59 months in Guinea-Bissau in 2017 (November 24–27) and 2018 (April 20– 23). Both campaigns were implemented as door-to-door cam- paigns where vaccination teams administered OPV. Vaccinated children had a fingernail painted. BHP field-assistants collected participation data among children <36 months at routine home visits after the campaigns by interviewing the mother/caretaker whether the child had participated in the campaign or not. We reg- istered whether the information was provided by the mother or another caretaker. 3.3.4. Prior reception of polio vaccines Among children with an inspected vaccination card between 10 weeks of age and the campaign, 98% in 2017 (3443/3510) and 99% in 2018 (3129/3168) had received OPV1. Timely routine OPV (doses 0–3) were all associated with higher coverage in the uni- variate regression models (Table 1). However, timely age- appropriate routine OPV1-3 was only statistically significant in the 2017-campaign. Including the variable in the multivariate analyses, restricted the sample to the older age-groups and timely OPV1-3 was not associated with campaign coverage (Fig. 2). Few children had received IPV. Timely IPV was not associated with campaign coverage in the univariate regression models (Table 1). 2519 children eligible for both the 2017-campaign and the 2018-campaign had participation data collected. Among these chil- dren, 97% (2454/2519) were reached at least once; coverage in 2018 was 94% (2022/2146) for children who had also participated in the 2017-campaign and 83% (308/373) among those who had not (RR = 1.14 (1.09–1.20). Adjusting for background factors included in the multivariate regression model, participants in the 2017-campaign were 8% (4–12%) more likely to participate in the 2018-campaign, than 2017-non-participants were. Among children with an inspected vaccination card between 10 weeks of age and the campaign, 98% in 2017 (3443/3510) and 99% in 2018 (3129/3168) had received OPV1. Timely routine OPV (doses 0–3) were all associated with higher coverage in the uni- variate regression models (Table 1). However, timely age- appropriate routine OPV1-3 was only statistically significant in the 2017-campaign. Including the variable in the multivariate analyses, restricted the sample to the older age-groups and timely OPV1-3 was not associated with campaign coverage (Fig. 2). Few children had received IPV. Timely IPV was not associated with campaign coverage in the univariate regression models (Table 1). 3.3.2. Maternal factors In the univariate models children of older mothers and mothers with more years of schooling were more likely to receive campaign vaccinations than children of younger mothers and mothers with no formal education. These associations were weakened in the multivariate regression model, where only maternal education was positively associated with campaign participation in the 2017-campaign (Table 1). Not having a co-wife and working out- side the household were also associated with higher coverage, but only in the univariate models. 2519 children eligible for both the 2017-campaign and the 2018-campaign had participation data collected. Among these chil- dren, 97% (2454/2519) were reached at least once; coverage in 2018 was 94% (2022/2146) for children who had also participated in the 2017-campaign and 83% (308/373) among those who had not (RR = 1.14 (1.09–1.20). Adjusting for background factors included in the multivariate regression model, participants in the 2017-campaign were 8% (4–12%) more likely to participate in the 2018-campaign, than 2017-non-participants were. 2.4. Statistical analysis Compared with Fula children who had the lowest coverage in both the 2017-campaign (75%) and the 2018-campaign (79%), chil- dren from other ethnic groups were 16–21% more likely to have received the campaign OPV in the 2017-campaign and 15–17% more likely in the 2018-campaign. Ethnicity remained strongly associated with receiving campaign vaccinations in both cam- paigns in the multivariate analyses, with only slight differences between adjusted and unadjusted estimates (Table 1). We excluded children without information on participation sta- tus from the coverage estimates and calculated coverage among included children. We assessed if background factors for children 0–35 months eligible for the campaigns differed depending on whether they were included or excluded in the study using chi2, Kruskal-Wallis and t-tests. Associations between background fac- tors and campaign participation were studied using univariate 6721 Vaccine 39 (2021) 6720–6726 M. Buus, I. da Silva, S. Nielsen et al. Fig. 1. Flowchart of children eligible for campaign participation in 2017 (Fig. 1a) and 2018 (Fig. 1b). Note: Polio eradication campaigns were conducted as door-to-doo campaigns in Bissau, Guinea-Bissau. Campaigns targeted children aged 0–59 months and administered OPV (0–59 months) + Vitamin A (6–59 months) + Mebendazole (12– 59 months) from November 24–27, 2017 and April 20–23, 2018. Analyses were restricted to children aged 0–35 months who were covered the Bandim Health Project (BHP routine data collection. Campaign participation status was assessed on the first routine home visit after the campaign. Fig. 1. Flowchart of children eligible for campaign participation in 2017 (Fig. 1a) and 2018 (Fig. 1b). Note: Polio eradication campaigns were conducted as door-to-door campaigns in Bissau, Guinea-Bissau. Campaigns targeted children aged 0–59 months and administered OPV (0–59 months) + Vitamin A (6–59 months) + Mebendazole (12– 59 months) from November 24–27, 2017 and April 20–23, 2018. Analyses were restricted to children aged 0–35 months who were covered the Bandim Health Project (BHP) routine data collection. Campaign participation status was assessed on the first routine home visit after the campaign. None of the measures of nutritional status (MUAC-for-age z- score, birthweight and composite nutrition z-score) showed a strong association with campaign participation (Table 1). campaign. Adjusted for other background factors, area of residence remained strongly associated with campaign coverage. 3.3.3. Household factors Sanitary facilities and electricity were not associated with coverage. Area of residence was strongly associated with participation status in both the 2017-campaign and the 2018-campaign (Table 1): Compared with children from the Bandim area, children from other areas were 15% more likely to receive OPV in the 2017- campaign and 2–9% more likely to receive OPV in the 2018- 6722 Vaccine 39 (2021) 6720–6726 M. Buus, I. da Silva, S. Nielsen et al. Table 1 Background variables by campaign participation status in children 0–35 months. Table 1 Background variables by campaign participation stat 2017-campaign1 Background Variables Coverage % (Participa (n)/ Children(n)) Child factors Sex Female 84.8 (1741/2052) Male 84.0 (1803/2147) Age at time of campaign 0–8 months 74.0 (781/1056) 9–17 months 85.4 (913/1069) 18–26 months 87.8 (959/1092) 27–35 months 90.7 (891/982) Twin Yes 83.0 (122/147) No 84.4 (3414/4043) Number of siblings 0 83.9 (1144/1364) 1 85.4 (922/1079) 2 85.8 (676/788) 3 82.3 (391/475) >3 83.0 (385/464) MUAC-for-age z-score >=-1 87.4 (2770/3170) <-1 89.3 (260/291) Birth weight (gram) <2500 83.7 (241/288) 2500–3000 84.8 (767/904) 3000–3500 86.0 (1106/1286) >3500 86.6 (632/730) Nutrition z-score >=-1 85.5 (3087/3609) <-1 83.0 (347/418) Ethnicity Fula 74.5 (834/1119) Balanta 86.2 (262/304) Mandinga 88.0 (285/324) Manjaco 90.4 (386/427) Pepel 86.7 (874/1008) Others 88.8 (881/992) Maternal factors Maternal age at birth of child (years) <22 81.9 (751/917) 22–27 83.0 (930/1121) 27–32 85.5 (946/1106) >32 86.9 (916/1054) Maternal education None 76.3 (757/992) Primary 85.2 (709/832) Secondary or higher 87.5 (2007/2294) Mother works outside the household Yes 87.1 (568/652) No 83.9 (2928/3488) Co-wife5 Yes 78.6 (467/594) No 85.3 (3018/3537) Household factors Sanitary facilities Inside the house 86.7 (875/1009) Latrine 83.7 (2645/3160) Electricity Yes 84.5 (2152/2547) No 84.3 (1381/1638) Area of residence Bandim 78.0 (1453/1862) Belem/Mindara 89.5 (663/741) Cuntum 89.5 (1428/1596) Polio vaccinations6 OPV-0 Timely 88.8 (2573/2946) Not timely 83.2 (708/851) Table 1 Background variables by campaign participation status in children 0–35 months. 3.3.3. Household factors g p g 4 Multivariate regression model 1 adjusting for sex, child age at time of campaign, nutrition z-score, maternal age at birth of child, maternal education, maternal employment, co-wife status, sanitary facilities, electricity, ethnicity and area of residence. We excluded variables with > 10% missing information (MUAC-for-age z-score; 18% (738/4199) in 2017 and 19% (739/3836) in 2018) and selected between the two highly correlated variables, maternal age and number of siblings. ( / ) ( / ) ) g y , g g 5 Co-wife: Living in a polygamic household where the husband has at least one other wife. Household and childcare tasks frequently shared between the ‘‘co-wifes”. 6 OPV-0 at birth and OPV primary series consisting of OPV-1 at 6 weeks, OPV-2 at 10 weeks and OPV-3 at 14 weeks of age. Timeliness of OPV1-3 defined as OPV dose administered within the interval 4 days prior to and 4 weeks after the scheduled age. Timely OPV-0 defined as OPV dose administered within 14 days from birth. 7 Combined measure of timely OPV1-3 primary series vaccination for age of the child, thus excluding OPV-0. ( / ) ( / ) ) g y g g 5 Co-wife: Living in a polygamic household where the husband has at least one other wife. Household and childcare tasks frequently shared between the ‘‘co-wifes”. 6 OPV-0 at birth and OPV primary series consisting of OPV-1 at 6 weeks, OPV-2 at 10 weeks and OPV-3 at 14 weeks of age. Timeliness of OPV1-3 defined as OPV dose administered within the interval 4 days prior to and 4 weeks after the scheduled age. Timely OPV-0 defined as OPV dose administered within 14 days from birth. 7 Combined measure of timely OPV1-3 primary series vaccination for age of the child, thus excluding OPV-0. 8 Fig. 2. Multivariable regression model1 among all children with complete information on included covariates. Model 2 with addition of the covariate timely OPV1-3. Note: 1Multivariate regression model 1 adjusting for sex, child age at time of the campaign, nutrition z-score, maternal age at birth of child, maternal education, maternal employment, co-wife status, sanitary facilities, electricity, ethnicity and area of residence. (n = 3852 in 2017; n = 3518 in 2018). 2Multivariate regression model 2 adjusting for variables included in model 1 and timely OPV1-3. (n = 3334 in 2017; n = 3008 in 2018). 3.3.3. Household factors 2017-campaign1 2018-campaign1 Background Variables Coverage % (Participants (n)/ Children(n)) [p-value]2 Crude RR(95% CI)3 [p-value]2 Adjusted RR (95% CI)4 Coverage % (Participants (n)/ Children(n)) [p-value]2 Crude RR(95% CI)3 [p-value]2 Adjusted RR(95% CI)4 Child factors Sex [0.44] [0.30] [0.95] [0.59] Female 84.8 (1741/2052) 1 1 88.0 (1650/1875) 1 1 Male 84.0 (1803/2147) 0.99 (0.96–1.02) 0.99 (0.96–1.01) 88.1 (1727/1961) 1.00 (0.98–1.02) 1.01 (0.98–1.03) Age at time of campaign [<0.001] [<0.001] [<0.001] [<0.001] 0–8 months 74.0 (781/1056) 1 1 76.5 (729/953) 1 1 9–17 months 85.4 (913/1069) 1.15 (1.11–1.21) 1.16 (1.10–1.21) 88.7 (906/1021) 1.16 (1.11–1.21) 1.15 (1.10–1.20) 18–26 months 87.8 (959/1092) 1.19 (1.14–1.24) 1.19 (1.13–1.24) 93.0 (851/915) 1.22 (1.17–1.26) 1.20 (1.15–1.25) 27–35 months 90.7 (891/982) 1.23 (1.18–1.28) 1.23 (1.17–1.29) 94.1 (891/947) 1.23 (1.18–1.28) 1.22 (1.16–1.27) Twin [0.63] [0.45] Yes 83.0 (122/147) 1 90.2 (119/132) 1 No 84.4 (3414/4043) 0.98 (0.91–1.06) 88.0 (3253/3698) 1.02 (0.97–1.09) Number of siblings [0.33] [0.32] 0 83.9 (1144/1364) 1 87.4 (1076/1231) 1 1 85.4 (922/1079) 1.02 (0.98–1.05) 88.4 (880/995) 1.01 (0.98–1.04) 2 85.8 (676/788) 1.02 (0.99–1.06) 87.5 (629/719) 1.00 (0.97–1.04) 3 82.3 (391/475) 0.98 (0.94–1.03) 87.2 (390/447) 1.00 (0.96–1.04) >3 83.0 (385/464) 0.99 (0.94–1.04) 91.1 (369/405) 1.04 (1.00–1.08) MUAC-for-age z-score [0.33] [0.34] >=-1 87.4 (2770/3170) 1 92.0 (2603/2828) 1 <-1 89.3 (260/291) 1.02 (0.98–1.07) 93.7 (252/269) 1.02 (0.98–1.05) Birth weight (gram) [0.57] [0.73] <2500 83.7 (241/288) 1 90.4 (236/261) 1 2500–3000 84.8 (767/904) 1.01 (0.96–1.07) 88.2 (718/814) 0.98 (0.93–1.02) 3000–3500 86.0 (1106/1286) 1.03 (0.97–1.09) 89.4 (1074/1201) 0.99 (0.95–1.03) >3500 86.6 (632/730) 1.03 (0.98–1.10) 89.2 (637/714) 0.99 (0.94–1.03) Nutrition z-score [0.17] [0.87] [0.026] [0.63] >=-1 85.5 (3087/3609) 1 1 89.6 (2914/3251) 1 1 <-1 83.0 (347/418) 0.97 (0.93–1.02) 1.00 (0.95–1.04) 86.0 (350/407) 0.96 (0.92–1.00) 0.99 (0.95–1.03) Ethnicity [<0.001] [<0.001] [<0.001] [<0.001] Fula 74.5 (834/1119) 1 1 79.1 (819/1035) 1 1 Balanta 86.2 (262/304) 1.16 (1.09–1.22) 1.11 (1.05–1.19) 90.8 (248/273) 1.15 (1.09–1.21) 1.12 (1.06–1.18) Mandinga 88.0 (285/324) 1.18 (1.12–1.24) 1.11 (1.04–1.18) 92.7 (281/303) 1.17 (1.12–1.23) 1.13 (1.08–1.19) Manjaco 90.4 (386/427) 1.21 (1.16–1.27) 1.16 (1.10–1.23) 91.2 (374/410) 1.15 (1.10–1.20) 1.13 (1.07–1.19) Pepel 86.7 (874/1008) 1.16 (1.12–1.21) 1.15 (1.09–1.21) 91.3 (829/908) 1.15 (1.11–1.20) 1.15 (1.10–1.20) Others 88.8 (881/992) 1.19 (1.14–1.24) 1.14 (1.08–1.20) 91.1 (800/878) 1.15 (1.11–1.20) 1.11 (1.06–1.17) Maternal factors Maternal age at birth of child (years) [0.007] [0.22] [<0.001] [0.10] <22 81.9 (751/917) 1 1 85.1 (717/843) 1 1 22–27 83.0 (930/1121) 1.01 (0.97–1.05) 1.01 (0.97–1.05) 87.2 (892/1023) 1.03 (0.99–1.06) 1.02 (0.99–1.06) 27–32 85.5 (946/1106) 1.04 (1.00–1.09) 1.03 (0.99–1.08) 88.1 (861/977) 1.04 (1.00–1.07) 1.01 (0.97–1.05) >32 86.9 (916/1054) 1.06 (1.02–1.10) 1.04 (1.00–1.09) 91.3 (905/991) 1.07 (1.04–1.11) 1.05 (1.01–1.09) Maternal education [<0.001] [0.018] [<0.001] [0.85] None 76.3 (757/992) 1 1 82.4 (757/919) 1 1 Primary 85.2 (709/832) 1.12 (1.07–1.17) 1.04 (0.99–1.09) 89.7 (636/709) 1.09 (1.05–1.13) 1.00 (0.96–1.04) Secondary or higher 87.5 (2007/2294) 1.15 (1.10–1.19) 1.06 (1.02–1.10) 90.0 (1914/2126) 1.09 (1.06–1.13) 1.01 (0.98–1.04) Mother works outside the household [0.04] [0.84] [0.05] [0.43] Yes 87.1 (568/652) 1 1 90.2 (592/656) 1 1 No 83.9 (2928/3488) 0.96 (0.93–1.00) 1.00 (0.96–1.04) 87.5 (2736/3126) 0.97 (0.94–1.00) 0.99 (0.95–1.02) Co-wife5 [<0.001] [0.22] [0.007] [0.12] Yes 78.6 (467/594) 1 1 84.5 (464/549) 1 1 No 85.3 (3018/3537) 1.09 (1.04–1.13) 1.03 (0.98–1.07) 88.6 (2856/3225) 1.05 (1.01–1.09) 1.03 (0.99–1.07) Household factors Sanitary facilities [0.021] [0.56] [0.31] [0.21] Inside the house 86.7 (875/1009) 1 1 89.0 (839/943) 1 1 Latrine 83.7 (2645/3160) 0.97 (0.94–0.99) 0.99 (0.95–1.03) 87.7 (2519/2871) 0.99 (0.96–1.01) 1.02 (0.99–1.06) Electricity [0.87] [0.15] [0.35] [0.44] Yes 84.5 (2152/2547) 1 1 88.4 (2135/2414) 1 1 No 84.3 (1381/1638) 1.00 (0.97–1.02) 1.02 (0.99–1.05) 87.4 (1230/1407) 0.99 (0.96–1.01) 0.99 (0.96–1.02) Area of residence [<0.001] [<0.001] [<0.001] [<0.001] Bandim 78.0 (1453/1862) 1 1 84.8 (1455/1716) 1 1 Belem/Mindara 89.5 (663/741) 1.15 (1.11–1.19) 1.17 (1.12–1.21) 86.6 (570/658) 1.02 (0.99–1.06) 1.03 (1.00–1.08) Cuntum 89.5 (1428/1596) 1.15 (1.11–1.18) 1.17 (1.13–1.21) 92.5 (1352/1462) 1.09 (1.06–1.12) 1.10 (1.07–1.13) Polio vaccinations6 OPV-0 [0.002] [0.06] Timely 88.8 (2573/2946) 1 91.0 (2469/2712) 1 6723 M. 3.3.3. Household factors Buus, I. da Silva, S. Nielsen et al. Vaccine 39 (2021) 6720–6726 Table 1 (continued) 2017-campaign1 2018-campaign1 Background Variables Coverage % (Participants (n)/ Children(n)) [p-value]2 Crude RR(95% CI)3 [p-value]2 Adjusted RR (95% CI)4 Coverage % (Participants (n)/ Children(n)) [p-value]2 Crude RR(95% CI)3 [p-value]2 Adjusted RR(95% CI)4 OPV-1 [<0.001] [0.009] Timely 88.4 (2687/3039) 1 92.5 (2551/2757) 1 Not timely 80.9 (381/471) 0.91 (0.87–0.96) 88.8 (365/411) 0.96 (0.93–0.99) OPV-2 [0.002] [0.013] Timely 89.0 (2128/2391) 1 93.2 (2029/2178) 1 Not timely 85.1 (852/1001) 0.96 (0.93–0.98) 90.5 (781/863) 0.97 (0.95–1.00) OPV-3 [<0.001] [0.029] Timely 89.9 (1683/1873) 1 93.6 (1588/1697) 1 Not timely 85.4 (1203/1408) 0.95 (0.93–0.98) 91.4 (1110/1214) 0.98 (0.96–1.00) Timely OPV1-3 vaccinations for age7 [0.003] [0.06] Timely 88.8 (1795/2021) 1 92.8 (1742/1877) 1 Not timely 85.5 (1273/1489) 0.96 (0.94–0.99) 90.9 (1174/1291) 0.98 (0.96–1.00) Polio vaccinations6 IPV8 [0.82] [0.49] Timely 88.2 (569/645) 1 93.3 (584/626) 1 Not timely 87.9 (2332/2653) 1.00 (0.97–1.03) 92.5 (2126/2299) 0.99 (0.97–1.02) 1 Polio eradication campaigns conducted in Guinea-Bissau from November 24 to 27 in 2017 and April 20 to 23 in 2018, targeting children 0–59 months, administering Vitamin A (6–59 months), OPV (0–59 months) and Mebendazol (12–59 months). Analyses restricted to children aged < 36 months that were covered by the Bandim Health Project HDSS data collection. 2 1 Polio eradication campaigns conducted in Guinea-Bissau from November 24 to 27 in 2017 and April 20 to 23 in 2018, targeting children 0–59 months, administering Vitamin A (6–59 months), OPV (0–59 months) and Mebendazol (12–59 months). Analyses restricted to children aged < 36 months that were covered by the Bandim Health Project HDSS data collection. 2 T t f diff i ti i t ti i t i hi d K k ll W lli d t t t j 2 Test for no difference in participants vs non-participants using chi-squared, Kruskall-Wallis and t-tests. g p g 4 Multivariate regression model 1 adjusting for sex, child age at time of campaign, nutrition z-score, maternal age at birth of child, maternal education, maternal employment, co-wife status, sanitary facilities, electricity, ethnicity and area of residence. We excluded variables with > 10% missing information (MUAC-for-age z-score; 18% (738/4199) in 2017 and 19% (739/3836) in 2018) and selected between the two highly correlated variables, maternal age and number of siblings. j 2 Test for no difference in participants vs non-participants using chi-squared, Kruskall-Wallis and t-tests. 3 4.2. Interpretation and implications [2] Sutter RW, Kew OM, Cochi SL, Aylward RB. In: Plotkin Stanley A, Orenstein Walter A, Offit Paul A, Edwards Kathryn M, editors. Plotkin’s vaccines, 7th ed. Elsevier; 2018, p. 866–917.e816. Coverage in the 2018-campaign was higher among children who participated in the 2017-campaign five months earlier and coverage was higher among children with timely routine OPV in both the 2017-campaign and the 2018-campaign, thereby sup- porting that campaigns reach more of the already immunized children. However, coverage was >80% in the 2018-campaign among eligible children not reached by the 2017-campaign, sup- porting that campaigns may still have large effects on population immunity. [3] GPEI - Global Polio Eradication Initiative. Polio Now, <http://polioeradication. org/polio-today/polio-now/>; 2020. [4] Mogensen SW, Andersen A, Rodrigues A, Benn CS, Aaby P. The introduction of diphtheria-tetanus-pertussis and oral polio vaccine among young infants in an [4] Mogensen SW, Andersen A, Rodrigues A, Benn CS, Aaby P. The introduction of diphtheria-tetanus-pertussis and oral polio vaccine among young infants in an urban African community: a natural experiment. EBioMedicine 2017;17:192–8. https://doi.org/10.1016/j.ebiom.2017.01.041. urban African community: a natural experiment. EBioMedicine 2017;17:192–8. https://doi.org/10.1016/j.ebiom.2017.01.041. [5] Helleringer S, Frimpong JA, Abdelwahab J, Asuming P, Touré H, Awoonor- Williams JK, et al. Supplementary polio immunization activities and prior use of routine immunization services in non-polio-endemic sub-Saharan Africa. Bull World Health Organ 2012;90(7):495–503. https://doi.org/10.2471/ BLT.11.092494. However, our study also identifies that coverage could be fur- ther increased by targeting efforts: Although area and ethnicity were somewhat correlated, both remained strong predictors of participation in the multivariate models and could imply that mes- sages targeted at the Fula community could have an impact. Since the same ethnic community had lower coverage 10 years previ- ously, where data were collected by special teams, rather than the BHP routine data collection[20], this is unlikely to be an arte- fact caused by a particular data collector. Hence, targeting informa- tion or deploying more vaccination teams to particular areas with ability to communicate in the spoken language, Fulani, may improve campaign coverage in urban Guinea-Bissau. [6] Minetti A, Hurtado N, Grais RF, Ferrari M. Reaching hard-to-reach individuals: nonselective versus targeted outbreak response vaccination for measles. Am J Epidemiol 2014;179:245–51. https://doi.org/10.1093/aje/kwt236. p p // g/ / j / [7] Portnoy A, Jit M, Helleringer S, Verguet S. Impact of measles supplementary immunization activities on reaching children missed by routine programs. Vaccine 2018;36(1):170–8. https://doi.org/10.1016/j.vaccine.2017.10.080. 3.3.3. Household factors 3Timely OPV1-3: Timeliness defined as OPV dose administered within the interval 4 days prior to and 4 weeks after the scheduled age of OPV1 at 6 weeks, OPV2 at 10 weeks and OPV3 at 14 weeks of age. Fig. 2. Multivariable regression model1 among all children with complete information on included covariates. Model 2 with addition of the covariate timely OPV1-3. Note: 1Multivariate regression model 1 adjusting for sex, child age at time of the campaign, nutrition z-score, maternal age at birth of child, maternal education, maternal employment, co-wife status, sanitary facilities, electricity, ethnicity and area of residence. (n = 3852 in 2017; n = 3518 in 2018). 2Multivariate regression model 2 adjusting for variables included in model 1 and timely OPV1-3. (n = 3334 in 2017; n = 3008 in 2018). 3Timely OPV1-3: Timeliness defined as OPV dose administered within the interval 4 days prior to and 4 weeks after the scheduled age of OPV1 at 6 weeks, OPV2 at 10 weeks and OPV3 at 14 weeks of age. 6724 M. Buus, I. da Silva, S. Nielsen et al. Vaccine 39 (2021) 6720–6726 Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2021.08.090. Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2021.08.090. 4. Discussion Oral polio campaign coverage reached 84% in the 2017- campaign and 88% in the 2018-campaign. Our study shows that coverage was lower among children of younger mothers and moth- ers who had no formal education, whereas coverage was higher for older children and in certain ethnic groups. Polio eradication campaigns in urban Guinea-Bissau reach a large proportion of children, including those who were not timely vaccinated and unreached in prior campaigns. Potentially higher coverage could be obtained by targeting messages to younger mothers, mothers of infants, mothers with no formal education and from the Fula ethnic group. The interviews used to assess campaign coverage were con- ducted approximately 2 months after the campaigns and may therefore have been subject to recall bias. However, there was no difference in the interval between campaign and interview for par- ticipants and non-participants after the two campaigns. Vaccinated children had a fingernail painted, which may have facilitated recall of campaign participation. Declaration of Competing Interest The authors declare that they have no competing financial inter- ests or personal relationships that could have appeared to influence the work reported in this paper. 4.1. Consistency with other studies This work was supported by Aase og Ejnar Danielsens Fond [10- 10-0526], Else og Mogens Wedell-Wedellsborgs Fond [20-18-2], Fonden til Lægevidenskabens Fremme [18-L-0078], Jyske Bank Almennyttige Fond and Aarhus Universitets ForskningsFond [28604]. ABFs research is supported through a Sapere Aude Research Leader grant from Independent Research Fund Denmark [9060-00018B] and an Ascending Investigator grant from Lund- beck Foundation [R313-2019-635]. The funding agencies had no role in the study design, data collection, data analysis and interpre- tation of data, the writing of the article or the decision of submis- sion for publication. Consistent with studies of both routine[15,16] and campaign[5] OPV coverage, coverage was lower among children of mothers with no formal education in both the 2017-campaign and the 2018- campaign, though the association was not evident in the 2018- campaign multivariate model. Also, in line with data from other African countries[5], campaign coverage was higher among older children. Contrary to other studies that found that wealth was associated with campaign coverage[5,7–10], our study generally showed no association between coverage and household factors, suggesting that inequity in access to campaign vaccination in urban Guinea- Bissau may be better targeted by other background factors than wealth. References [1] World Health Organization (WHO). Polio Endgame Strategy 2019-2023: Eradication, integration, certification and containment. Geneva: World Health Organization; 2019 (WHO/Polio/19.04) (2019). Appendix A. Supplementary material Children of the Fula ethnicity had significantly lower campaign coverage in both the 2017-campaign and the 2018-campaign, as also observed in vitamin A campaigns in the same area 10 years earlier[20]. 4.2. Interpretation and implications ( ) p // g/ /j [8] Helleringer S, Abdelwahab J, Vandenent M. Polio supplementary immunization activities and equity in access to vaccination: evidence from the demographic and health surveys. J Infect Dis 2014;210(Suppl 1):S531–9. https://doi.org/ 10.1093/infdis/jiu278. [9] Vijayaraghavan M, Martin RM, Sangrujee N, Kimani GN, Oyombe S, Kalu A, et al. Measles supplemental immunization activities improve measles vaccine coverage and equity: evidence from Kenya, 2002. Health Policy 2007;83 (1):27–36. https://doi.org/10.1016/j.healthpol.2006.11.008. [10] Portnoy A, Jit M, Helleringer S, Verguet S. Comparative distributional impact of routine immunization and supplementary immunization activities in delivery of measles vaccine in low- and middle-income countries. Value Health 2020;23(7):891–7. https://doi.org/10.1016/j.jval.2020.03.012. The youngest children (<9 months) had the lowest coverage, though they are commonly in closer contact with the health cen- ters for completion of the EPI schedule and growth monitoring through monthly weighing sessions. Potentially, these contacts may be used to disseminate information on upcoming campaigns. [11] Mutua MK, Kimani-Murage E, Ettarh RR. Childhood vaccination in informal urban settlements in Nairobi, Kenya: who gets vaccinated? BMC Public Health 2011;11:6. https://doi.org/10.1186/1471-2458-11-6. 6725 M. Buus, I. da Silva, S. Nielsen et al. Vaccine 39 (2021) 6720–6726 complete vaccination schedule in Burkina Faso. Vaccine 2013;32(1):96–102. https://doi.org/10.1016/j.vaccine.2013.10.063. complete vaccination schedule in Burkina Faso. Vaccine 2013;32(1):96–102. https://doi.org/10.1016/j.vaccine.2013.10.063. [12] Crocker-Buque T, Mindra G, Duncan R, Mounier-Jack S. Immunization, urbanization and slums - a systematic review of factors and interventions. BMC Public Health 2017;17(7):556. https://doi.org/10.1186/s12889-017- 4473-7. [17] WHO. Recommended Routine Immunizations for Children, <https://www. who.int/immunization/policy/Immunization_routine_table2.pdf?ua=1>; 2017. [13] World Health Organization. Regional Office for Africa. Reaching every district (RED): A guide to increasing coverage and equity in all communities in the African Region. World Health Organization. Regional Office for Africa; 2017. [18] Ezeanolue EHK, Hunter P, Kroger A, Pellegrini C. Best Practices Guidance of the Advisory Committee on Immunization Practices (ACIP), <https://www. cdc.gov/vaccines/hcp/acip-recs/general-recs/downloads/general-recs.pdf>; 2017. [14] UNICEF/Gavi. Urban Immunization Toolkit, http://gotlife.gavi.org/wp-content/ uploads/2018/12/Urban-immunization-toolkit.pdf; 2018. uploads/2018/12/Urban-immunization-toolkit.pdf; 201 [19] WHO. Polio immunization coverage among one-year-olds (%), <https://www. who.int/data/gho/indicator-metadata-registry/imr-details/3339>; 2020. [15] Mekonnen ZA, Gelaye KA, Were MC, Tilahun B. Timely completion of vaccination and its determinants among children in northwest, Ethiopia: a multilevel analysis. BMC Public Health 2020;20:908. https://doi.org/10.1186/ s12889-020-08935-8. [20] Fisker AB, Aaby P, Bale C, Balde I, Biering-Sørensen S, Agergaard J, et al. Does the effect of vitamin A supplements depend on vaccination status? An observational study from Guinea-Bissau. BMJ Open 2012;2(1):e000448. https://doi.org/10.1136/bmjopen-2011-000448. [16] Schoeps A, Ouédraogo N, Kagoné M, Sié A, Müller O, Becher H. 4.2. Interpretation and implications Socio- demographic determinants of timely adherence to BCG, Penta3, measles, and 6726
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https://www.joghr.org/article/29042.pdf
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Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs
Journal of global health reports
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Background The Meningitis Vaccine Project (MVP) was an international partnership that began in 2010 and focused on the development, testing, licensure, and widespread introduction of a conjugate vaccine with the promise of protecting millions of lives from group A meningococcal meningitis. To bridge gaps upstream of vaccine administration and in understanding the project’s planning, delivery and outcomes, the primary objective of this study was to delineate the barriers to and facilitators of sustainable implementation in an upstream context of the delivery of the MenAfriVac vaccine and to draw critical lessons for other vaccination programs. Results There were multiple barriers and facilitators to implementing the vaccine under all five domains of the CFIR framework, including intervention characteristics, outer setting, inner setting, characteristics of individuals, and process. The most common domains mentioned by stakeholders that facilitated the success of the MVP were inner setting and process; specifically, three constructs were most representative—Tension for Change, Formally Appointed Implementation Leaders, and External Change Agents. Journal of Global Health Reports Vol. 5, 2021 Methods We conducted a qualitative study with key project partners and leaders who worked on th MVP project. The interviewees were initially identified through a literature review and a search of publications and evaluations of the project. Eighteen key stakeholders were interviewed. The interviewees included individuals in the roles of implementation oversight, champion, partner, funder, and frontline implementer. The Consolidated Framework for Implementation Research (CFIR) informed the identification of a priori codes for the directed content analysis. Ezezika O, Mengistu M, Lear T. Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs. Journal of Global Health Reports. 2021;5:e2021097. doi:10.29392/001c.29042 Ezezika O, Mengistu M, Lear T. Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs. Journal of Global Health Reports. 2021;5:e2021097. doi:10.29392/001c.29042 Ezezika O, Mengistu M, Lear T. Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs. Journal of Global Health Reports. 2021;5:e2021097. doi:10.29392/001c.29042 Ezezika O, Mengistu M, Lear T. Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs. Journal of Global Health Reports. 2021;5:e2021097. doi:10.29392/001c.29042 Obidimma Ezezika 1 , Meron Mengistu 2 , Thomas Lear 3 Obidimma Ezezika 1 , Meron Mengistu 2 , Thomas Lear 3 1 Department of Health and Society, University of Toronto Scarborough; Dalla Lana School of Public Health, University of Toronto; African Centre for Innovation & Leadership Development, 2 Department of Health and Society, University of Toronto Scarborough; London School of Hygiene & Tropical Medicine, 3 Department of Health and Society, University of Toronto Scarborough 1 Department of Health and Society, University of Toronto Scarborough; Dalla Lana School of Public Health, University of Toronto; African Centre for Innovation & Leadership Development, 2 Department of Health and Society, University of Toronto Scarborough; London School of Hygiene & Tropical Medicine, 3 Department of Health and Society, University of Toronto Scarborough Keywords: meningitis, implementation, vaccine, immunization https://doi.org/10.29392/001c.29042 Conclusions The tension for change—that is, the degree to which stakeholders perceive the current situation as intolerable or needing change— advocated by African leaders served as a crucial foundation for solid leadership for the MPV. The leadership came in the form of the project director and other formally appointed leaders, who could plan, engage and execute the project’s goals effectively. This leadership translated into consistent messaging, information sharing, and multiple levels of engagement with external change agents (including technical experts), which promoted a shared sense of readiness contributing to significant change investments in implementing MVP. Meningococcal meningitis is found in many populations worldwide, and its subtypes are typically confined to spe­ cific geographical regions. Neisseria meningitidis serogroup A (hereafter referred to as meningitis A) accounted for 90% of meningitis cases in sub-Saharan Africa.1 Meningitis A causes infection of the central nervous system, in the lining of the brain and spinal cord, and its transmission is fa­ cilitated through respiratory droplets or throat secretions. Despite rapid diagnosis and treatment, there remains a 5%–10% fatality rate, typically within 24–48 hours of the onset of symptoms.2 The urgency for a more effective vac­ cine was evident after the 1996 outbreak of meningitis A in the meningitis belt, which spans from Senegal to Ethiopia, infecting over 250 000 people and killing 25 000 within a few Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs months.3 Several African leaders called on the World Health Organization (WHO) to resolve the meningitis epidemic. In 2001, the Bill and Melinda Gates Foundation (BMGF) of­ ficially funded the Meningitis Vaccine Project (MVP) with a partnership between the WHO and the Program for Ap­ propriate Technology in Health (PATH). By 2003, after al­ most a year-long delay in identifying a suitable pharmaceu­ tical partner to produce the vaccine at the requisite cost, the Serum Institute of India agreed to produce the menin­ gitis A vaccine, called MenAfriVac, at US$0.50 a dose.4 In response to this catastrophic outbreak, the BMGF awarded US$70 million to the WHO and the PATH to establish the MVP. Conclusions qualitative studies on the MVP do well at examining down­ stream perspectives of the MVP, including the viewpoints of health care workers13 and individual perceptions of re­ current meningococcal epidemics in Ghana.14 Therefore, questions on upstream delivery—such as how stakeholders and partners interacted, how information was shared, what kinds of decisions were made between different organisa­ tions and institutions, and how these decisions were imple­ mented—were left unanswered. To this end, we employed a conceptual framework known as the Consolidated Framework For Implementation Re­ search (CFIR), which has been developed to guide the sys­ tematic assessment of implementation contexts and factors that influence effective intervention implementation.15 In­ corporating CFIR during the analysis and synthesis phase of this review is beneficial, as integrating a conceptual frame­ work increases the generalizability and interpretability of study results. To further explain the results of this study, we used the organisational readiness for change (ORC) the­ ory, a multifaceted construct that describes change com­ mitment and change efficacy.9 The theory effectively char­ acterises organisational views on implementation and may be used to advise the changes required to yield the desired outcomes. A recent study utilised the ORC theory to un­ derstand the association between readiness for change and psychosocial conditions of health care professionals (HCP).16 Ever since, MenAfriVac has witnessed widespread adop­ tion to over 300 million Africans across 22 of 26 target countries in the meningitis belt, including Burkina Faso, Niger, Mali, and surrounding areas.5,6 Collaborative efforts in establishing the MVP led to a large-scale vaccine cam­ paign in December 2010 in Burkina Faso, Mali, and Niger.7 One of the greatest success stories can be found in Burkina Faso, which suffered a major epidemic of meningitis A with 45 000 cases between 2006 and 2008. Since the introduction of MenAfriVac, there have been only eight cases in Burkina Faso between 2011 and 2017.1 Burkina Faso is one of the few countries that has conducted a catch-up campaign among children and infants to accelerate the timeline for the intro­ duction of MenAfriVac into the Expanded Program on Im­ munization (EPI). However, gaps continue to persist, with unvaccinated cohorts increasing due to a lack of integration of routine national immunisation programs and catch-up campaigns.4 Our aim was to address the following research question: "Which are the upstream facilitators of and barriers to the effective implementation of MenAfriVac? Conclusions The findings of this study may inform generalisable quality improvement strategies for vaccinations in SSA, create a better qualitative understanding of the needs and resources of stakeholders, and promote the complete eradication of meningitis by highlighting directions for advocacy. The MVP is a valuable initiative to study, with crucial lessons to be drawn from it for future implementation pro­ jects and initiatives. This study aims to identify the barriers to and facilitators of the implementation of MenAfriVac through the MVP. Despite the intervention’s effectiveness, a host of social determinants or upstream contextual gaps decreased diffusion.8,9 As is becoming evident through growing implementation literature, it is not sufficient to know that an intervention is effective but rather what con­ texts it works best in and how.10 In Burkina Faso, there are vaccination coverage gaps among regions, thereby prompt­ ing further inquiry regarding attaining sustainable imple­ mentation.5 To this end, we aim to identify the contexts of MenAfriVac at the level of multilateral organisations, gov­ ernments, decision-makers, and academics that lead to challenges or successes in implementing the vaccine. Journal of Global Health Reports CFIR CODING After consensus was reached, MM and TL coded the barriers and facilitators using the CFIR (see Table 2). The CFIR is a conceptual framework developed to guide the systematic assessment of implementation contexts and factors that in­ fluence the implementation of effective intervention.18 In­ corporating CFIR during the analysis and synthesis phase is beneficial, as integrating a conceptual framework increases the generalizability and interpretability of the study results. In addition, in systematic research, this framework is known to support the implementation of health care deliv­ ery interventions to produce an actionable evaluation for the improvement of implementation.18 The CFIR includes five major domains (intervention characteristics, outer set­ ting, internal setting, characteristics of individuals, and process) with 39 underlying constructs and sub-constructs that can potentially influence efforts to change the prac­ tice.15 To reach a consensus, MM and TL met twice: the first time to go over three barriers and facilitators as a pilot to establish coding criteria and the second time to reach a con­ sensus for all barriers and facilitators and calculate the in­ ter-reliability score. STUDY PURPOSE AND SAMPLING STRATEGY This study aimed to identify the barriers to and facilitators of implementing MenAfriVac through the MVP. The leaders and partners of the MVP were initially identified through a literature review of publications related to the project and were interviewed as key informants. Potential stakeholders for this study included those in­ volved in the upstream processes of the project, including planning, decision-making, and implementation. Eligibility included direct involvement – either individually or through an organisation/affiliation - in the MVP initiative. Key informants also needed to have experience of the pro­ ject’s initiative in the context of the Meningitis Belt coun­ tries. In this regard, several implementation science studies are of significance as they attempt to fill a specific research gap through the analysis of primary data. The implemen­ tation science studies relevant to MenAfriVac have high­ lighted the numerous downstream consequences of imple­ mentation, such as demonstrating the potential socio-cultural, economic, and environmental factors that enhance the seasonal transmission of meningitis A.11 Moreover, a 2019 study analysed routine coverage of MenAfriVac and the continued socio-economic obstacles to achieving high coverage of both MenAfriVac and the measles-containing-vaccine second dose (MCV2).12 Fur­ thermore, several studies have examined the MVP, but none have done so from an upstream perspective. Two recent Eighty-nine individuals were identified as potential stakeholders and were contacted for an interview. Two-way communication was established among forty-seven of the potential stakeholders, and out of them, twenty-six stake­ holders did not meet the eligibility criteria and therefore could not be interviewed. Of the remaining twenty-one eligible stakeholders who responded, three declined due to schedule constraints. Ul­ 2 Journal of Global Health Reports Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs timately, 18 stakeholders agreed to participate in this study and were interviewed. and further breakdown and reorganise the barriers and fa­ cilitators. In these meetings, the names given to the barriers and facilitators were revised, a few were combined, and quotations were moved around and condensed for accuracy (Table 1). This process was an extensive one. INTERVENTION CHARACTERISTICS This domain describes the critical attributes of the inter­ vention—in this case, the vaccine MenAfriVac—and its in­ fluence on implementation success. DATA COLLECTION OE & MM met on two separate occasions to review the in­ terview questions and run through a mock interview. This was done to ensure that MM was trained and that interview etiquette was established. Interviews were conducted by MM between July 14, 2020, and October 14, 2020, on Zoom. Interviews lasted for approximately 20–80 minutes. A semi- structured questionnaire was employed, and interviews were recorded to be subsequently transcribed and analysed. During the interview, each stakeholder was asked to de­ scribe their role and responsibility in the project, including critical barriers to and facilitators of distributing MenAfriVac that they came across in their capacity. Fur­ thermore, stakeholders were asked to identify any processes that could have been strengthened to denote the significant lessons learned from implementing the project. All 18 in­ terviews were transcribed manually using the audio files saved and prepared as a Word document for data analysis. Further, MM and OE had two meetings to establish cod­ ing criteria, test the criteria using pilot examples, and reach a consensus by collapsing similar codes under more accu­ rate categories and reorganising the CFIR results. These two meetings followed the same exhaustive process as the first analysis to ensure validity and consistency. Moreover, we reached out to participants with a summary of the results and received feedback, which was done to improve the va­ lidity of the data analyses process. PARTICIPANTS Stakeholders who participated in the interviews played var­ ious roles that contributed to the MVP initiative. These roles included technical, regulatory governance, capacity building, deployment, communication, and charitable con­ tributions. Participants responsible for the technical and regulatory processes of MenAfriVac contributed to full-scale manufacturing, exploratory and pre-clinical research, vac­ cine trials and surveillance, and post-licensure stages. Moreover, participants in governance roles were responsi­ ble for overseeing these various stages of the value chain to provide support, resources, and consultation. The distri­ bution and delivery of MenAfriVac was the responsibility of the participants in the deployment role, and participants responsible for communication led various sensitisation campaigns. Lastly, some participants were affiliated with charitable organisations that led the finance aspect of the project and funded the manufacturing stage. Journal of Global Health Reports DATA ANALYSIS The transcripts were analysed using NVivo, a qualitative re­ search analysis tool.17 The 18 interview transcripts were in­ dividually assessed by MM and TL to identify the barriers and facilitators mentioned. For this stage, MM and TL met four times: once to go over the first three barriers and fa­ cilitators to establish coding criteria, the second time to reach a consensus regarding all the facilitators of and bar­ riers to each interview; and finally, twice to condense all facilitators to and barriers of from all interviews into one table. During each meeting, MM and TL shared their results and created a new table with the consensus and the inter- reliability score. The inter-reliability score for the barriers and facilitators after identification was 80% and 85%, re­ spectively. Disagreements in data extraction were resolved through discussion facilitated by OE. In addition, MM and OE had four meetings to validate the inter-reliability scores MenAfriVac itself was discussed as a critical facilitator in the effective reach of populations within the meningitis belt. According to four key informants, this was facilitated by the adaptability of the vaccine through the FDA-ap­ proved technology transfer to the Serum Institute of India, the trialability through the acceptance of clinical trials in a few African countries, and the design of the MenAfriVac that led to the development of a cold-chain independent vac­ cine. As highlighted by one of the interviewees, “this vaccine is capable of sustaining four hours at room temperature. So, it doesn’t need the cold chain infrastructure.” (Interviewee 7). MenAfriVac and its associated MVP initiative encoun­ tered a few barriers that hindered or slowed its implemen­ tation success. According to one stakeholder, an obstacle to implementation was region-wide contention on external vaccine inspections and regulations. In addition, the design 3 Journal of Global Health Reports Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs Table 1. Barriers and facilitators with sample excerpts and their respective Consolidated Framework for Implementation Research (CFIR) constructs CFIR Domain and Construct: Intervention Source (Intervention Characteristics) Barriers Facilitators Region-wide contention on external vaccine inspections and regulations (1) "[...] trying to get [trials] done for the first time was a challenge, it wasn't easy at all. Getting countries to agree that they could do something together, both ethics committees and national regulatory authorities. DATA ANALYSIS So there, again, because it was the first time that was quite a challenge" (Int #13) CFIR Domain and Construct: Adaptability (Intervention Characteristics) Barriers Facilitators FDA-approved technology transfer for the vaccine to Serum Institute of India (1) "So, the technology was actually made available from the FDA. A group working extensively with the FDA had developed a technology for conjugation of the protein to the polysaccharide, which is the foundation for the vaccine, and that technology was made available to Marc and his crew, put together for the creation of this vaccine, at no cost" (Int #10) CFIR Domain and Construct: Trialability (Intervention Characteristics) Barriers Facilitators Acceptance of clinical trials in a few African countries (2) "I would say colleagues at WHO, the partnership with WHO, was a great facilitator for the clinical trials, for the acceptance of the clinical trials in Africa. [...] we have to say that facilitators were also many African doctors who really helped trusted us and really made us a lot of credit, they had credit" (Int #18) CFIR Domain and Construct: Complexity (Intervention Characteristics) Barriers Facilitators Complexities and delays in production of vaccines (2) "So only one manufacturer, one manufacturer is producing the vaccine. We had the challenge, at the beginning was that we don't have enough vaccine to cover all the 26 countries." (Int #17) Complexities and delays in vaccine development (2) "[...] Some of the challenges were with the actual vaccine development. [...] the challenge with, you know, starting off with one kind of technology and they left the project and we had to find another one." (Int #11) CFIR Domain and Construct: Design Quality and Packaging (Intervention Characteristics) Barriers Facilitators Narrow-scope vaccine immunity (Single serogroup) (2) "Yes, the other challenge also is that the vaccine protects against one serogroup, the meningococcal Meningitis A. Delay in EPI uptake due to lack of funding from Foundation and GAVI (1) DATA ANALYSIS So doesn't protect against B, C, X, Y and Z [...] Even now that we have eliminated MenA, we still have Meningitis cases" (Int #7) Cold-chain independent vaccine (1) "It was a vaccine that was able to be provided outside of the cold-chain, which was critical in terms of getting it out to people who needed it in remote areas" (Int #16) CFIR Domain and Construct: Cost (Intervention Characteristics) Barriers Facilitators Delay in EPI uptake due to lack of funding from the Gates Foundation and GAVI (1) "Some of the issues is that near the end we ran out of additional grants from the Gates Foundation and therefore became much more defeated to find money and by that time the Gates Foundation had grown and were not willing to give us money without strings attached. So we had a hard time, we struggled to get the money for the completion pediatric program. We had difficulties also with Gavi" (Int #14) CFIR Domain and Construct: External Policy and Incentives (Outer Setting) Barriers Facilitators Increasing control of funds from Gates Foundation (1) Funding flexibility and autonomy early in project (1) Foundation and GAVI (1) 4 Journal of Global Health Reports 4 Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs "[…] adapting to the changing environment and approach of the Gates Foundation as the project grew and also became more complex, was a challenge, definitely, was a challenge" (Int #16) "The Gates Foundation, which was its beginning at that time because they didn't have human capacity at the foundation, decided to give us money in one go. So from the beginning the money was available. There was no chance from the donor to micromanage, to try to influence, to whatever. The money was out" (Int #14) De-recognition of Indian regulators (2) "Unfortunately, I think there was a situation in India at that time in 2007 or 2008, when the national control authority was de-recognised by the WHO, and that was one of the major setbacks" (Int #1) Delay in individual country licensing/vaccine registration (1) "We brought the countries together to do their reviews of these assessment reports with the support of WHO prequalification, and it came to going back now to the countries for each individual country to license the vaccine or register its use to be in their campaigns which were used to introduce the vaccine. Of course, some of the countries took a while to do this" (Int #13) CFIR Domain and Construct: Structural Characteristics (Inner Setting) Barriers Facilitators WHO's respected reputation (1) "The African Regional Offices and Country Offices for WHO are highly respected and have a lot of authority in those areas. So working closely with them and being based in Geneva and having them as a partner was critical" (Int #16) CFIR Domain and Construct: Tension for Change (Inner Setting) Barriers Facilitators Unwillingness of neighbouring governments (low-risk countries) to adopt the vaccine (1) l was to eliminate, so to also vaccinate all the surrounding countries of all the risk countries. For example, Uganda is a high-risk country for meningitis, so Tanzania was supposed to also be vaccinated. Foundation and GAVI (1) [...] They say that the risk is low" (Int #3) Commitment and advocacy of African leaders (8) "The partnership with the leaders of the African countries who made a commitment, a verbal commitment to see this forward because of the magnitude and threat this had for their nations and their peoples" (Int #2) Delays in routine vaccine introduction (1) "[...] So we really there was a lot of momentum to get that vaccine out there in the form of a mass preventive campaign but the problem was that now that we see that decoupling, we have countries that even though the WHO recommendation is to introduce in their routine the maximum five years after they've done the mass preventive campaign, we're seeing countries that are delaying the introduction for much longer than that" (Int #9) CFIR Domain and Construct: Compatibility (Inner Setting) Barriers Facilitators Competing ideas between MVP stakeholders (2) "We were very different partners – PATH and WHO – so we had our agreement and disagreement" (Int #14) Willingness of manufacturer (Serum Institute of India) to develop cost-effective vaccine (1) "I would also say that Serum Institute is a very good partners because they accepted to develop at this low cost. So this vaccine is a cost effectiveness vaccine" (Int #16) CFIR Domain and Construct: Leadership Engagement (Inner Setting) Barriers Facilitators Regular involvement of recipient countries and stakeholders in planning/development process (1) "[...] the discussion was open, and everybody knew the discussion was going on and knew the final decision and the rational for that final decision. So, it's a bit of mix of management, clear, detailed project" (Int #5) CFIR Domain and Construct: Available Resources (Inner Setting) Barriers Facilitators Inadequate funding by country governments (3) Country-level (Burkina Faso) training of health workers (1) "[…] adapting to the changing environment and approach of the Gates Foundation as the project grew and also became more complex, was a challenge, definitely, was a challenge" (Int #16) g g f y license the vaccine or register its use to be in their campaigns which were used to introduce the vaccine. Of course, some of the countries took a while to do this" (Int #13) CFIR Domain and Construct: Structural Characteristics (Inner Setting) Barriers Facilitators WHO's respected reputation (1) "The African Regional Offices and Country Offices for WHO are highly respected and have a lot of authority in those areas. Foundation and GAVI (1) So working closely with them and being based in Geneva and having them as a partner was critical" (Int #16) CFIR Domain and Construct: Tension for Change (Inner Setting) Barriers Facilitators Unwillingness of neighbouring governments (low-risk countries) to adopt the vaccine (1) l was to eliminate, so to also vaccinate all the surrounding countries of all the risk countries. For example, Uganda is a high-risk country for meningitis, so Tanzania was supposed to also be vaccinated. [...] They say that the risk is low" (Int #3) Commitment and advocacy of African leaders (8) "The partnership with the leaders of the African countries who made a commitment, a verbal commitment to see this forward because of the magnitude and threat this had for their nations and their peoples" (Int #2) Delays in routine vaccine introduction (1) "[...] So we really there was a lot of momentum to get that vaccine out there in the form of a mass preventive campaign but the problem was that now that we see that decoupling, we have countries that even though the WHO recommendation is to introduce in their routine the maximum five years after they've done the mass preventive campaign, we're seeing countries that are delaying the introduction for much longer than that" (Int #9) CFIR Domain and Construct: Compatibility (Inner Setting) Barriers Facilitators Competing ideas between MVP stakeholders (2) "We were very different partners – PATH and WHO – so we had our agreement and disagreement" (Int #14) Willingness of manufacturer (Serum Institute of India) to develop cost-effective vaccine (1) "I would also say that Serum Institute is a very good partners because they accepted to develop at this low cost. So this vaccine is a cost effectiveness vaccine" (Int #16) CFIR Domain and Construct: Leadership Engagement (Inner Setting) Barriers Facilitators Regular involvement of recipient countries and stakeholders in planning/development process (1) "[...] the discussion was open, and everybody knew the discussion was going on and knew the final decision and the rational for that final decision. Foundation and GAVI (1) So, it's a bit of mix of management, clear, detailed project" (Int #5) CFIR Domain and Construct: Available Resources (Inner Setting) Barriers Facilitators Inadequate funding by country governments (3) "Of course there were also challenges in terms of funding, in terms of, even though most of the operational costs were provided by partners, Gavi, when it came to getting the government at all levels to participate or contribute to the funding it was also challenging in some countries" Country-level (Burkina Faso) training of health workers (1) "I must say that I think my full compliments to Burkina, because they were the first ones to introduce country wide, [...] they had activated the machinery and the health workers, given them training sufficiently, to ensure that this target is completed, and 5 Journal of Global Health Reports (Int #17) which they did complete" (Int #1) Insufficient staff and training (3) "[...] So we could have invested a little bit more in human resources. I think that would have been a plus. It would have made it easier to implement because you can't be everywhere" (Int #8) Infrastructure support (1) "Lastly, I would say that the development of infrastructure to support vaccine introduction, which was part of the program, was critical" (Int #16) Logistical gaps in health systems (3) "The health systems, most of the time, are not very strong enough to sustain large-scale interventions. So, we have logistical issues. [...] So it's an issue of logistics and reliance on external support" (Int #12) CFIR Domain and Construct: Access to Knowledge and Information (Inner Setting) Barriers Facilitators Insufficient sensitisation at local and national level (1) "The other thing is insufficient sensitisation. Sometimes the government does rush, they skip some of the key steps toward the proper implementation, proper acceptance of the vaccine from the communities" (Int #12) Strong communication strategy including crisis communication (5) "But we had developed a crisis communication strategy to deal with this situation where we said scenario 1. If someone dies during the campaign. Foundation and GAVI (1) Scenario 2 if, for example, we are working with meningococcal A vaccine and during our campaign there are people who have an outbreak or in a country with meningococcal C or another, serogroup" (Int #8) CFIR Domain and Construct: Knowledge and Beliefs about the Intervention (Characteristics of Individuals) Barriers Facilitators Complacency of MenA eradication (1) "[...] there was not a single case of meningitis A in the vaccinated population. So that gave some sort of a false complacency to the people, health authorities and the public in general, that now the Meningitis A is gone" (Int #1) Vaccine misinformation (3) "And we often had difficult situations, the most difficult situations. It was in Mauritania, in Chad, in 2011 or at one point, we had newspapers in the media that wrote that there were people who were paralysed after being vaccinated with MenAfriVac vaccine" (Int #8) CFIR Domain and Construct: Planning (Process) Barriers Facilitators Adequate time for planning project (2) "We had started working on the introduction framework for the vaccine three years before the vaccine was ready to be introduced, so, the lesson being the secret is always planning, planning, planning, planning, and then planning" (Int #4) Well-defined scope of project (2) "Perhaps one of the reasons for success was this was a very focused project, we are creating a vaccine against one group of meningococcus A, which is the main cause of these epidemics" (Int #15) CFIR Domain and Construct: Formally Appointed Internal Implementation Leaders (Process) Barriers Facilitators Leadership of Project Director (10) "I would say, is very strong and consistent leadership. [...]He is, and certainly was, and is, a remarkable leader. [...] he really led that project with a strong focus and a sort of relentless drive to get it done, and I think that was very, very helpful to the project" (Int #16) CFIR Domain and Construct: External Change Agents (Process) Barriers Facilitators Early product cancellation by contracted lab (1) "I think I can tell you another process which made the job very difficult, that the lab who was contracted initially by PATH who developed the conjugate technology in Sienna. Strong technical expertise (3) "So, I think I would say for the development of the vaccine, the help of consultants that were highly expert in the field. Facilitators Capacity building integration in national systems (1) "Developing, testing and introducing the vaccine by giving a lot of capacity building in the country and we make sure that it was not a vertical program even when they show when the time of introducing the vaccine came, he put to develop the project into the internal structure of WHO as well as the country organisation. I think all these together make a lot of awareness and engagement" (Int #10) Note. One sample excerpt is listed for each barrier and facilitator, and the number in brackets following the name of each barrier or facilitator indicates the number of interviewees who cited each barrier and facilitator. Note. One sample excerpt is listed for each barrier and facilitator, and the number in brackets following the name of each barrier or facilitator indicates the number of interviewees h i d h b i d f ili fluence implementation. For this study, the implementing organisations/partners include the WHO, PATH, and coun­ try governments. quality and cost of MenAfriVac were barriers mentioned by three stakeholders. Lastly, four key informants listed com­ plexities such as licensing and vaccine registration chal­ lenges, slow production of vaccines, and delays in vaccine development in the earlier stages of the project. For exam­ ple, one interviewee discussed the delays in vaccine devel­ opment, mentioning “[…] the challenge with, you know, start­ ing off with one kind of technology and they left the project, and we had to find another one.” (Interviewee 11). These bar­ riers under the intervention characteristics domain provide insight into the challenges that arose from the production, scaling-up, licensing, and registration of MenAfriVac. Under the internal setting domain, stakeholders men­ tioned facilitators under three constructs. One key infor­ mant discussed the WHO’s structural characteristic as fa­ cilitating factor as well as the advantages of working and partnering with a large global health organisation. Second, nine key informants mentioned facilitators that fall under implementation climate, including the commitment and advocacy of African leaders and the willingness of the man­ ufacturer to develop cost-effective vaccines. Further, one interviewee discussed how the “[African governments] were completely on board from the start. They were regularly up­ dated because it was a yearly meningitis partners meeting with the country, and there were yearly updates on the development of the project.” (Interviewee 5). Facilitators Lastly, eight key informants discussed the regular involvement of recipient countries and stakeholders in the planning and development process, country-level training of health workers, and strong com­ munication strategies, including crisis communication as facilitators, that led to the success of the MVP initiative. EXTERNAL SETTING This domain described changes in the external setting—de­ cisions and changes made outside of the MVP—that can positively or negatively influence implementation. One stakeholder discussed the early structure of the Gates Foundation and the funding flexibility and autonomy granted to the MVP project, which was key facilitators of the project. As stated by the key informant, “[…] the Gates Foundation had a relatively small staff, and there was a large amount of funding that was provided for this project that really allowed the leaders of the project and the partners of the pro­ ject to be creative in moving forward. So that was another im­ portant, I think, the driver for success.” (Interviewee 16) The MVP initiative involved numerous stakeholders and networks from various countries, organisations, and disci­ plines. With multiple stakeholders involved, the challenges that four stakeholders mentioned included the unwilling­ ness of neighbouring governments (low-risk countries) to adopt the vaccine, delays in routine vaccine introduction, priority setting from several country governments, and compatibility challenges in balancing several groups and their ideas. One key informant stated, “we were seeing a lot of these countries losing the momentum to actually intro­ duce the vaccine in their routine program.” (Interviewee 9). Moreover, eight stakeholders indicated inadequate funding by country governments, unavailable resources (i.e., insuf­ ficient staff and training), and lack of access to knowledge and information as challenges to the seamless implementa­ tion of MVP goals. Four key informants were in consensus regarding the barriers related to the external strategies for spreading the intervention. These included policies and incentives that influenced the implementation of MenAfriVac and the MVP initiative in general. The de-recognition of Indian regula­ tors by the WHO and the increasing control of funds from the BMGF much later in the project were mentioned as key challenges to the effective and timely implementation of MVP goals. Foundation and GAVI (1) These helped enormously" (Int #18) (Int #17) Early product cancellation by contracted lab (1) "I think I can tell you another process which made the job very difficult, that the lab who was contracted initially by PATH who developed the conjugate technology in Sienna. […] they get a message from this lab that the project is cancelled. And they whatever technology they had developed, they will not transfer to MVP." (Int #1) 6 Journal of Global Health Reports Disinterest of Big Pharmaceuticals to partner for MenA vaccine (3) "The barriers I think has been relatively difficult to identify among a manufacturer that would accept to manufacture for that price." (Int #5) Involvement of variety of partners/stakeholders (9) "I think the fact that the project, the MVP, was a very large consultation of partners, made it to be an opportunity for partners and governments to come together in a collaborative way to work together and look at the same voice, same object. So, this happened and contributed to the success of the project" (Int #17) CFIR Domain and Construct: Executing (Process) Barriers Facilitators Security threats from epidemics and civil war (1) "You have this one in South Sudan, we were obliged to postpone because of insecurities, civil war" (Int #3) Capacity building integration in national systems (1) "Developing, testing and introducing the vaccine by giving a lot of capacity building in the country and we make sure that it was not a vertical program even when they show when the time of introducing the vaccine came, he put to develop the project into the internal structure of WHO as well as the country organisation. I think all these together make a lot of awareness and engagement" (Int #10) Note. One sample excerpt is listed for each barrier and facilitator, and the number in brackets following the name of each barrier or facilitator indicates the number of interviewees who cited each barrier and facilitator. Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs CFIR Domain and Construct: Executing (Process) PROCESS The process domain included the strategies that influenced the implementation at all stages of the project. Most of the facilitators discussed by stakeholders are found under this domain, including the planning, engage­ ment, and execution processes. Three key informants dis­ cussed the constant and organised planning process that was implemented for the MVP initiative. In addition, ten key informants praised the effective and strong leadership of the project director and the strong technical expertise that was sought as crucial facilitators to the initiative’s suc­ cess. Moreover, twelve stakeholders described the impor­ tant role that external organisations, affiliates, and other supporters played in rendering this initiative successful. Lastly, one key informant highlighted the capacity building prioritised to integrate the MVP initiative into national health systems. Most informants collectively mentioned the planning, engagement, and execution processes as the key facilitator to the success of the MVP initiative. One inter­ viewee shared that “the MVP was a very large consultation of partners, made it to be an opportunity for partners and gov­ ernments to come together in a collaborative way to work to­ gether and look at the same voice, the same object. So, this happened and contributed to the success of the project.” (In­ terviewee 17). Regarding inner setting characteristics, our study re­ vealed both the advocacy of African leaders and the willing­ ness of the Serum Institute of India to develop a cost-effec­ tive vaccine as key facilitators were important facilitators to the implementation of the MVP. PATH released a report identifying the advocacy and call for action from African ministries of health to find a solution to the 1996-1997 meningitis A epidemics.20 The report described the plea from African leaders and public health officials to create a cost-effective vaccine, leading to the unique partnership with the Serum Institute of India as the MVP’s vaccine man­ ufacturer. As stated by most stakeholders, the various part­ nerships affiliated with the MVP contributed significantly to the implementation across the Meningitis Belt. This sen­ timent is complimented by one study analysing the multiple stages of MVP governance, development, and partnership accruement.4 Four stakeholders discussed barriers that occurred at the onset of the project, such as the early product cancellation by a contracted lab and the disinterest of Big Pharmaceuti­ cals to partner with the MVP to create a cost-effective vac­ cine. PROCESS Moreover, one stakeholder also described barriers re­ lated to the execution process due to security threats (such as war and other epidemics). Given the frequency of results around the inner setting and process CFIR domains, we find that organisational the­ ories best capture the goal of upstream sustainability and the scope of participant responses. Since 2009, increased attention has been paid to the organisational theory for implementation—the ORC theory.9 Our results map well to three core aspects of this theory: change valence, task assessment, change efficacy, and drivers of organisational readiness. First, change valence in ORC describes motiva­ tional theory-based group value of an impending change.9 CFIR domains (n = 5) and constructs (n = 39) D. Reflecting and Evaluating formation on the processes that have rendered the efforts of the MVP successful. We have revealed multiple facilita­ tors and barriers to a large-scale vaccine project in which numerous stakeholders are involved and display the im­ portance of the preparatory stages before and during im­ plementation. The key informants stated various barriers and facilitators, falling under all five domains of the CFIR framework, including intervention characteristics, external setting, internal setting, characteristics of individuals, and process. The key and most common facilitators to MVP re­ volved around the inner setting and the process domains of the CFIR. CHARACTERISTICS OF INDIVIDUALS This domain focused on the actions and behaviours of in­ dividuals and their effect through their teams, units, net­ works, and organisations on implementation. No facilitators were mentioned under this domain. Knowledge and beliefs regarding the intervention was the single construct listed under this domain and was men­ tioned by four key informants as a barrier to the initiative. The complacency related to the eradication of meningitis A and vaccine misinformation were challenges in the uptake of MenAfriVac by authorities and the public in general, re­ spectively. As highlighted by one interviewee, “There was a lot of writing on the web and even in the newspapers saying that Bill Gates was killing children in Africa. […] So, it was one of the most difficult situations.” (Interviewee 8). Our findings complement existing literature as key in­ formants cited critical facilitators that were echoed by pre­ vious studies. The cold chain infrastructure of the MenAfriVac was mentioned as a contributing factor to the success of the MVP initiative, as was the case in a previous study analysing the benefits of using vaccines out of the cold chain during the mass immunisation campaign in Benin.19 This study emphasised the benefits of MenAfriVac’s cold-chain independence for cases in which health centres and target populations are far from each other. In addition, the funding flexibility and autonomy early in the MVP initiative were mentioned as a facilitator to the project’s success. One study supports the notion of funding flexibility, as BMGF started to seek new global health projects to invest around the time the MVP was ini­ tiated.7 INTERNAL SETTING Executing 8 Journal of Global Health Reports Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs INTERNAL SETTING The internal setting domain included characteristics of the implementing organisation and its partners that might in­ 7 Journal of Global Health Reports Journal of Global Health Reports Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs Table 2. Frequency table of cited Consolidated Framework for Implementation Research (CFIR) constructs (barriers and facilitators) related to the implementation of MenAfriVac through the Meningitis Vaccine Project (MVP) (n = 18) CFIR domains (n = 5) and constructs (n = 39) Barrier n (%) of interviews Facilitator n (%) of interviews I. Intervention characteristics     1 (6%) None identified None identified None identified None identified None identified None identified 1 (6%) None identified 2 (11%) 4 (22%) None identified 2 (11%) 1 (6%) 1 (6%) None identified II. Outer Setting     None identified None identified None identified None identified None identified None identified 4 (22%) 1 (6%) III. Inner Setting     None identified 1 (6%) None identified None identified None identified None identified 2 (11%) 8 (50%) 2 (11%) 1 (6%) None identified None identified None identified None identified None identified None identified None identified None identified None identified 1 (6%) 7 (39%) 2 (11%) 1 (6%) 5 (28%) IV. Characteristics of Individuals     4 (22%) None identified None identified None identified None identified None identified None identified None identified None identified None identified V. Process     None identified 3 (17%) None identified None identified None identified 10 (56%) None identified None identified 4 (22%) 12 (67%) 1 (6%) 1 (6%) A. Intervention Source B. Evidence Strength and Quality C. Relative Advantage D. Adaptability E. Trialability  F. Complexity G. Design Quality and Packaging H. Cost A. Patient Needs and Resources B. Cosmopolitanism C. Peer Pressure D. External Policy and Incentives A. Structural Characteristics B. Network and Communications C. Culture D. Implementation Climate 1. Tension for Change 2. Compatibility 3. Relative Priority 4. Organisational Incentives and Rewards 5. Goals and Feedback 6. Learning Climate E. Readiness for Implementation 1. Leadership Engagement 2. Available Resources 3. Access to Knowledge and Information A. Knowledge and Beliefs about the Intervention B. Self-Efficacy C. Individual Stage of Change D. Individual Identification with Organisation E. Other Personal Attributes A. Planning B. Engaging 1. Opinion Leaders 2. Formally Appointed Internal Implementation Leaders 3. Champions 4. External Change Agents C. IMPLICATIONS FOR IMPLEMENTATION PRACTITIONERS It illustrates that an organisation’s readiness will align with its interests and perceived organisational value, down to the individual. Climate and readiness in the MVP were fre­ quently reported across barriers and facilitators alike, indi­ cating a spectrum of change valence across recipient coun­ tries. Indeed, in 2009, Weiner first postulated that organisational readiness might be related in scope to Klein and Sorra’s (1996) construct of implementation climate. Therefore, this maps well to Weiner’s views on change va­ lence.21 The view of the implementation landscape revealed in this analysis advances knowledge regarding how to maximise the implementation of vaccination programs in Africa in ways that can immediately inform ongoing program deliv­ ery and the design of future vaccination programs. First, there was a strong tension for change—the degree to which stakeholders perceive the current situation as intolerable or requiring change. The palpable tension was translated into the commitment and advocacy of African leaders to bring attention and urgency to address the burden of meningi­ tis A. Second, formally appointed internal implementation leaders were critical in successful vaccine implementation. The project director of the MVP provided excellent leader­ ship and expertise, thereby rendering the initiative success­ ful. The technical expertise sought and provided through­ out the implementation stages was a critical facilitator to successfully implementing the vaccine program. This was achieved through the engagement of product experts, the EPI immunisation department of the WHO, and epidemi­ ologists. Third, external change agents were vital. The in­ volvement of multiple partners and stakeholders—such as donors, agencies, government, and communities—was highlighted as a critical facilitator to the success of the MVP. These agents formally influenced and facilitated the intervention decisions in a desirable direction. Finally, key attributes of the vaccine were critical for successful imple­ mentation, including stability of the freeze-dried vaccine and its adaptability through the FDA-approved technology transfer to the Serum Institute of India. These, among oth­ ers, were crucial facilitators of successful development, pro­ duction, and implementation of the vaccine program. DISCUSSION Stakeholders involved in the implementation of the MVP are the focus of this study, as they provide valuable in­ 9 Journal of Global Health Reports Journal of Global Health Reports Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs IMPLICATIONS FOR IMPLEMENTATION PRACTITIONERS Second, change efficacy in ORC draws on social theories to identify three aspects of a task—its demands, resources, and situational factors—that comprise an organisation’s as­ sessment of its adoption.9 This is in line with our findings that complexity, resource unavailability, and a range of country-specific situational factors were associated with poorer readiness and low change-associated effort in spe­ cific country cases.21 i Finally, our results align with what ORC identifies as positive influences on an organisation’s readiness: leader­ ship, consistent messaging, and a shared sense of project value. Organisational readiness posits that consistent lead­ ership, information sharing, messaging, and social inter­ action—or engagement in this context—promote a shared sense of readiness that contributes to a shared experience and more significant change investments.9 Similarly, in this study, we identified leadership, multiple levels of engage­ ment, and access to information as critical facilitators. One key informant spoke in favour of the project director’s lead­ ership, stating that “[…] he really led that project with a strong focus and a sort of relentless drive to get it done, and I think that was very, very helpful to the project.” (Interviewee 16). Overall, we observed that leadership, engagement, and consistency in advocacy and information sharing upstream do facilitate success, whereas poor readiness and climate or complexity implied challenges. Given our results being similar to the ORC, we suggest further investigation and mapping of engagement and readiness/climate constructs as they relate to the theory for great accuracy. A novel retro­ spective follow-up on the MVP initiative could examine our exploratory themes and constructs regarding stakeholder or decision-maker contexts, policies, and actions that (i) have practical implications to organisational readiness, (ii) pro­ mote positive task assessment according to the ORC, and (iii) are associated with greater engagement. LIMITATIONS This study presents several limitations that must be taken into consideration when interpreting the results. A poten­ tial limitation was our selection of a determinant frame­ work (CFIR), as such frameworks have been criticised for their inadequacy in addressing how change takes place.22 Nevertheless, the framework served as a valuable tool for organising and contextually understanding data.23 Further, the CFIR assisted in triangulating information, allowed us to conceptualise the barriers and facilitators in a more or­ ganised manner, resulted in the identification of 16 out of 39 CFIR constructs, and allowed the findings to be placed in the context of the wider implementation research litera­ ture.24,25 Practically, organisational readiness may be further dri­ ven through open information sharing and further com­ munity engagement. Given existing strong implementation leadership, engagement may benefit from diversifying its source. This can be achieved through investing in commu­ nication campaigns, specifically involving communities as external change agents to their own implementation and involving community-based implementation champions and opinion leaders in rollouts. These benefit from addi­ tional community resource mobilisation and intervention ownership, thereby further increasing the probability of embeddedness. Importantly, follow-ups to the MVP and ad­ ditional vaccine rollouts can leverage a strong sense of pro­ ject value and engage actors toward greater readiness for change through consistently strong leadership, open infor­ mation-sharing and messaging, and partnership building. In addition, our interviewee pool was limited by the number of key implementation stakeholders that we dis­ covered through our initial literature review, referrals, and snowball sampling. Although our initial list of potential stakeholders to be interviewed was high, only twenty-one met the eligibility criteria established. The outcome of this small sample size may be the inability to determine if our study has considered all factors that would have impacted the scaling of the project. Despite this, our sample size in­ cluded stakeholders who took on various roles, including 10 Journal of Global Health Reports Journal of Global Health Reports Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs technical, regulatory, governance, capacity building, de­ ployment, communication, and charitable contributions. This variety of stakeholders proved to be critical in provid­ ing many essential perspectives in the upstream implemen­ tation of the MenAfriVac across the Meningitis Belt. fectively. LIMITATIONS This leadership translated into consistent mes­ saging, information sharing, and multiple levels of engage­ ment with external change agents (including technical experts), which promoted a shared sense of readiness con­ tributing to significant change investments in implement­ ing MVP. Finally, the decision to narrow the study sample to only partners and implementers of the MVP program means that the experiences and perspectives of citizens and healthcare workers administering the MenAfriVac were excluded. We acknowledge that this may serve as a limitation to our study, as these perspectives may have informed the down­ stream barriers and facilitators of the MVP initiative. How­ ever, two recent studies have examined the downstream perspectives of the MVP. The first study highlights the chal­ lenges expressed by healthcare workers in implementing the MenAfriVac in the routine childhood immunisation schedule in Burkina Faso.13 In the second study, perspec­ tives were the focal point in determining the perspectives of Meningitis A in Ghana.14 The niche of the research pur­ pose of our research was focused on organisations, govern­ ments, and key individuals involved in upstream processes, compared to existing studies that focused on citizens’ and healthcare workers’ perspectives. Therefore, our study fills a gap in the existing literature by investigating stakehold­ ers’ perspectives around the upstream processes of the MVP initiative. AUTHORSHIP CONTRIBUTIONS Study conception and design: OE. Data Collection: MM. Analysis and interpretation of data: MM, TL, and OE. Draft of the manuscript: MM, TL, and OE. Critical revision of the manuscript for important intellectual content: OE, TL and MM. All authors read and approved the final manuscript. No funding was received for this research project. No funding was received for this research project. ACKNOWLEDGEMENTS The authors are grateful to Shaathana Ratnasingam for sup­ porting the transcription of interview audio files. CONCLUSIONS The authors completed the Unified Competing Interest form at http://www.icmje.org/disclosure-of-interest/ (available upon request from the corresponding author) and declare no conflicts of interest. To bridge gaps upstream in understanding the delivery and outcomes of the global MVP, we conducted 18 in-depth, semi-structured interviews with stakeholders involved in the project. All five domains from the CFIR framework were critical in implementing the MVP, including intervention characteristics, the outer and inner settings, the characteris­ tics of individuals, and the process. However, two of these domains (inner setting and process) were most representa­ tive as facilitators for MPV. Specifically, the tension for change—the degree to which stakeholders perceive the cur­ rent situation as intolerable or needing change— advocated by African leaders served as a crucial foundation for solid leadership for the MPV. The leadership came in the form of the project director and other formally appointed leaders, who could plan, engage and execute the project’s goals ef­ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CCBY-4.0). View this license’s legal deed at http://creativecommons.org/licenses/by/4.0 and legal code at http://creativecom­ mons.org/licenses/by/4.0/legalcode for more information. Submitted: August 29, 2021 GMT, Accepted: September 18, 2021 GMT CORRESPONDENCE TO: Obidimma Ezezika Department of Health and Society, University of Toronto, Scarborough, 1265 Military Trail, Toronto, Ontario, M1C 1A4, Canada. obidimma.ezezika@utoronto.ca Submitted: August 29, 2021 GMT, Accepted: September 18, 2021 GMT This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CCBY-4.0). View this license’s legal deed at http://creativecommons.org/licenses/by/4.0 and legal code at http://creativecom­ mons.org/licenses/by/4.0/legalcode for more information. 11 Journal of Global Health Reports Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs REFERENCES 10. Ridde V. Need for more and better implementation science in global health. BMJ Glob Health. 2016;1(2):e000115. doi:10.1136/bmjgh-201 6-000115 1. Diallo AO, Soeters HM, Yameogo I, et al. 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Keith RE, Crosson JC, O’Malley AS, Cromp D, Taylor EF. Using the Consolidated Framework for Implementation Research (Cfir) to produce actionable findings: A rapid-cycle evaluation approach to improving implementation. Implementation Sci. 2017;12(1). doi:10.1186/s13012-017-0550-7 9. Weiner BJ. A theory of organizational readiness for change. Implementation Sci. 2009;4(1). doi:10.1186/17 48-5908-4-67 9. Weiner BJ. A theory of organizational readiness for change. 25. Kirk MA, Kelley C, Yankey N, Birken SA, Abadie B, Damschroder L. A systematic review of the use of the consolidated framework for implementation research. Implementation Sci. 2015;11(1):72. doi:10.1186/s1301 2-016-0437-z REFERENCES Implementation Sci. 2009;4(1). doi:10.1186/17 48-5908-4-67 12 Journal of Global Health Reports mplementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs 19. Zipursky S, Djingarey MH, Lodjo JC, Olodo L, Tiendrebeogo S, Ronveaux O. 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Implementation Sci. 2019;14(1):21. doi:10.1186/s13012-019-0867-5 24. Means AR, Kemp CG, Gwayi-Chore MC, et al. Evaluating and optimizing the consolidated framework for implementation research (CFIR) for use in low- and middle-income countries: A systematic review. Implementation Sci. 2020;15(1):17. doi:10.1186/s13012-020-0977-0 25. Kirk MA, Kelley C, Yankey N, Birken SA, Abadie B, Damschroder L. A systematic review of the use of the consolidated framework for implementation research. Implementation Sci. 2015;11(1):72. doi:10.1186/s1301 2-016-0437-z Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs 19. Zipursky S, Djingarey MH, Lodjo JC, Olodo L, Tiendrebeogo S, Ronveaux O. Benefits of using vaccines out of the cold chain: Delivering Meningitis A vaccine in a controlled temperature chain during the mass immunization campaign in Benin. Vaccine. 2014;32(13):1431-1435. doi:10.1016/j.vaccine.2014.0 1.038 19. Zipursky S, Djingarey MH, Lodjo JC, Olodo L, Tiendrebeogo S, Ronveaux O. Benefits of using vaccines out of the cold chain: Delivering Meningitis A vaccine in a controlled temperature chain during the mass immunization campaign in Benin. Vaccine. 2014;32(13):1431-1435. doi:10.1016/j.vaccine.2014.0 1.038 23. Weir NM, Newham R, Dunlop E, Bennie M. Factors influencing national implementation of innovations within community pharmacy: A systematic review applying the Consolidated Framework for Implementation Research. Implementation Sci. 2019;14(1):21. doi:10.1186/s13012-019-0867-5 19. Zipursky S, Djingarey MH, Lodjo JC, Olodo L, Tiendrebeogo S, Ronveaux O. Benefits of using vaccines out of the cold chain: Delivering Meningitis A vaccine in a controlled temperature chain during the mass immunization campaign in Benin. Vaccine. 2014;32(13):1431-1435. doi:10.1016/j.vaccine.2014.0 1.038 20. The meningitis vaccine project: A groundbreaking partnership. 21. Damschroder LJ, Lowery JC. Evaluation of a large- scale weight management program using the consolidated framework for implementation research (CFIR). Implementation Sci. 2013;8(1). doi:10.1186/17 48-5908-8-51 REFERENCES Accessed September 17, 2021. https://ww w.path.org/articles/about-meningitis-vaccine-projec t/ 21. Damschroder LJ, Lowery JC. Evaluation of a large- scale weight management program using the consolidated framework for implementation research (CFIR). Implementation Sci. 2013;8(1). doi:10.1186/17 48-5908-8-51 22. Nilsen P. Making sense of implementation theories, models and frameworks. Implementation Sci. 2015;10(1):53. doi:10.1186/s13012-015-0242-0 24. Means AR, Kemp CG, Gwayi-Chore MC, et al. Evaluating and optimizing the consolidated framework for implementation research (CFIR) for use in low- and middle-income countries: A systematic review. Implementation Sci. 2020;15(1):17. doi:10.1186/s13012-020-0977-0 24. Means AR, Kemp CG, Gwayi-Chore MC, et al. Evaluating and optimizing the consolidated framework for implementation research (CFIR) for use in low- and middle-income countries: A systematic review. Implementation Sci. 2020;15(1):17. doi:10.1186/s13012-020-0977-0 20. The meningitis vaccine project: A groundbreaking partnership. Accessed September 17, 2021. https://ww w.path.org/articles/about-meningitis-vaccine-projec t/ 21. Damschroder LJ, Lowery JC. Evaluation of a large- scale weight management program using the consolidated framework for implementation research (CFIR). Implementation Sci. 2013;8(1). doi:10.1186/17 48-5908-8-51 21. Damschroder LJ, Lowery JC. Evaluation of a large- scale weight management program using the consolidated framework for implementation research (CFIR). Implementation Sci. 2013;8(1). doi:10.1186/17 48-5908-8-51 22. Nilsen P. Making sense of implementation theories, models and frameworks. Implementation Sci. 2015;10(1):53. doi:10.1186/s13012-015-0242-0 13 13 Journal of Global Health Reports Journal of Global Health Reports
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Developing emulsion gels by incorporating Jerusalem artichoke inulin and investigating their lipid oxidative stability
Food production, processing and nutrition
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RESEARCH Open Access © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract This study investigated physical, chemical and lipid oxidative properties of emulsion gels (W/O) incorporating Jerusalem artichoke (JA) inulin. Primary purified inulin extract (PPIE, 1%) improved the homogeneity of emulsion gel (with no syneresis) and developed smaller particle size droplets (average 40 μm) than control (average size 60 μm). HPLC revealed that PPIE had 80.28% inulin content compared with commercial inulin (CI, 100%). Crude inulin extract (CIE, 0.08–0.33 mg/mL) delayed linoleic acid oxidation because of higher total phenolic content (4.96 ± 0.01, mg GAE/g), compared with PPIE (0.72 ± 0.03). Lipid oxidative stability of emulsion gels with inulin samples was in the order of CI > PPIE > CIE (P < 0.05) by Rancimat analysis, which agreed with volumetric gel index results. This study suggests that emulsion gels with JA inulin (PPIE) could act as a potential fat replacement in food systems. Keywords: Inulin, Emulsion gel, Reduced-fat, Liposome, Lipid oxidation, Rancimat Highlights products (Dave 2012). Low-fat or reduced-fat foods with grainy and undesirable texture have less organoleptic properties than full-fat foods (Hamilton et al. 2000). One of the promising strategies is using fat replacers that have similar physical and organoleptic properties as fat. It is reported that succinyl chitosan in cakes formula- tions (Rios et al. 2018), hydroxypropyl methylcellulose in muffins (Oh & Lee 2018), soy protein hydrolysate/xan- than gum in low-fat ice cream (Liu et al. 2018), chia and oat emulsion gels in low-fat sausages (Pintado et al. 2018) were used as fat replacers.  Emulsion gel with 1% primary purified inulin extract (PPIE) developed smaller droplets size and had no syneresis.  Emulsion gel with 1% primary purified inulin extract (PPIE) developed smaller droplets size and had no syneresis.  Emulsion gels with higher inulin content meant longer induction time.  Crude inulin extract could delay linoleic acid oxidation using liposome model.  Crude inulin extract contained higher amount of phenolics than PPIE. Emulsion gel comprised of inulin and monoglycerides is a potential fat replacer. Inulin could form a gel in aqueous solution at high concentrations (> 15%) throughout the heating-cooling process (Kim et al. 2001). In addition, monoglycerides and vegetable oil could form a semi-solid aggregated network structure when cooling from heating (Ojijo et al. 2004). Developing emulsion gels by incorporating Jerusalem artichoke inulin and investigating their lipid oxidative stability Feijie Li1, Aynur Gunenc1 and Farah Hosseinian1,2* Feijie Li1, Aynur Gunenc1 and Farah Hosseinian1,2* © The Author(s). 2020 Open Access This a International License (http://creativecomm reproduction in any medium, provided yo the Creative Commons license, and indica * Correspondence: farah.hosseinian@carleton.ca 1Food Science and Nutrition, Chemistry Department, Carleton University, Ottawa, ON, Canada 2Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada Food Production, Processing and Nutrition Food Production, Processing and Nutrition Li et al. Food Production, Processing and Nutrition https://doi.org/10.1186/s43014-019-0017-0 * Correspondence: farah.hosseinian@carleton.ca 1Food Science and Nutrition, Chemistry Department, Carleton University, Ottawa, ON, Canada 2Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa ON K1S 5B6, Canada Introduction Traditionally, solid fat is derived from saturated animal or vegetable fat (Hartel et al. 2018), or from partially hy- drogenated oil which might be associated with an in- creased risk of coronary heart disease (Mozaffarian et al. 2006). With increased health concerns, people prefer to choose low-fat or reduced-fat food. However, fat has a significant effect on the physical properties of food Inulin (Fig. 1) is a dietary fiber (polysaccharide) in which D-fructose units linked by β (2 →1) linkages with the end of glucose residue (Li et al. 2015). The degree of polymerization (DP) of inulin varies from 2 to 60, de- pending on plant resources, harvest time, storage and process conditions (Saengthongpinit & Sajjaanantakul 2005), which determines the biological and physical * Correspondence: farah.hosseinian@carleton.ca 1Food Science and Nutrition, Chemistry Department, Carleton University, Ottawa, ON, Canada 2Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada * Correspondence: farah.hosseinian@carleton.ca 1Food Science and Nutrition, Chemistry Department, Carleton University, Ottawa, ON, Canada 2Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada Page 2 of 11 Page 2 of 11 Li et al. Food Production, Processing and Nutrition Page 2 of 11 (2020) 2:2 Sample preparation Fresh Jerusalem artichoke (JA) tubers were sliced, freeze-dried and milled to obtain powdered tubers. It was stored in a desiccator at room temperature until fur- ther analysis (Srinameb et al. 2015). Materials and methods Chemicals and reagents Fig. 1 Chemical structure of inulin, drawn by BIOVIA Draw Soy lecithin and monoglycerides (MGs) (Alphadim® 90 SBK) were kindly provided by Grain Process Enterprises limited (Scarborough, ON, CA) and Caravan Ingredients, Inc. (Lenexa, Kansas, USA), respectively. Extra virgin olive oil was purchased from Amazon.ca. Tween 80 (polyox- yethylene-20-sorbitan monooleate) and sucrose (ultra- pure) were from Fisher Scientific (CA). Activated charcoal (powder, 100 particle size), Trolox (6-hydroxy-2,5,7,8-tet- ramethylchroman-2-carboxylic acid), linoleic acid (≥99%), Tween 20 (polyoxyethylene sorbitan monolaurate), inulin from chicory (commercial inulin) and Folin & Ciocalteu phenol reagent were from Sigma-Aldrich (St Louis, MO, USA). CaCl2 was from Sigma Chemical Co. (St. Louis, MO, USA). Na2CO3 was from VWR International Co. (Mississauga, ON, CA). Glucose (CAS: 50–99-7) was from Sigma-Aldrich (UK). D-fructose was from BioShop Canada Inc. AAPH (2,2′-Azobis (2-amidinopropane) dihydrochloride, 98%) was from ACROS ORGANICS (New Jersey, USA). Gallic acid was from Sigma Chemical (St Louis, MO, USA). Fig. 1 Chemical structure of inulin, drawn by BIOVIA Draw properties of inulin (Rubel et al. 2018). High DP inulin has good ability to form gels (Kim et al. 2001). Also, inu- lin has amazing water-binding activity, and which could reduce syneresis in spread (Fadaei et al. 2012). Inulin extraction Sample preparation On the other hand, sugars and sugar-like compounds play an important role in defending oxidative stress in plant cells (Bolouri-Moghaddam et al. 2010). Stoyanova et al. (2011) re- vealed that inulin and stevioside have super capacity of scav- enging hydroxyl and superoxide radicals, which were more effective than mannitol that is well-known as an antioxidant (Stoyanova et al. 2011). Microscopic observations Microstructures of inulin gel and oleogel were observed under PLM (polarized light microscope, Axioplan 2 im- aging and Axiophot 2 universal microscope). Emulsion gels (with 1% PPIE and control) were visualized both by PLM and cryo-SEM (cryo-scanning electron microscope, Nano Imaging Facility Laboratory of Carleton University, Ottawa, ON) after prepared immediately to see their morphological differences, so that effect of 1% PPIE was achieved. The mass spectrometry (MS) analysis of inulin extract (PPIE) was conducted in Carleton Mass Spectrometry Center. Four microliters inulin sample was placed into the Proxeon nanoelectrospray emitter and detected by a hy- brid quadrupole TOF MS. The spectra of samples were recorded with the voltage of ESI (electrospray ionization), and the data was analyzed by ChemStation software. HPLC-MS form emulsion gel at room temperature (23 °C) until fur- ther analysis. form emulsion gel at room temperature (23 °C) until fur- ther analysis. Chromatographic analysis was carried out using high- performance liquid chromatography (HPLC, Waters e2695, Waters Corporation, Milford, USA) with a re- fractive index (RI) detector (Waters 2414, Waters Cor- poration, Milford, USA). Commercial inulin (CI, 0.25 mg/mL), PPIE (1.0 mg/mL) and SPIE (1.0 mg/mL) were separated by Sugar-Pak™I column (6.5 × 300 mm, 10 μm, Waters, USA) at 60 °C with 10 μL injection volume. Milli-Q water (Millipore) was used as mobile phase with a flow rate of 0.6 mL/min. Glucose, fructose and sucrose were used as standards. Emulsion gel preparations Inulin gel CI, PPIE and CIE were dissolved in distilled water (20%, w/v) separately at 80 °C for 5 min, and then cooled to room temperature (23 °C) for 24 h to set gel structure. In order to compare gel formation degree, volumetric gel index (VGI) was calculated for each sample. VGI is de- fined as the ratio of gel volume to total volume (Eq. 1) (Kim et al. 2001). Volumetric gel index VGI ð Þ Volumetric gel index VGI ð Þ ¼ gel volume ð Þ= total volume ð Þ x 100% ð1Þ ð Þ ¼ gel volume ð Þ= total volume ð Þ x 100% Emulsion gel Four emulsion gels (A, B, C, D) consisted of CI, PPIE, CIE and control were prepared as in Inulin emulsion gel section. They were diluted 4 times with phosphate buffer solution (10 mL of 0.05 mol/L, pH 7.4) containing 0.8% (v/v) Tween 20 until further use. Extractions Lipid oxidation can be measured with different methods including spectrophotometry for conjugated dienes (Liégeois et al. 2000) and 2-thiobarbituric acid (TBA) value (Zeng et al. 2017), Rancimat method for oil stability index (Tavakoli et al. 2019), chromatographic analysis for changes in reactants (Bazina & He 2018), Fou- rier transform infrared (FTIR) method for peroxide value; differential scanning calorimetry (DSC), iodometric titra- tion, and others (Shahidi & Zhong 2005). Conjugated di- enes with strong absorption of UV radiation at 234 nm are produced during oxidation of lipid that contains dienes or polyenes, which is a good index for measuring lipid oxida- tion (Vieira & Regitano-D’arce 1998). Rancimat method is normally used to test oil stability under accelerated stor- age conditions to estimate the shelf life of food products (Farhoosh et al. 2008). Crude inulin extract (CIE) JA tuber powder was ex- tracted with distilled water (1:20 w/v) at 80 °C for 2 h. After cooling to room temperature, the mixture was filtered and centrifuged (Thermo Fisher Scientific, Langenselbold, Germany) at a speed of 4500 g at 23 °C for 15 min. Super- natant was freeze-dried (Labconco, Kansas, USA) and stored in the fridge until further analysis (Srinameb et al. 2015). Primary purified inulin extract (PPIE) CIE was further purified by deproteinization (Huang et al. 2011) and decolouration (Hongxin 2008). Briefly, CaCl2 (0.1%, w/v) was added into CIE solution and mixed until flocci were no longer produced. Na2CO3 was added to precipitate redundant CaCl2. Color was removed by adding 2.0% (w/v) activated charcoal at 80 °C for 15 min, then fil- trated and freeze-dried to obtain PPIE. The objectives of this work were to: (i) analyze JA inu- lin extracts by HPLC-IR; (ii) formulate emulsion gels by incorporating JA inulin; (iii) evaluate the effects of inulin extracts against lipid oxidation using liposome model and emulsion gel; (iv) inspect total phenolic content in inulin extracts; (v) investigate oxidative stability of emul- sion gels by Rancimat method. Secondary purified inulin extract (SPIE) For further purification, 4 parts of ethanol were added to 1 part of PPIE water solution (5%) to get precipitated inulin (SPIE) by filtration (Ku et al. 2003). Li et al. Food Production, Processing and Nutrition (2020) 2:2 Page 3 of 11 Li et al. Food Production, Processing and Nutrition (2020) 2:2 Li et al. Food Production, Processing and Nutrition (2020) 2:2 Li et al. Food Production, Processing and Nutrition Antioxidant activity Conjugated dienes Aqueous dispersion of liposome model Liposome dis- persion was prepared by the film hydration method (Hosseinian et al. 2006). Specifically, 0.5% (v/v) lipid dis- persion was prepared by dissolving 20 μL linoleic acid and 30 μL soy lecithin into a vial containing 2 mL chloroform. A thin lipid film was formed at the bottom of the vial after chloroform evaporated in a fume hood overnight. Phos- phate buffer solution (10 mL of 0.05 mol/L, pH 7.4) con- taining 0.5% (v/v) Tween 20 was added into the vial (Liégeois et al. 2000) and agitated in ultrasonic bath (PS- 20, Qingdao, Shandong, China) for 15 min, mixed by a mini-vortexer until it became cloudy. Unilamellar lipo- some vesicles (ULVs) were produced by the extrusion method using Liposofast mini-extruder (Avestin, Inc., Ottawa, ON, CA) as explained by Hosseinian et al. (2006) and established in this lab. Oleogel Monoglycerides were dissolved in olive oil (5%, w/v) at 80 °C until totally melted, and then cooled at room temperature to form oleogel (23 °C). Inulin emulsion gel Antioxidant test Antioxidant activities were determined by the method of Liégeois et al. (2000) with modifica- tion. As for the liposome model, 50 μL of 0.5% ULVs dispersion and 50 μL of 5 mg/mL sample (either PPIE, CIE, CI, trolox or control) were added into a quartz cu- vette which contained 2.75 mL phosphate buffer solution (pH 7.4, 50 mmol/L). For the emulsion gel, 100 μL 4-fold diluted emulsion gel (containing either PPIE, CIE, CI or control) was added to the quartz cuvette and other con- ditions were the same. The oxidation reaction was initi- ated at ambient temperature by adding 150 μL of 40 mmol/L AAPH which acted as a free radical generator. Antioxidant activities were tested kinetically using a Cary 50 Bio UV-visible spectrophotometer (Varian Inc., Inulin emulsion gel The ingredients of emulsion gels included 57.0% olive oil, 3.0% monoglycerides, 0.8% Tween 80, 39% distilled water and 1% inulin samples (CI, PPIE, CIE). An equal amount of distilled water instead of inulin samples acted as a control. These formulations A (CI), B (PPIE), C (CIE) and D (control) of emulsion gels were designed and modified according to preliminary experimental re- sults. Specifically, monoglycerides were dissolved in oil completely at 80 °C, and then the water phase including inulin was added to the oil phase at the same temperature, followed by strong vortex (Mini-vortexer VM-3000, VWR, Radnor, USA) until a desired consistency was achieved. Emulsions were cooled to Li et al. Food Production, Processing and Nutrition (2020) 2:2 Page 4 of 11 Li et al. Food Production, Processing and Nutrition (2020) 2:2 Li et al. Food Production, Processing and Nutrition (2020) 2:2 Page 4 of 11 Li et al. Food Production, Processing and Nutrition Gen 5 software (BioTek, USA). Each sample was per- formed and analyzed in separate triplicates, and the av- erages were used as final results expressed as mg gallic acid equivalency per gram sample (mg GAE/g sample) (Gunenc et al. 2015). Gen 5 software (BioTek, USA). Each sample was per- formed and analyzed in separate triplicates, and the av- erages were used as final results expressed as mg gallic acid equivalency per gram sample (mg GAE/g sample) (Gunenc et al. 2015). Australia). In the liposome model without any antioxi- dant, lipid oxidation was carried out in the presence of the same amount of phosphate buffer solution (PBS, blank control). Statistics analysis One-way ANCOVA (analysis of covariance) followed by post-hoc Tukey’s test was used to find significant differences (α = 0.05). Results were significantly different when P < 0.05. Inulin emulsion gel Absorbances of samples themselves and AAPH in buffer at 234 nm were all subtracted (Liégeois et al. 2000). Each test was performed in triplicate and the averages were used as final results. TPC assay Crude inulin extract (CIE), primary purified inulin ex- tract (PPIE) and commercial inulin (CI) were dissolved in distilled water separately and formed 5 mg/ml sample solutions. Fifty microliters of standard (gallic acid), sam- ple solution or blank (distilled water) was added into a 1.5 mL Eppendorf tube, followed by 475 μL of 10-time diluted Folin & Ciocalteu phenol solution, and mixed thoroughly. Then 475 μL of 6% sodium carbonate solu- tion was added and mixed again. All these mixtures were incubated in darkness at ambient temperature for 2 h and measured at 725 nm using a microplate reader with Rancimat Accelerated oxidative stability of emulsion gels (CIE, PPIE, CI) was tested by Rancimat (Metrohm). Stability parameters were set at 121 °C and 20 mL/h airflow rate. This test was performed in duplicate. Results and discussion HPLC-MS analysis HPLC-IR chromatograms of the samples (PPIE: primary purified inulin extract, SPIE: secondary purified inulin Fig. 2 HPLC chromatograms and mass spectra of inulin. a 0.25 mg/mL commercial inulin (CI) from chicory root; b 1 mg/mL primary purified inulin extract (PPIE) from Jerusalem artichoke (JA); c 1 mg/ml secondary purified inulin extract (SPIE) from JA; and d Partial mass spectra of PPIE Fig. 2 HPLC chromatograms and mass spectra of inulin. a 0.25 mg/mL commercial inulin (CI) from chicory root; b 1 mg/mL primary purified inulin extract (PPIE) from Jerusalem artichoke (JA); c 1 mg/ml secondary purified inulin extract (SPIE) from JA; and d Partial mass spectra of PPIE ig. 2 HPLC chromatograms and mass spectra of inulin. a 0.25 mg/mL commercial inulin (CI) from chicory root; b 1 mg/mL nulin extract (PPIE) from Jerusalem artichoke (JA); c 1 mg/ml secondary purified inulin extract (SPIE) from JA; and d Partial m Page 5 of 11 Li et al. Food Production, Processing and Nutrition (2020) 2:2 Li et al. Food Production, Processing and Nutrition (2020) 2:2 Li et al. Food Production, Processing and Nutrition (2020) 2:2 extract) and control (CI: commercial inulin) were shown in Fig. 2. The chromatogram of crude inulin extract (CIE) was not shown because CIE contains many impur- ities. Figure 2a showed only one peak at 4.4 min, which could be inulin with several DPs (degree of polymeriza- tions) that had close retention time. DP of inulin varies from 2 to 60, depending on plant resources, harvest time, storage and process conditions (Saengthongpinit & Sajjaanantakul 2005). It was reported that commercial chicory inulin (from Sigma-Aldrich the same as CI used in this study) had an average DP between 13 and 30 ana- lyzed by HPAEC-PAD (Böhm et al. 2005). and glucose came out at last (Beirão-da-costa et al. 2005). Other peaks in Fig. 2b were possibly inulin with higher DPs. In Fig. 2d, the MS analysis confirmed PPIE contained DP 3 (503 Da) and DP 4 (665 Da). In general, PPIE had wider distributed peaks than CI. Interestingly, the chromatogram of SPIE (Fig. 2c) had almost the same peak with CI (Fig. 2a). This confirmed that addition of ethanol to PPIE solution precipitated inulin with higher DP, which agreed with the literature (Temkov et al. 2015). Although SPIE had higher purity than PPIE, its yield (7.85%) was negligible. Results and discussion HPLC-MS analysis Therefore, PPIE was chosen to prepare emulsion gel instead of SPIE. The chromatogram of PPIE sample (Fig. 2b) mainly showed five peaks (a, b, c, d and e); peak a to d (higher DP inulin, DP > 2) constituted 80.28%, and peak e (su- crose) took up 17.92% according to their peak areas. From all those peaks, the peak “b” that accounted for 45.48% had the same retention time as the one in CI (Fig. 2a). Similarly, (Beirão-da-costa et al. 2005) analyzed inulin with the same detector and column (HPLC-RI with Sugar pak I), and found that higher DP came out from the column earlier than lower DP, and fructose Inulin emulsion gel Figure 3 shows powders, gels of CI, PPIE, CIE, MGs, and emulsion gels (A, B, C, D) after heating-cooling process. Inulin gel and oleogel Gel can be defined as an intermediate (semi-solid) ma- terial between liquid and solid (Nishinari 2009). Inulin solution formed a gel slowly as temperature decreased, Fig. 3 The powders, gels and emulsion gels formed by inulin and/or MGs. The first row from the top shows the pictures of CI (commercial inulin from chicory root), PPIE (primary purified inulin extract from JA), CIE (crude inulin extract from JA) and MGs (monoglycerides) powders; The middle row shows the pictures of gels formed by corresponding powders solution through the heating-cooling process. The bottom row shows the pictures of emulsion gels; a contains CI; b contains PPIE; c contains CIE; d is control (no inulin). Black arrow shows separations in emulsion gel a, c and d Fig. 3 The powders, gels and emulsion gels formed by inulin and/or MGs. The first row from the top shows the pictures of CI (commercial inulin from chicory root), PPIE (primary purified inulin extract from JA), CIE (crude inulin extract from JA) and MGs (monoglycerides) powders; The middle row shows the pictures of gels formed by corresponding powders solution through the heating-cooling process. The bottom row shows the pictures of emulsion gels; a contains CI; b contains PPIE; c contains CIE; d is control (no inulin). Black arrow shows separations in emulsion gel a, c and d Li et al. Food Production, Processing and Nutrition (2020) 2:2 Li et al. Food Production, Processing and Nutrition (2020) 2:2 Li et al. Food Production, Processing and Nutrition Page 6 of 11 Page 6 of 11 Inulin emulsion gel displaying a typical sol-gel transition (Kim et al. 2001). In the middle row of Fig. 3, PPIE and CIE gels were dar- ker than CI, possibly due to remaining inherent color or products of enzymatic browning reactions in JA tuber (Tchoné et al. 2005). As mentioned earlier, the degree of gel formation was expressed as volumetric gel index (VGI). The VGI of CI gel (26%) was higher than PPIE gel (22%) and CIE gel (18%), which was likely due to different DPs and inulin content. Monoglycerides structured oil and formed gel-like material (oleogel). In this process, oil transferred into a thermo-reversible three-dimensional gel net- work in the presence of monoglycerides (Mert & Demirkesen 2016). Emulsion gel can be regarded as a gelled emulsion. Emulsion gel with PPIE (B) had better appearance than the other three. Emulsion gel with CI (A) had slight separation, whereas those with CIE (C) and control (D) presented apparent sepa- rations. PPIE improved the homogeneity of emulsion gel with no syneresis, which might be due to its wider DP distri- bution (inulin peaks a-d in HPLC). Microstructure PLM Inulin gel and oleogel Microscopic images of inulin (CI, PPIE and CIE) gels and oleogel were shown in Fig. 4. CI Inulin gel and oleogel Microscopic images of inulin (CI, PPIE and CIE) gels and oleogel were shown in Fig. 4. CI Fig. 4 Polarized light microscope images of gels and emulsion gels. The first two rows from the top indicate inulin gels and/or oleogel; the third row shows emulsion gel with 1% PPIE and without PPIE (control). CI: commercial inulin from chicory root; PPIE: primary purified inulin extract from JA; CIE: crude inulin extract from JA; MGs: monoglycerides; oleogel is formed by MGs; magnification: 200x; scale bar =100 μm Fig. 4 Polarized light microscope images of gels and emulsion gels. The first two rows from the top indicate inulin gels and/or oleogel; the third row shows emulsion gel with 1% PPIE and without PPIE (control). CI: commercial inulin from chicory root; PPIE: primary purified inulin extract from JA; CIE: crude inulin extract from JA; MGs: monoglycerides; oleogel is formed by MGs; magnification: 200x; scale bar =100 μm Fig. 4 Polarized light microscope images of gels and emulsion gels. The first two rows from the top indicate inulin gels and/or oleogel; the third row shows emulsion gel with 1% PPIE and without PPIE (control). CI: commercial inulin from chicory root; PPIE: primary purified inulin extract from JA; CIE: crude inulin extract from JA; MGs: monoglycerides; oleogel is formed by MGs; magnification: 200x; scale bar =100 μm Page 7 of 11 Li et al. Food Production, Processing and Nutrition (2020) 2:2 Page 7 of 11 Li et al. Food Production, Processing and Nutrition (2020) 2:2 gel had homogeneous crystals with approximate 10 μm particle size which was two times larger than the small crystals (5 μm) in PPIE gel. Besides small crystals, PPIE gel also consisted of large bulky crystals with 20~200 μm par- ticle size. It was possibly due to inulin (PPIE) with differ- ent chain lengths that dispersed and packed efficiently in spatial arrangement and connected to become large crys- tals. Interestingly, CIE gel had large pieces of crystals simi- lar to that in oleogel. It was likely attributed to the connection of inulin with some other compounds, such as peptides remaining in CIE (Rubel et al. 2018). pressure-temperature phase diagram of water. Liposome model Different inulin samples with the same concentration Antioxidant properties of CIE, PPIE and CI were eval- uated by measuring their capacity to inhibit or delay linoleic acid oxidation, which was detected kinetically by increased absorbance of products (conjugated dienes hy- droperoxides) at 234 nm (Vieira & Regitano-D’arce 1998), and results were presented in Fig. 6a. Emulsion gel Microstructures of emulsion gel with 1% PPIE and control (without 1% PPIE) were shown in the third row of Fig. 4. Full and round droplets in emulsion gel with PPIE were smaller (average size 40 μm) and more homogeneous than the droplets that partially crumpled in control (average size 60 μm). This phenomenon indicated that addition of 1% PPIE could prevent emulsion from syneresis and made it more homogenous, which also cor- responded with the observation on a macro level: emul- sion gel with PPIE had better appearance. It could be due to the excellent water-binding ability of inulin (Fadaei et al. 2012). CIE had the highest antioxidant activity amongst all inu- lin samples and was similar to trolox as there was no sig- nificant difference (P < 0.05). CIE started to differentiate from the control at ~ 50 min, and its absorbance was lower than control throughout the tested time. Whereas PPIE had a similar but weaker antioxidant activity com- pared with CIE as statistical analysis showed no significant differences, but PPIE had significantly lower activity than trolox (Fig. 6a). This result was parallel to total phenolic content (TPC) results: CIE had approximately 7 times as many TPC values as PPIE. Antioxidant activity of CIE and PPIE might be from remaining phenolic compounds (Tchoné et al. 2005) or peptides (Bhagia et al. 2018). Be- sides phenolics that remained in CIE and PPIE, another possible mechanism could be due to the interactions of CIE with other compounds, acting as physical barriers that encapsulated linoleic acid (de Barros Fernandes et al. 2016). Hincha et al. (2000) revealed inulin had a capacity of stabilizing liposome in combination with glucose (Hincha et al. 2000). Microstructure PLM In this case, it was assumed that the remaining matrix was mainly formed by inulin, monoglyceride, Tween 80, as well as partial fat and ice. Incorporation of PPIE inhibited ice from forming large crystals, which could have potential applications on ice cream (Aleong et al. 2008). Cryo-SEM The morphological characteristics of emulsion gels with 1% PPIE and control (without 1% PPIE) were also ob- served by cryo-SEM and shown in Fig. 5. Emulsion gel in- corporating PPIE displayed a porous network with smaller pore sizes, which probably contributed to its creamy and smooth texture, whereas the control exhibited an uneven surface with large pore sizes. When emulsion gels were observed by cryo-SEM, temperature and pressure were − 65 °C and 18~30 Pa, respectively. Water underwent par- tially frozen and sublimated according to the typical Fig. 5 Cryo-scanning electron microscope images of emulsion gels containing 1% PPIE and control (without PPIE); magnification: 1kx; scale bar = 20 μm; PPIE: primary purified inulin extract from JA Fig. 5 Cryo-scanning electron microscope images of emulsion gels containing 1% PPIE and control (without PPIE); magnification: 1kx; scale bar = 20 μm; PPIE: primary purified inulin extract from JA Li et al. Food Production, Processing and Nutrition (2020) 2:2 Page 8 of 11 Li et al. Food Production, Processing and Nutrition (2020) 2:2 Page 8 of 11 Fig. 6 Conjugated dienes formation from lipid oxidation in aqueous system influenced by adding inulin. a CIE (crude inulin extract from JA), PPIE (primary purified inulin extract from JA) and CI (commercial inulin from chicory root) solutions with the same concentration; b Different concentrations of CIE; c Different concentrations of PPIE. Each data point represents mean (n = 3). One-way ANCOVA followed by post-hoc Tukey’s test is used to find significant differences (α = 0.05). Results are different when P < 0.05 Fig. 6 Conjugated dienes formation from lipid oxidation in aqueous system influenced by adding inulin. a CIE (crude inulin extract from JA), PPIE Fig. 6 Conjugated dienes formation from lipid oxidation in aqueous system influenced by adding inulin. a CIE (crude inulin extract from JA), PPIE (primary purified inulin extract from JA) and CI (commercial inulin from chicory root) solutions with the same concentration; b Different concentrations of CIE; c Different concentrations of PPIE. Each data point represents mean (n = 3). One-way ANCOVA followed by post-hoc Tukey’s test is used to find significant differences (α = 0.05). Results are different when P < 0.05 Fig. 6 Conjugated dienes formation from lipid oxidation in aqueous system influenced by adding inulin. CIE and PPIE-different concentrations Rancimat Accelerated oxidative stability of emulsion gels (con- taining CI, PPIE, or CIE) were tested by Rancimat, and results were displayed in Fig. 8. Emulsion gel containing CI had longer induction time (14.83 ± 0.69 h) than that containing PPIE (5.19 ± 0.64 h) or CIE (2.92 ± 0.21 h) (P < 0.05). It is probably because of complex components (Bhagia et al. 2018) in PPIE and CIE. Some impurities inside were possibly oxidized very fast under the accelerated oxidation condition, which produced more volatile compounds and in- creased conductivity. Therefore, emulsion gel contain- ing inulin with higher purity had longer induction time. Rancimat result was in agreement with the volumetric gel index result. g p Antioxidant assessment of emulsion gels (PPIE, CIE, CI and control) were performed by the conjugated dienes method (the same method as liposome model), and the re- sult was presented in Fig. 7. In order to eliminate spectral interference by emulsion gel itself, absorbances at 22 h were subtracted since emulsion gels had the lowest absor- bances at that point. For all samples, absorbances began to increase at 22 h, indicating that oxidative reactions started at this time and more conjugated dienes hydroxy- peroxides accumulated. Compared with Fig. 6a in which oxidation started at ~ 50 min, emulsion gels delay lipid oxidation much longer (oxidation started at ~ 22 h) than the liposome model. In addition, emulsion gel with PPIE had lower absorbance than control after 94 h, meaning that it slowed down the oxidation at that time. It was pos- sibly related to the increased stability and homogeneity of emulsion gel with PPIE (Fig. 3). Cryo-SEM The CIE sample had a higher TPC value (4.96 ± 0.01) than PPIE (0.72 ± 0.03), and commercial inulin had no TPC data to present, which was parallel to the results of antioxidant activity. Our result was close to the study of Petkova et al. (2014) in which TPC value was between 4 and 10 mg GAE/g dry weight in JA tuber water extraction (Petkova et al. 2014) but lower than the work of Yuan et al. (2012) in which TPC value was 22.40 ± 0.63 mg GAE/g dry weight (Yuan et al. 2012). 20 mg/mL) were run in the same assay, and results were shown in Fig. 6c. PPIE with 5 mg/mL showed antioxidant activity, and it was the only treatment concentration that was significantly (P < 0.05) different than control. Emulsion gel model The above measurements were based on the liposome model in which linoleic acid was dispersed in aqueous buffer solution stabilized by soy lecithin, and inulin samples were dissolved in buffer so- lution and added to the reaction system separately. In the following experiment, emulsion gel was used in which oil and inulin samples were incorporated, and the whole emulsion gel was dispersed in buffer solution. Conclusion This work examined the formulation and lipid oxidative stability of emulsion filled gels developed by incorporat- ing Jerusalem artichoke inulin. HPLC results showed that the chromatogram of primary purified inulin extract (PPIE) had wider distributed peaks than commercial inu- lin (CI) and secondary purified inulin extract (SPIE). Physical investigation showed that addition of 1% PPIE Cryo-SEM a CIE (crude inulin extract from JA), PPIE (primary purified inulin extract from JA) and CI (commercial inulin from chicory root) solutions with the same concentration; b Different concentrations of CIE; c Different concentrations of PPIE. Each data point represents mean (n = 3). One-way ANCOVA followed by post-hoc Tukey’s test is used to find significant differences (α = 0.05). Results are different when P < 0.05 It is well-known that activity is generally dose- dependent (Karadag et al. 2009). To investigate the effect of concentration, CIE samples with five different con- centrations (in between 2.5–20 mg/mL) were tested in the same assay, and results were shown in Fig. 6b. All tested CIE were significantly different from blank con- trol, but similar with trolox (Fig. 6b). It indicated that CIE had antioxidant activity in the tested concentration range. Trolox curve was almost stable and increased slightly during 120 min run. For blank control (PBS), it increased dramatically after 50 min, indicating that lots of conjugated dienes were produced. Whereas this curve kept stable dur- ing the first 50 min, which was likely because linoleic acid was encapsulated with lecithin (liposome model) and pro- tected from oxidation at the beginning of reaction. In summary, CIE had stronger antioxidant activity than other tested inulin samples, and this result was in agreement with the TPC results. Similarly, different concentrations of PPIE were also in- vestigated. Five different concentrations (in between 2.5– CIE and PPIE-different concentrations CIE and PPIE-different concentrations Page 9 of 11 Li et al. Food Production, Processing and Nutrition (2020) 2:2 Page 9 of 11 Li et al. Food Production, Processing and Nutrition (2020) 2:2 (2020) 2:2 Li et al. Food Production, Processing and Nutrition (2020) 2:2 Li et al. Food Production, Processing and Nutrition GAE/g sample). The CIE sample had a higher TPC value (4.96 ± 0.01) than PPIE (0.72 ± 0.03), and commercial inulin had no TPC data to present, which was parallel to the results of antioxidant activity. Our result was close to the study of Petkova et al. (2014) in which TPC value was between 4 and 10 mg GAE/g dry weight in JA tuber water extraction (Petkova et al. 2014) but lower than the work of Yuan et al. (2012) in which TPC value was 22.40 ± 0.63 mg GAE/g dry weight (Yuan et al. 2012). GAE/g sample). References Aleong, J. M., Frochot, S., & Goff, H. D. (2008). Ice recrystallization inhibition in ice cream by propylene glycol monostearate. Journal of Food Science, 73(9), E463–E468. https://doi.org/10.1111/j.1750-3841.2008.00954.x. Fig. 8 Induction times of emulsion gels containing CI, PPIE, or CIE tested by Rancimat. PPIE: primary purified inulin extract from JA; CIE: crude inulin extract from JA; CI: commercial inulin from chicory root. Results represent mean (n = 2), and standard deviation value is used as error bar. Values with different superscript letters are significantly different (p < 0.05) Bazina, N., & He, J. (2018). Analysis of fatty acid profiles of free fatty acids generated in deep-frying process. Journal of Food Science and Technology, 55(8), 3085–3092. https://doi.org/10.1007/s13197-018-3232-9. Beirão-da-costa, M. L., Januario, M. I. N., Simao, F. M. S., & Leitao, A. E. B. (2005). Characterisation of inulin from chicory and salsify cultivated in Portugal. Alimentos E Nutrição Araraquara, 16(3), 221–225. Bhagia, S., Ferreira, J. F. S., Kothari, N., Nunez, A., Liu, X., da Silva Dias, N., et al. (2018). Sugar yield and composition of tubers from Jerusalem Artichoke (Helianthus tuberosus) irrigated with saline waters. Biotechnology and Bioengineering, 115(6), 1475–1484. https://doi.org/10.1002/bit.26582. improved the appearance and stability of emulsion gel, which characterized by smaller droplets size (average 40 μm) than control (average size 60 μm). Rancimat test indicated that emulsion gel with high purity inulin (CI) had longer induction time than those with PPIE and CIE, which was in agreement with volumetric gel index results. Most importantly, CIE had antioxidant ability against linoleic acid oxidation using liposome model by delaying or inhibiting the production of conjugated di- enes with a tested range of 2.5–20 mg/mL (final concen- tration 0.04–0.33 mg/mL). It might be attributed to remaining phenolics as the TPC assay showed that CIE had high total phenolic content. In the future study, ap- plications and characterization of emulsion gel with JA inulin (PPIE) used in food system as a potential fat re- placement need to be investigated. Böhm, A., Kaiser, I., Trebstein, A., & Henle, T. (2005). Heat-induced degradation of inulin. European Food Research and Technology. https://doi.org/10.1007/ s00217-004-1098-8. Bolouri-Moghaddam, M. R., Le Roy, K., Xiang, L., Rolland, F., & Van Den Ende, W. (2010). Sugar signalling and antioxidant network connections in plant cells. FEBS Journal, 277(9), 2022–2037. https://doi.org/10.1111/j.1742-4658.2010. 07633.x. Dave, P. (2012). Rheological properties of low-fat processed cheese spread made with inulin as a fat replacer. Authors’ contributions d h d Hosseinian, F. S., Muir, A. D., Westcott, N. D., & Krol, E. S. (2006). Antioxidant capacity of flaxseed lignans in two model systems. Journal of the American Oil Chemists’ Society, 83(10), 835–840. FL carried out this study and drafted the manuscript. AG participated in modification and discussion of the manuscript. FH designed the study and modified the manuscript. All authors read and approved the final manuscript. Huang, G., Chen, Y., & Wang, X. (2011). Extraction and deproteinization of pumpkin polysaccharide. International Journal of Food Sciences and Nutrition, 62(6), 568–571. https://doi.org/10.3109/09637486.2011.560566. Acknowledgements Hincha, D. K., Hellwege, E. M., Heyer, A. G., & Crowe, J. H. (2000). Plant fructans stabilize phosphatidylcholine liposomes during freeze- drying. European Journal of Biochemistry, 267(2), 535–540. https://doi.org/10.1046/j.1432-1327. 2000.01028.x. Thanks for funding by Natural Science and Engineering Research Council of Canada [grant number 315080]. The authors would like to thank Dr. Jianqun Wang for his help with cryo-SEM. Hongxin, W. (2008). Study on the decoloration of chicory inulin by active carbon method. Journal of Anhui Agricultural Sciences, 36(7), 2626–2628. Availability of data and materials All data supporting this study are included in this article. Further details are available from the corresponding author on reasonable request. References (Doctoral dissertation, University of Wisconsin-- Stout), Menomonie, WI 54751, USA). de Barros Fernandes, R. V., Botrel, D. A., Silva, E. K., Borges, S. V., de Oliveira, C. R., Yoshida, M. I., et al. (2016). Cashew gum and inulin: new alternative for ginger essential oil microencapsulation. Carbohydrate Polymers, 153, 133–142. https://doi.org/10.1016/J.CARBPOL.2016.07.096. Fadaei, V., Poursharif, K., Daneshi, M., & Honarvar, M. (2012). Chemical characteristics of low-fat wheyless cream cheese containing inulin as fat replacer. European Journal of Experimental Biology, 2(3), 690–694. Farhoosh, R., Niazmand, R., Rezaei, M., & Sarabi, M. (2008). Kinetic parameter determination of vegetable oil oxidation under Rancimat test conditions. European Journal of Lipid Science and Technology, 110, 587–592. Gunenc, A., HadiNezhad, M., Farah, I., Hashem, A., & Hosseinian, F. (2015). Impact of supercritical CO2and traditional solvent extraction systems on the extractability of alkylresorcinols, phenolic profile and their antioxidant activity in wheat bran. Journal of Functional Foods, 12, 109–119. https://doi.org/10. 1016/j.jff.2014.10.024. Abbreviations CI C i l i CI: Commercial inulin from chicory root; CIE: Crude inulin extract; DP: Degree of polymerization; GAE: Gallic acid equivalency; HPLC: High performance liquid chromatography; JA: Jerusalem artichoke; MGs: Monoglycerides; MS: Mass spectrometry; PBS: Phosphate buffer solution; PLM: Polarized light microscope; PPIE: Primary purified inulin extract; RI: Refractive index; SEM: Scanning electron microscope; SPIE: Secondary purified inulin extract; TPC: Total phenolic content; VGI: Volumetric gel index; W/O: Water in oil Hamilton, J., Knox, B., Hill, D., & Parr, H. (2000). Reduced fat products – Consumer perceptions and preferences. British Food Journal, 102(7), 494–506. https://doi. org/10.1108/00070700010336454. Hartel, R. d. W., von Elbe, J. H., & Hofberger, R. (2018). Fats, oils and emulsifiers. In Confectionery science and technology (pp. 85–124). Cham: Springer. https:// doi.org/10.1007/978-3-319-61742-8_4. Competing interests Th h d l h The authors declare that they have no competing interests. Received: 12 July 2019 Accepted: 29 December 2019 TPC TPC values of PPIE and CIE were expressed as milli- gram gallic acid equivalency per gram of sample (mg Fig. 7 Conjugated dienes formation from lipid oxidation in emulsion gels influenced by adding inulin. Emulsion gel containing PPIE (primary purified inulin extract from JA); CI (commercial inulin from chicory root); CIE (crude inulin extract from JA) and control (without inulin). Data points represent means (n = 3). One-way ANCOVA followed by post-hoc Tukey’s test is used to find significant differences (α = 0.05). Results are different when P < 0.05 Fig. 7 Conjugated dienes formation from lipid oxidation in emulsion gels influenced by adding inulin. Emulsion gel containing PPIE (primary purified inulin extract from JA); CI (commercial inulin from chicory root); CIE (crude inulin extract from JA) and control (without inulin). Data points represent means (n = 3). One-way ANCOVA followed by post-hoc Tukey’s test is used to find significant differences (α = 0.05). Results are different when P < 0.05 Page 10 of 11 Page 10 of 11 Li et al. Food Production, Processing and Nutrition (2020) 2:2 Fig. 8 Induction times of emulsion gels containing CI, PPIE, or CIE tested by Rancimat. PPIE: primary purified inulin extract from JA; CIE: crude inulin extract from JA; CI: commercial inulin from chicory root. Results represent mean (n = 2), and standard deviation value is used as error bar. Values with different superscript letters are significantly different (p < 0.05) Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Mert, B., & Demirkesen, I. (2016). Reducing saturated fat with oleogel / shortening blends in a baked product. Food Chemistry, 199, 809–816. https://doi.org/10. 1016/j.foodchem.2015.12.087. Mozaffarian, D., Katan, M. B., Ascherio, A., Stampfer, M. J., & Willett, W. C. (2006). Trans fatty acids and cardiovascular disease. The New England Journal of Medicine, 354(15), 1601–1613. Nishinari, K. (2009). Some thoughts on the definition of a gel. In Gels: Structures, properties, and functions (Vol. 136, pp. 87–94). Berlin: Springer. Oh, I. K., & Lee, S. (2018). Utilization of foam structured hydroxypropyl methylcellulose for oleogels and their application as a solid fat replacer in muffins. Food Hydrocolloids, 77, 796–802. https://doi.org/10.1016/J.FOODHYD. 2017.11.022. Ojijo, N. K. O., Neeman, I., Eger, S., & Shimoni, E. (2004). Effects of monoglyceride content, cooling rate and shear on the rheological properties of olive oil/ monoglyceride gel networks. Journal of the Science of Food and Agriculture, 84(12), 1585–1593. https://doi.org/10.1002/jsfa.1831. Petkova, N., Ivanov, I., Denev, P., & Pavlov, A. (2014). Bioactive substance and free radical scavenging activities of flour from Jerusalem artichoke (Helianthus tuberosus L.) tubers–a comparative study. Turkish Journal of Agricultural and Natural Sciences, 1(2), 1773-1778. Pintado, T., Herrero, A. M., Jiménez-Colmenero, F., Cavalheiro, C. P., & Ruiz-Capillas, C. (2018). Chia and oat emulsion gels as new animal fat replacers and healthy bioactive sources in fresh sausage formulation. Meat Science, 135, 6– 13. https://doi.org/10.1016/J.MEATSCI.2017.08.004. Rios, R. V., Garzón, R., Lannes, S. C. S., & Rosell, C. M. (2018). Use of succinyl chitosan as fat replacer on cake formulations. LWT-Food Science and Technology, 96, 260–265. https://doi.org/10.1016/J.LWT.2018.05.041. Rubel, I. A., Iraporda, C., Novosad, R., Cabrera, F. A., Genovese, D. B., & Manrique, G. D. (2018). Inulin rich carbohydrates extraction from Jerusalem artichoke (Helianthus tuberosus L.) tubers and application of different drying methods. Food Research International, 103, 226–233. https://doi.org/10.1016/J.FOODRES. 2017.10.041. Saengthongpinit, W., & Sajjaanantakul, T. (2005). Influence of harvest time and storage temperature on characteristics of inulin from Jerusalem artichoke (Helianthus tuberosus L.) tubers. Postharvest Biology and Technology, 37(1), 93– 100. https://doi.org/10.1016/j.postharvbio.2005.03.004. Shahidi, F., & Zhong, Y. (2005). Lipid oxidation: Measurement methods. 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Reflections on graduate professor training
Escola Anna Nery
2,019
cc-by
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Resumo Objetivo: Refletir acerca de atividades voltadas para o desenvolvimento de pós-graduandos, em programa de estágio docente, na perspectiva de ensino e formação na pós-graduação Stricto sensu. Método: Trata-se de estudo reflexivo, fundamentado na formulação discursiva acerca de competências requeridas e diretrizes propostas pelo Plano Nacional de Pós-Graduação para a capacitação docente articulada à pós-graduação. Resultados: O programa de estágio docente é uma via para a formação de professores universitários por meio do preparo do pós-graduando. Além disso, mostra a relevância do papel docente supervisor na construção da aprendizagem desses indivíduos. Conclusão e implicações para a prática: O primeiro contato com a prática da docência, por meio do programa de estágio docente, mostra-se relevante para a construção do olhar com criticidade do processo de ensino-aprendizagem. Nesse tipo de programa, é possível aprender a ser e a fazer a docência de forma coparticipativa, valorizar as relações interativas, impulsionar a autonomia, estimular a autoavaliação e autorreflexão do pós-graduando, permitindo que participe, ativamente, do processo de aprendizagem. Palavras-chave: Prática Profissional; Docentes de Enfermagem; Educação de Pós-Graduação em Enfermagem. Reflections on graduate professor training Reflexões sobre a formação docente na pós-graduação Reflexiones sobre la formación docente en el postgrado Larissa Roberta Alves1 Márcia Aparecida Giacomini1 Verônica Modolo Teixeira1 Silvia Helena Henriques1 Lucieli Dias Pedreschi Chaves1 Abstract Larissa Roberta Alves1 Márcia Aparecida Giacomini1 Verônica Modolo Teixeira1 Silvia Helena Henriques1 Lucieli Dias Pedreschi Chaves1 1. Universidade de São Paulo. Ribeirão Preto, São Paulo, Brasil. Objective: To reflect on activities aimed at the development of graduation students, in a teaching internship program, in the perspective of master’s degree and doctoral graduate education and training. Method: It is a reflexive study, based on the discursive formulation about required competencies and guidelines proposed by the National Graduation Plan (named PNPG, Plano Nacional de Pós-Graduação) for professor training articulated to the graduate. Results: The teaching internship program is a way for the formation of university professors, through the preparation of the graduate student. In addition, it shows the relevance of the professor supervisor role in the construction of the learning of these individuals. Conclusion and implications for practice: The first contact with teaching practice, through the teaching internship program, is relevant for constructing a critical look at the teaching-learning process. In this type of Program, it is possible to learn to be and make teaching in a participatory manner, to value interactive relations, to boost autonomy, to stimulate self-evaluation and self-reflection of the graduate student, allowing them to participate actively in the learning process. 1. Universidade de São Paulo. Ribeirão Preto, São Paulo, Brasil. 1. Universidade de São Paulo. Ribeirão Preto, São Paulo, Brasil. Keywords: Professional Practice; Faculty, Nursing; Education, Nursing, Graduate. REFLECTION | REFLEXÃO Esc Anna Nery 2019;23(3):e20180366 REFLECTION | REFLEXÃO Esc Anna Nery 2019;23(3):e20180366 Esc Anna Nery 2019;23(3):e20180366 Corresponding author: Márcia Aparecida Giacomini. E-mail: marcia.giacomini77@gmail.com Submitted on 12/06/2018. Accepted on 05/02/2019. DOI: 10.1590/2177-9465-EAN-2018-0366 Larissa Roberta Alves1 Márcia Aparecida Giacomini1 Verônica Modolo Teixeira1 Silvia Helena Henriques1 Lucieli Dias Pedreschi Chaves1 Contextualizing the theme It is noteworthy that the approach on specific competences for the development of scientific research, in different areas, includes themes that necessarily do not permeate more specific aspects of the competencies required for the practice of Teaching, i.e. master’s degree and doctoral graduate courses can form excellent researchers, without having their training and experience of professor training. g The impasse of universities in their attributions, whether in the sense of training for research or pedagogical training for teaching, and also the training of professionals to respond to the demands of the labor market, is not a fact present only in Brazilian scenario. In the USA and Canada, there is already a segmentation of the universities and graduates activities, some focusing on the production of scientific research and others whose scope includes the training of professionals to serve the labor market. This fragmentation is considered to be harmful, since the pedagogical competences for teaching and for research are essential for the exercise of university teaching. 4 In this sense, it is understood the importance of the interface between training in a specific area and the particularity of profes- sor education, which requires specific references and, because it is a professor's education, it is believed to be pertinent the concept of competence, based in Perrenoud, which is pointed out as an effective way to cope with analogous situations in order to articulate consciousness and cognitive resources with knowledge, capacities, attitudes, information and values, all of this in a fast, creative and connected way.5 The impasse of universities in their attributions, whether in the sense of training for research or pedagogical training for teaching, and also the training of professionals to respond to the demands of the labor market, is not a fact present only in Brazilian scenario. In the USA and Canada, there is already a segmentation of the universities and graduates activities, some focusing on the production of scientific research and others whose scope includes the training of professionals to serve the labor market. This fragmentation is considered to be harmful, since the pedagogical competences for teaching and for research are essential for the exercise of university teaching. Contextualizing the theme College Degree Education in Brazil has undergone an accelerated expansion in the last two decades, with an important increase in access, which allowed the continuity of the process of training a large contingent of students from high school. This growth had the incentive of programs for the promotion of College Degree Education, such as the Fund for College Degree Education Students (named FIES, Fundo de Financiamento ao Estudante do Ensino Superior) and the University for All Program (named PROUNI, Programa Universidade para Todos).1 With regard to the graduate course, there was an increase in the offer of master’s degree and doctoral courses derived, in part, from governmental stimuli through scholarships and, on the other hand, the offer requirements of these courses for the Institution of College Degree Education (named IES, Instituição de Ensino Superior) to reach the status of university. In addition, there was also a significant increase in society's demand for a higher level of schooling, understood as a differential for the labor market.2 Given these questions, we need to report to the National Graduation Plan (named PNPG, Plano Nacional de Pós- graduação), which establishes guidelines, strategies and targets for directing actions in this area, among which is the Institutional Professor Training Program (named PIQD, Programa Institucional de Qualificação Docente) for the qualification of the teaching staff in attention to the needs of improvement and expansion of teaching, research and extension activities of Institution of College Degree Education (named IES, Instituições de Ensino Superior).7 Master’s degree and doctoral graduate courses, in addition to the main objective of scientific research development, constitute a pathway for the formation of university professors, meeting the demand for training of these professionals due Expansion of college degree education.3i However, this approach to professor training, coupled with graduation, is not recent in Brazil and in the world, since several teaching institutions adopted different strategies, namely: the insertion of the graduate student in scenarios of university teaching practice in class room, laboratories and/or practical work field. As well as participation in graduate courses with content focused on university and/or college degree education topics, core activities involving the preparation of didactic material, among other strategies, which are succeed by means of programs for the further training of the IES. Resumen Objetivo: Reflexionar sobre las actividades dirigidas al desarrollo de estudiantes de post-graduación en un programa de prácticas de enseñanza, en la perspectiva de stricto sensu graduado de educación y formación. Método: Se trata de un estudio reflexivo, fundamentado en la formulación discursiva acerca de competencias requeridas y directrices propuestas por el Plan Nacional de Postgrado para capacitación docente articulada al posgrado. Resultados: El programa de práctica docente es una vía para la formación de profesores universitarios, a través de la preparación del estudiante de postgrado. Además, muestra la relevancia del papel docente supervisor en la construcción del aprendizaje de estos individuos. Conclusión e implicaciones para la práctica: El primer contacto con la práctica de la docencia, a través del programa de práctica docente, se muestra relevante para la construcción de una mirada con criticidad del proceso enseñanza-aprendizaje. En este tipo de programa es posible aprender a ser y hacer la docencia de forma coparticipativa, valorar relaciones interactivas, impulsar la autonomía, estimular la autoevaluación y autorreflexión del postgrado permitiendo que participen activamente en el proceso de aprendizaje. Palabras clave: Práctica Profesional; Docente de Enfermería; Educación de Postgrado en Enfermería. Corresponding author: Márcia Aparecida Giacomini. E-mail: marcia.giacomini77@gmail.com Submitted on 12/06/2018. Accepted on 05/02/2019. DOI: 10.1590/2177-9465-EAN-2018-0366 Escola Anna Nery 23(3) 2019 1 1 Reflections on graduate professor training Alves LR, Giacomini MA, Teixeira VM, Henriques SH, Chaves LDP Reflections on graduate professor training INTRODUCTION In the teaching work, one can highlight competences related to the organization of learning situations; to the administration of learning progression; the use of differentiation devices; the involvement of students in their learning and work; focus on teamwork; the use of new technologies; to the confrontation of duties and to the ethical dilemmas of the professor training, the profession and the administration of the own continuous formation. The importance not only of scientific domain, but of pedagogical foundations necessary for good teaching practice, not distanced from practice and respect for ethical-moral commitment, is important in order to meet the needs of the various actors in the educational system.6 Contextualizing the theme 4 In the perspective of professor development, the Coordi- nation of Improvement of Higher Education Personnel (named CAPES, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) adopted, in 1999, a measure for the improvement of the graduate student focused on professor education, since this may be responsible for College Degree Education in the country, making the teaching internship compulsory, as part of the activi- ties of the masters and doctoral fellows under the supervision of a professor, with a maximum working load of four hours per week, leaving the scholars have the autonomy to organize the format of the said stage, which reinforces the need for their pedagogical training.8 In this sense, it is understood the importance of the interface between training in a specific area and the particularity of profes- sor education, which requires specific references and, because it is a professor's education, it is believed to be pertinent the concept of competence, based in Perrenoud, which is pointed out as an effective way to cope with analogous situations in order to articulate consciousness and cognitive resources with knowledge, capacities, attitudes, information and values, all of this in a fast, creative and connected way.5 Institutions such as the Universidade Federal do Rio de Ja- neiro (UFRJ), a Universidade Federal de Minas Gerais (UFMG) e a Pontifícia Universidade Católica de São Paulo (PUC-SP) adopt the teaching internship, a compulsory activity for doctor- ates with the financial assistance of CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), with the pos- sibility of dispensing from the internship those who prove to have developed activity in College Degree Education. Escola Anna Nery 23(3) 2019 2 Reflections on graduate professor training Alves LR, Giacomini MA, Teixeira VM, Henriques SH, Chaves LDP Reflections on graduate professor training Alves LR, Giacomini MA, Teixeira VM, Henriques SH, Chaves LDP It is noteworthy that, at the Universidade de São Paulo (USP), even before the standardization of the CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), the graduation Dean established the Teaching Improvement Program (named PAE, Programa de Aperfeiçoamento do Ensino) through the provision of monitoring scholarships for graduate students as part of their training. Contextualizing the theme In view of the standardization of the CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), the aforementioned program was regulated by the Ordinance GR 3588 with the objective of contributing to the improvement and training of graduate students enrolled in master's and doctorate levels, in didactic activities of undergraduate teaching, in order to enable a first approximation with political, theoretical and methodological aspects that permeate the teaching and learning actions to be coordinated by them in college degree education institutions. It is understood that, in order to cover the pedagogical and didactic aspects of student training in undergraduate courses, future professors must experience the two stages of the PAE (Programa de Aperfeiçoamento do Ensino) - Pedagogical Preparation and Supervised Internship - that are necessarily articulated and interdependent.9 Reflect on the teaching internship appears to be relevant as a possibility to understand the different impact of the perfor- mance of professors and doctorates. It is a challenge to equate the various approaches that are embedded in the training of graduate students, since the emphasis of the graduate in Bra- zil is on the training of researchers, considering the specifics and competences inherent in the training for the production of scientific research. However, due to a contingency, these gradu- ate students become professors of College Degree Education, without necessarily having the pedagogical training required to act in the training of undergraduate students, according to pe- culiarities inherent to the group of undergraduates themselves, as well as the national scenario of health system. In this sense, in order to overcome the misunderstanding that the scientific competence certified in a Master’s degree and Doctoral course is equivalent to the declaration of Pedagogical competence, the experience of the internship in teaching is shown as a potent strategy, even though limited in its workload, to provide the pedagogical development of masters and doctors. Contextualizing the theme Given the emphasis and specificity of the training of researchers in Master’s degree and Doctoral, graduate courses of the challenges of teaching in the contemporary world, the possibility of graduation is a means of professor training, but that necessarily does not encompass desirable pedagogical requirements, the importance of skilled teaching practice and Instrumental to favor the teaching-learning process, of the various technologies and innovations in the teaching available in the current context, of the power created by articulating the look of the graduate student and the supervising professor, providing an opportunity to focus differentiated from activities aimed at the development of graduate students in a teaching internship program, justify the reflection that sometimes presents itself. It should enable the student to experience and participate in multiple dimensions of teaching: organizational, which concerns the selection of content, didactic resources, among others; technique, which is related to the monitoring of activities, notes, presence list, among others; pedagogical, linked to the organization and development of classes; professor/student relationship and evaluative dimension.10 This stage can only be developed after the pedagogical preparation, being the stage performed in undergraduate disciplines, both theoretical and theoretical-practical. It creates the opportunity to experience, in real context, teaching-learning situations, with different approaches, focuses, scenarios and strategies, together with the supervising professor. This study is expected to contribute to the professor training of the graduate student, as well as to present weighting elements for the supervising professor in a continuous learning process. The objective was to reflect on activities aimed at the development of graduate students, in a teaching internship program, in the perspective of teaching and training in Master’s degree and Doctoral graduate studies. The teaching internship, similar to PAE (Programa de Aperfeiçoamento do Ensino), can have different characteristics. Depending on the structure and the unit in which it applies, the student can participate in disciplines, conferences with specialists in the area of education or core of activities that address topics and issues relevant to college degree education, and the activities must have organizational, technical and didactic-pedagogical approach. Professor training in the teaching of graduate students Finally, depending on established articulations and interactions, this process can constitute an active learning possibility, both for the supervising professor and for the graduate student. It should be pointed out that, in this stage of pedagogical preparation, it is fundamental that the offered course be able to, in fact, qualify the graduate student to the teaching practice, given that each unit or department provides this step according to its Political-pedagogical context, although the objective is common: to fill the existing gaps in professor training. It is understood that teaching, articulated to education and research, is an integrated vision and assumed as an intrinsic pedagogical principle, in which it is perceived that the university essentially needs research, not only for the purpose of generating new knowledge, but to develop qualified education and provide the extension as a way to respond to the demands of society.11 In this context, the teaching internship can be understood as a relevant part of the training for graduate student teaching. The supervised internship, developed in theoretical and practical discipline, inserted in a practice scenario, favors meaningful learning, both in terms of structuring and the dynamics adopted for the interaction between the supervising professor, the graduate students. Although the proposal for the improvement of the graduate student, focused on professor education, contemplates both stages, the process of professor education is broader, complex, dynamic and lasting. In this sense, there are questions about the feasibility and scope of a specific pedagogical training discipline to fill gaps in professor education, supplying the needs of different courses.3 It is understood that it can be a trigger in training, which does not end in graduate school, but should be a continuous process. This experience enables learning about aspects of the organization of the discipline, of professor-student-service relationship and of conducting meaningful learning, in order to bring new meanings to graduate students. One of the reflections that emerge about this theme is about the form of professor- student communication as a strategy to enhance the teaching- learning process. The professor acquires attributes to play the role in multiple ways, such as school education and social influences, in addition to continuing education processes, among others,12 which extend to the graduate student, as far as professional training is concerned, both in the disciplines of graduate courses focused on didactics and/or College Degree Education as in participation in teaching internship. Professor training in the teaching of graduate students In the course of the teaching internship, the graduate student also has the opportunity to participate in formative, continuous and shared evaluation processes with the supervising professor and other students, whether in theoretical disciplines and/or in the scenario of professional practice, by means of feedback, supervision, case studies, etc. The formation of the professor for college degree education has a dynamic character; it is built and based not only on pedagogical preparation and in supervised internships in teaching, but also in personal experiences.i The first stage of the teaching internship includes attend- ing specific preparatory discipline, which includes pedagogical approach and contact with specific references on the contents of the discipline program. The graduate student, when identify- ing and recognizing the issues of College Degree Education in Brazil, different teaching-learning strategies, as well as their facilities and difficulties, can develop skills for teaching practice. In this sense, the graduate student can assume an important role of mediator between undergraduate students and the supervising professor, as it contributes with a critical look at the situations that happen throughout the discipline, both in the perspective of the development of activities the work proposal/ professor approach.l Graduate students, during the insertion in the disciplines, have the possibility to reflect on the professional teaching praxis, besides sharing experiences with other students. The classes with differentiated format, based on different active methods of teaching, favor another approach for College Degree Education. Throughout the classes, the graduate students play the role of professor, by explaining the theme of the class according to the program of the discipline, using active teaching proposals and discussion with other professors on the subject itself, about the performance of the group of graduate students and what could be improved, according to the classes taught, in an action- reflection approach in order to instrumental future action. This mediation can trigger relevant reflections, since the graduate student can approach both the professor's and the undergraduate's eyes in an exercise in learning the complexity and multidimensionality of the teaching-learning relationship. It is a relational, communicative and learning process for the three parties involved. However, it is not something simple, since it can bring some movement and/or restlessness in established roles and that is exactly where one of the riches of this experience can be. RESULTS AND DISCUSSION of knowledge, stimulation and reflection on the theoretical and the scenario of practice, as well as the co-responsibility of those involved in their own learning. METHOD This is a reflexive study, based on the discursive formulation of the required competencies and guidelines proposed by the PNPG (Plano Nacional de Pós-graduação) for professor training articulated with graduate studies, considering the views of graduates and supervisors of a Nursing College Degree Course at a Public Education Institution. The Masters and doctoral students experience multiple demands inherent to graduation studies, which makes it necessary to recognize that equating time for all activities is a challenge, as well as thinking about the specificity of the preparation for the exercise of teaching, and perhaps, therefore, this last question is relegated in the background. In this direction, questions arise about the insertion of the graduate student in the labor market, specifically in the area of education, which encourages discussing aspects inherent to the academic formalities of teaching internship. From the context and relevance of the teaching internship to professor training, reflections on pedagogical preparation and the practical internship for teaching were considered, considering the process of meaningful learning. Escola Anna Nery 23(3) 2019 3 Reflections on graduate professor training g p g Alves LR, Giacomini MA, Teixeira VM, Henriques SH, Chaves LDP Alves LR, Giacomini MA, Teixeira VM, Henriques SH, Chaves LDP The supervising professor in the construction of learning This double aspect is an unparalleled opportunity to visualize, in an expanded way, the teaching role in College Degree Education, permeated by action-reflection-action and based on scientific and pedagogical knowledge, a situation that may favor the revision of the teaching performance itself, in a process where there can be meaningful learning for all involved. Through this approach, is stimulated, the formation of more critical and reflexive students, capable of seeking for knowledge and of learning to think in a more articulated way to the context in which they are inserted, understanding the meaning in the work and the potential of the knowledge to qualify practices. Subsequently to all the situations experienced and the activities carried out, it is possible to understand that the journey towards professor training is plural and dynamic. It is necessary to develop a critical posture in front of the teaching praxis and to enable students to become producers of knowledge and take a critical and reflexive attitude towards the proposed content, enabling them to learn to learn, mainly in their future professional practice. The stages of the improvement to the teaching practice favor the contact with professional practice, as well as the relationship between theory and practice, with the development of competencies. In addition, they offer graduates students elements to learn how to prepare classes and to use active teaching strategies in the classroom, to reduce the gap between a good theorist and a good professor, to create possibilities for experiencing and exploring the challenges of teaching, as well as evaluating and testing practices in the perspective of teaching, research and extension.3 The use of practice scenarios in student training enables the development of competencies with the necessary clinical reasoning for the exercise of safe professional practice in health. For this, we need not only technical elements, but also organizational and reflective planning, contextualizing the theoretical contents with the problems that emerge from the practice.13l The teaching challenge is to stimulate the action-reflection- action process among the students, to provide opportunities for cognitive development (know-how) and the operational ability (know-how) supported by ethics and commitment (knowing how to be), as well as knowing how to live. The supervising professor in the construction of learning It is essential to understand and know how to take advantage of the changes resulting from globalization in terms of the use of technological resources available to teaching. For the current generation of students, this is an unpostponable process, because existing technologies, when used correctly, can benefit teaching and learning. The development of teaching internship can favor the guidance process of the graduate student, by allowing the approximation with the supervising professor, in a perspective of articulation teaching-research not only when it relates to the research theme of the graduation, but also, as a possibility of favoring evidence-based practice, the perception of the relevance of keeping up to date for teaching practice, access to different bibliographic sources, also strengthening the aspect of research consumption and its incorporation into practice that can also raise questions about how to make your own research project viable For potentialized results in this strand. The path to be taken by graduate students is meandering and encouraging, and advancing in their training, focusing on the teaching-learning process based on the previous experience of traditional teaching, is a challenge. The adoption of a critical-reflexive attitude towards teaching, the understanding that the professor plays the role of facilitator and stimulator of the intellectual development of the student in the learning process and that the acquisition of knowledge does not, per se, characterize the aptitude for the are advances that require investments to be consolidated. This is a situation that can mobilize the supervising professor to review and/or seek different pedagogical strategies to discuss the teaching-research articulation and, in this sense, this professor is encouraged to seek new ways/methods to teach pedagogical strategies. In order to encourage learning, graduates students and supervisor professors should, in the course of theoretical and practical activities, support undergraduate students, whether in attitudinal or psychosocial and cognitive issues, by stimulating reflection from real situations and to solve doubts, encouraging them to seek the answers of their questions through scientific literature to subsequently discuss the outcome, building a cycle of action-reflection-action. Supervising the graduate student in the teaching internship can be quite enriching for the supervising professor when he understands that he is under different approaches of teaching- learning: that of undergraduate student in his professional training and the graduate student in his teaching training. Professor training in the teaching of graduate students Nonetheless, it is true that only attending preparatory courses and supervised internships in teaching does not make the graduate professor, i.e.: there is a constant need to update on the specific knowledge to be taught and the In the course of a program aimed at the improvement of future professors, especially in the teaching internship, at various times, the supervising professor has the possibility of providing reflections on the formation of undergraduate students, regarding the strategies of Learning, and the graduation itself can act in this process. Linked to the previous pedagogical preparation, it provides support for the practice and is fundamental for the improvement of teaching, since the teaching-learning process can be understood as something dependent on the valorization Escola Anna Nery 23(3) 2019 4 Reflections on graduate professor training Alves LR, Giacomini MA, Teixeira VM, Henriques SH, Chaves LDP Reflections on graduate professor training methods to be used, not to remain in the monotony and play the role of a mere content replicator. Reflections on graduate professor training Alves LR, Giacomini MA, Teixeira VM, Henriques SH, Chaves LDP Sharing with the graduate student the practice of teaching in practice scenarios with undergraduate students provides mutual learning, at each meeting with students and professionals of the scenario, favoring the daily reflection on the work itself. Given the limitations of the study of the type of reflection, which considers the focus of a specific group of researchers, highlights the contribution in order to allow the construction of multiple views to show the creative potential that experienced processes represent: in relation to graduate students, who when applying the theoretical knowledge acquired in the preparatory discipline, can modify the reality of the context of the theoretical- practical activities developed and, in relation to the supervisor professor, weights can emerge on the teaching-learning process, leading to improvements in professional practice.l l Thus, it is considered that teaching internship is important, both for the graduate student and the supervising professor who, while teaching, have the opportunity to review their own action, in a constant movement of (re)construction, to reinvent in that role to facilitate the teaching-learning process of the graduate student in the walk of teaching. This result is also found in another study,15 which highlights the relevance of the teaching internship in relation to the process of reconstruction of the meaning of the teaching work, to the reflection about the work of the professor in university education and, in as trainer of trainers. Thus, it presents itself as an aspect capable of favoring the problematic of teaching, in both undergraduate and graduate studies, as well as the construction of new resources for such practice. It is believed that this reflection highlights the potential of the teaching internship to be explored, but this is still a challenging, dynamic process, with gaps that must be overcome and better instrumental, emphasizing pedagogical practices that need better and more attention, as well as the appropriation of new technologies, the encouragement to build personal identity and constant renewal of their teaching practices, consistent with social transformations, cultivating criticality and reflection. REFERENCES 1. Tachibana TY, Menezes Filho N, Komatsu B. Ensino superior no Brasil. Policy Paper [Internet]. 2015 dec; [cited 2018 sep 05]; 4:1-53. Available from: https://www.insper.edu.br/wp-content/uploads/2018/09/Ensino- superior-no-Brasil.pdf 1. Tachibana TY, Menezes Filho N, Komatsu B. Ensino superior no Brasil. Policy Paper [Internet]. 2015 dec; [cited 2018 sep 05]; 4:1-53. Available from: https://www.insper.edu.br/wp-content/uploads/2018/09/Ensino- superior-no-Brasil.pdf After intense reflection, it is understood that the pedagogical preparation is a fundamental step to be included in the teaching internship, since the participation in theoretical disciplines focused on teaching in College Degree Education enables to understand different active teaching strategies as well as their practical use. Practical activities with nursing undergraduates, accompanied by a supervising professor, with the insertion in the theoretical-practical disciplines of the undergraduate, in the scenarios previously selected, instrument the graduate student for the teaching exercise. 2. Cirani CBS, Campanario MA, Silva HHM. A evolução do ensino da pós- graduação senso estrito no Brasil: análise exploratória e proposições para pesquisa. Avaliação (Campinas) [Internet]. 2015 mar; [cited 2018 jan 25]; 20(1):163-87. Available from: http://www.scielo.br/pdf/aval/ v20n1/1414-4077-aval-20-01-00163.pdf 3. Joaquim NF, Vilas Boas AA, Carrieri AP. Estágio docente: Formação profissional, preparação para o ensino ou docência em caráter precário?. Educ Pesqui [Internet]. 2013 abr/jun; [cited 2018 jan 22]; 39(2):351-65. Available from: http://www.scielo.br/pdf/ep/v39n2/ a05v39n2.pdf 3. Joaquim NF, Vilas Boas AA, Carrieri AP. Estágio docente: Formação profissional, preparação para o ensino ou docência em caráter precário?. Educ Pesqui [Internet]. 2013 abr/jun; [cited 2018 jan 22]; 39(2):351-65. Available from: http://www.scielo.br/pdf/ep/v39n2/ a05v39n2.pdf The teaching internship articulates theory and practice, demonstrates the dynamics and problematic of being a professor, through living together with the supervising professor and undergraduate students, enabling the sharing of knowledge among the actors involved, but the relational component can be challenging and compromise the use of this experience. The teaching internship can also provide those involved with reflective, joint and contextualized action to the health service scenario, raising questions in a dynamic teaching-learning relationship that, in addition to professor construction, can promote changes in the environment of theoretical and practical activities of undergraduate students and contribute to an improvement of health services that are practice scenarios.f 4. Tardif M. Questões sobre a docência universitária no Canadá. Entrevistado por Silvia Maria Nóbrega-Therrien. Traduzido do francês por Rosa dos Anjos Oliveira. Em Aberto, Brasília. 2016 set/dez; [acesso em 2019 mar 29]; 29(97):133-140. Disponível em: http://www.emaberto. inep.gov.br/index.php/emaberto/article/view/2953/2675 5. Perrenoud P. Construir competências é virar as costas aos saberes?. Pátio Rev Pedagógica [Internet]. The supervising professor in the construction of learning In this way, the possibility of changing reality emerges.14 From this perspective, the process by which the graduate student goes through, in the midst of teaching and learning, is arduous and intense, because he is no longer in a student position, but is not yet seen as the professor of the discipline in which he is inserted. Thus, the graduate student goes through periods of deconstruction and construction of his gaze and knowledge in view of the various situations that can be delineating in the daily life of teaching, supervision and learning, a fact that contributes positively to its professor training. It is necessary to think of a greater participation of the graduate students in the preparation of theoretical classes, since they can contribute with new teaching-learning strategies, sharing the learning obtained in the preparatory stage, which would benefit the supervising professor with new knowledge. Escola Anna Nery 23(3) 2019 5 10. Universidade de São Paulo - USP. Pró-Reitoria de Pós-Graduação. Programa de Aperfeiçoamento de Ensino - PAE. Diretrizes [Internet]. São Paulo: Universidade de São Paulo. 2010; [cited 2018 jan 17]. Available from: http://www.prpg.usp.br/attachments/article/631/Diretrizes_PAE.pdf 14. Chamlian HC. Docência na universidade: professores inovadores na USP. Cad Pesqui [Internet]. 2003 mar; [cited 2017 nov 25]; 118:41-64. Available from: http://www.scielo.br/pdf/cp/n118/16829.pdf REFERENCES 1999 nov; [cited 2018 oct 15]; 3(11):15-9. Available from: http://egov.ufsc.br/portal/sites/default/files/ anexos/29108-29126-1-PB.pdf 6. Perrenoud P. Dez novas competências para ensinar. Porto Alegre: Artmed Editora; 2000. 7. Ministério da Educação (BR). Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES. Plano Nacional de Pós-Graduação - (PNPG) 2005-2010 [Internet]. Brasília: Ministério da Educação. 2004 dez; [cited 2018 jun 15]. Available from: http://www.capes.gov.br/ images/stories/download/editais/PNPG_2005_2010.pdf In the view of the potential of the teaching internship, efforts must be made to create operational conditions conducive to favorable conditions for its development, offering it to graduate students, regardless of whether or not they are scholarship holders, since it allows a problematizing and participative approach that stands out in the construction of professionals in addition to research, developing skills and attitudes, preparing them for teaching. 8. Ministério da Educação (BR). Circular nº 28, de 1º de setembro de 1999. Estabelece requisitos para concessão de bolsas. Diário Oficial da União, Brasília (DF), 12 nov 1999. 9. Universidade de São Paulo - USP. Pró-Reitoria de Pós-Graduação. Programa de Aperfeiçoamento de Ensino - PAE. Diretrizes para a proposição das Disciplinas da Etapa de Preparação Pedagógica [Internet]. São Paulo: Universidade de São Paulo. 2017; [cited 2019 feb 12]. Available from: http://www.prpg.usp.br/attachments/article/631/ Diretrizes%20para%20disciplina%20de%20EPP%20-%2029-09- 2016.pdf Escola Anna Nery 23(3) 2019 6 Reflections on graduate professor training Reflections on graduate professor training 13. Florêncio PCS, Austrilino L, Medeiros ML. O processo ensino aprendizagem nos cenários de prática; concepções dos docentes do curso de graduação em enfermagem. ATAS CIAIQ [Internet]. 2016 jul; [cited 2018 nov 15]; 2(2016):1312-9. Available from: https://proceedings. ciaiq.org/index.php/ciaiq2016/issue/view/13 11. Severino AJ. Da docência no ensino superior: condições e exigências. Comunicações [Internet]. 2013 jan/jun; [cited 2018 jan 15]; 20(1):43- 52. Available from: http://sites.uepg.br/prograd/wp-content/themes/ PROGRAD/assetsDes/artigos/Artigo%20Severino.pdf 14. Chamlian HC. Docência na universidade: professores inovadores na USP. Cad Pesqui [Internet]. 2003 mar; [cited 2017 nov 25]; 118:41-64. Available from: http://www.scielo.br/pdf/cp/n118/16829.pdf 12. Iza DFV, Benites LC, Sanches Neto L, Cyrino M, Ananias EV, Arnosti RP, et al. Identidade docente: as várias faces da constituição do ser professor. Rev Eletr Educ [Internet]. 2014; [cited 2018 oct 12]; 8(2):273- 92. Available from: http://dx.doi.org/10.14244/198271999978 15. Kramer S, Souza MC, Oliveira A. Estágio de docência e metodologia de ensino de língua portuguesa: literatura como caminho para a formação. Leitura: Teoria & Prática [Internet]. 2015; [cited 2018 nov 12]; 33(64):65- 80. Available from: https://ltp.emnuvens.com.br/ltp/article/view/369/270 Escola Anna Nery 23(3) 2019 7 15. Kramer S, Souza MC, Oliveira A. Estágio de docência e metodologia de ensino de língua portuguesa: literatura como caminho para a formação. Leitura: Teoria & Prática [Internet]. 2015; [cited 2018 nov 12]; 33(64):65- 80. Available from: https://ltp.emnuvens.com.br/ltp/article/view/369/270
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Positron Emission Tomography and Magnetic Resonance Imaging of the Brain in Fabry Disease: A Nationwide, Long-Time, Prospective Follow-Up
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Editor: Raphael Schiffmann, Baylor Research Institute, UNITED STATES Received: September 24, 2015 Accepted: November 11, 2015 Published: December 2, 2015 Patients Forty patients with Fabry disease (14 males, 26 females, age at inclusion: 10–66 years, median: 39 years) underwent a brain F-18-FDG-PET-scan at inclusion, and 31 patients were followed with FDG-PET biannually for up to seven years. All patients (except one) had a brain MRI-scan at inclusion, and 34 patients were followed with MRI biannually for up to nine years. Data Availability Statement: All relevant data can be found in the manuscript, and the remaining data (PET- and MRI-scans and patient reports) cannot be made publicly available for ethical and legal reasons, as public availability would compromise patient confidentiality, and local (Danish) legislation prohibits public availability of the data. National Ethics Committee, De Videnskabsetiske Komiteer for Region Hovedstaden, Center for Sundhed, Kongens Vænge 2, 3400 Hillerød, Denmark, Website: www. regionh.dk/vek Email: vek@regionh.dk OPEN ACCESS Citation: Korsholm K, Feldt-Rasmussen U, Granqvist H, Højgaard L, Bollinger B, Rasmussen AK, et al. (2015) Positron Emission Tomography and Magnetic Resonance Imaging of the Brain in Fabry Disease: A Nationwide, Long-Time, Prospective Follow-Up. PLoS ONE 10(12): e0143940. doi:10.1371/journal. pone.0143940 Kirsten Korsholm1*, Ulla Feldt-Rasmussen2, Henrik Granqvist2, Liselotte Højgaard1, Birgit Bollinger3, Aase K. Rasmussen2, Ian Law1 Kirsten Korsholm1*, Ulla Feldt-Rasmussen2, Henrik Granqvist2, Liselotte Højgaard1, Birgit Bollinger3, Aase K. Rasmussen2, Ian Law1 1 Department of Clinical Physiology, Nuclear Medicine and PET, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark, 2 Department of Endocrinology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark, 3 Department of Radiology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark * korsholmk@gmail.com * korsholmk@gmail.com * korsholmk@gmail.com Objective The objective of this study was to assess brain function and structure in the Danish cohort of patients with Fabry disease in a prospective way using 18-fluoro-deoxyglucose (F-18 FDG) positron emission tomography (PET) and magnetic resonance imaging (MRI). Copyright: © 2015 Korsholm et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Background Fabry disease is a rare metabolic glycosphingolipid storage disease caused by deficiency of the lysosomal enzyme α-galactosidase A—leading to cellular accumulation of globotria- sylceramide in different organs, vessels, tissues, and nerves. The disease is associated with an increased risk of cerebrovascular disease at a young age in addition to heart and kidney failure. Conclusion Our data indicated that, in patients with Fabry disease, MRI is the preferable clinical modal- ity—if applicable—when monitoring cerebral status, as no additional major brain-pathology was detected with FDG-PET. Brain Imaging with PET and MRI in Fabry Disease Results Funding: The study has been partly sponsored by unrestricted research grants from Genzyme (https:// www.genzyme.com/) and Shire (https://www.shire. com)/, and UFR has received teaching honoraria and served in advisory boards in Shire, Genzyme and Amicus (http://www.amicusrx.com/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. In 28 patients brain-FDG-PET was normal; in 23 of these 28 patients brain MRI was normal —of the remaining five patients in this group, four patients had WMLs and one patient never had an MRI-scan. In 10 patients hypometabolic areas were observed on brain-FDG-PET; all of these patients had cerebral infarcts/hemorrhages visualized on MRI corresponding to the main hypometabolic areas. In two patients brain-FDG-PET was ambiguous, while MRI was normal in one and abnormal in the other. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Image Analysis The FDG-PET-images were inspected visually and analysed using a quantitative 3-dimen- sional stereotactic surface projection analysis (Neurostat). MRI images were also inspected visually and severity of white matter lesions (WMLs) was graded using a visual rating scale. 1 / 13 PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 Introduction Fabry disease is a rare X-linked recessive metabolic glycosphingolipid storage disease caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A) [1]. The glycosphingolipid substrate of α-gal A is globotriasylceramide (Gb3) which accumulates in e.g. endothelial cells, smooth muscles cells of the vascular system, renal epithelial cells, myocardial cells and central nervous system [2]. The most dominant clinical features of the patients include acroparaesthe- sia, angiokeratomas, corneal opacities and hypohidrosis, and with increasing age patients often develop cerebrovascular events (transient ischemic attacks and strokes) in addition to cardio- vascular disease and renal failure. A breakthrough in the treatment of patients with Fabry disease was the development of an effective and well-tolerated direct enzyme substitution in 2001[3,4], which shifted the manage- ment of the patients from a palliative to a causal active treatment. Conventional magnetic resonance imaging (MRI) of the brain have shown that Fabry patients are at risk of severe and progressive white matter lesions (WMLs) at an early age in addition to cerebral infarcts and hemorrhages [5,6]—especially in the posterior cerebral circu- lation, probably due to dolichoectatic arteries [7,8]. However, the mechanism by which defi- ciency of α-gal A and glycosphingolipid-accumulation causes the Fabry vasculopathy is not completely understood [9]; a protrombotic state in Fabry disease has been confirmed [10], however, endothelial dysfunction and altered cerebral blood flow may also play a role [11]. In addition, emboli as a consequence of cardiac arrhythmia may also contribute. The primary aim of our study was to assess long-term cerebral function in the nationwide Danish cohort of patients with Fabry disease using 18-fluoro-deoxyglucose (F-18 FDG) brain positron emission tomography (PET) scanning in addition to assessment of structural brain changes using MRI. The study was a descriptive, observational and prospective study initiated in relation to commencement of the treatment with enzyme substitution. FDG-PET-Brain-Imaging The distribution of the relative regional cerebral glucose metabolic rate (rCMRglc) was mea- sured using F-18 FDG-PET-scanning. PET scans were performed with an eighteen-ring GE-Advance scanner (General Electric Medical Systems, Milwaukee, WI, USA) operating in 3D-acquisition mode, producing 35 image slices with an interslice distance of 4.25 mm. The total axial field of view was 15.2 cm with an approximate in-plane resolution of 5 mm [15]. Each patient received an intravenous bolus injection of approximately 200 MBq F-18 FDG while resting in the supine position with eyes covered and noise level kept at a minimum. After 30 min the patient was placed in the scanner, and the head fixed to restrict movements. A 10 min transmission scan was performed for attenuation correction followed by a 10 min 3-D emission scan. PET images of the FDG distribution were reconstructed using a 4.0 mm Han- ning filter transaxially and an 8.5 mm Ramp filter axially. The same scanner was used in all patients for the sake of reproducibility, although the department acquired newer and better scanners during the study period. Magnetic Resonance Imaging All patients (except one) underwent MRI of the brain around the time of the PET scans. Before 2008 all MRI-scans were performed on a 1.5 T Siemens Vision scanner, however, in 2008 the scanner was replaced by a 1.5 T Siemens Avanto scanner. Sagittal T1-weighted images (TR 630 msec, TE 14 msec, flip-angle 90°, slice thickness 5 mm), axial double spin echo (TR 3703 msec, TE 22/90 msec, flip-angle 180°, slice thickness 5 mm) and coronal FLAIR-images (TR 9000 msec, TE 105 ms, flipangle 180°, slice thickness 3 mm) were obtained before contrast-injection, and sagittal and axial T1-weighted images (TR 588 msec, TE 17 ms, flip-angle 90°, slice thickness 5 mm) were obtained after contrast-injection (Multihance 0.2 ml/kg iv). Contrast was not given to patients with impaired kidney function. Patients We studied 40 patients with Fabry disease (14 males, 26 females, age: 10–66 years, median 35 years (men) and 43 years (women), at inclusion). The study was approved by the National 2 / 13 PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 Brain Imaging with PET and MRI in Fabry Disease Ethics Committee (02-038/02, H-3-2014-FSP8), and informed consent was obtained from all participating subjects. The patients were followed at the Department of Endocrinology at Rig- shospitalet, Copenhagen, Denmark, and the patients were examined with F-18 FDG-PET and MRI of the brain biannually for up to seven years (PET) / nine years (MRI) in addition to regu- lar and systematic examinations for manifestations of Fabry disease as a part of the normal fol- low-up procedure at our hospital. Fabry disease was confirmed in all patients by alpha-galactosidase A (GLA) mutation analy- sis, and the following mutations were seen: G85D (14 patients), A156T (11 patients), N34S (6 patients), G10694 (2 patients), I232T (2 patients), R342X (1 patient), D313Y (1 patient), G271S +D313Y (1 patient), c.369+3_c.547 (1 patient), G271S (1 patient) (see Table 1). Thirty-two patients were treated with enzyme replacement treatment (ERT). Initially all treated patients received agalsidase beta (Fabrazyme1, Genzyme (Cambridge, MA 02142)) 1 mg/kg intravenously every second week during the follow-up period. Because of a shortage of Fabrazyme1 in 2010/2011, most patients subsequently received agalsidase alfa (Replagal1, Shire (Hampshire, UK)) 0.2 mg/kg for the remaining follow-up period. One patient was treated with pharmacological chaperone therapy in a randomized trial. Seven patients did not receive any ERT (see Table 1). Part of the patient cohort has been described previously [12–14]. Age at inclusion Sex Mutation Cerebro- vascular disease before inclusion Cerebro- vascular disease during study Cardio- vascular disease before inclusion Cardio- vascular disease during study Renal event before inclusion Renal event during study ERT 1 36 M R342X - - - - - - + 2 10 M G85D - - - - - - + 3 60 F A156T + - U + - - + 4 37 M A156T + - - + D KT + 5 63 F A156T - - + - - - + 6 58 F A156T + - + + - - + 7 41 F A156T - - - + - - + 8 38 F G85D - - - - - - + 9 42 F A156T - + - - - - + 10 37 M A156T - - - - - - + 11 15 M N34S - - - - - - + 12 15 F A156T - - - - - - + 13 33 F A156T - - + - - - - 14 27 F G85D + - - - - - + 15 34 F N34S + - - - - - + 16 54 F A156T - - + - - - + 17 34 M G85D - - - - - - + 18 18 M N34S - - - - - - + 19 59 F G85D + - - + - - + 20 48 F N34S - - + - - - + 21 55 F G85D - - - + - - + 22 35 M G85D - - - - - D, KT + 23 24 M N34S - - - - - - + 24 58 F G85D + - + - - - + 25 58 F G85D - + - - - - + 26 26 M G85D - - - - - - + 27 39 F G85D - - - - - - - 28 33 F G85D - - - - - - + 29 18 F c.369 +3_c.547 - - - - - - + 30 50 F G10694 - - - - - - + 31 47 M G10694 - - - - - - + 32 40 F G85D - - - - - - + 33 66 F G271S + - + - - - + 34 29 M G85D - - - - D, KT - - 35 35 M N34S - - - - - - - 36 44 F D313Y - - - - - - - 37 39 F G271S + D313Y - - - - - - -* 38 40 F A156T - - - - - - - 39 64 F I232T - - - - - - - (Continued) Table 1. Data Analysis The FDG PET images were registered to the patients T1- and T2-weighted MRI scans, oriented parallel to the cranial base, and evaluated visually in coronal, sagittal and transaxial sections 3 / 13 PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 Age at inclusion Sex Mutation Cerebro- vascular disease before inclusion Cerebro- vascular disease during study Cardio- vascular disease before inclusion Cardio- vascular disease during study Renal event before inclusion Renal event during study ERT 1 36 M R342X - - - - - - + 2 10 M G85D - - - - - - + 3 60 F A156T + - U + - - + 4 37 M A156T + - - + D KT + 5 63 F A156T - - + - - - + 6 58 F A156T + - + + - - + 7 41 F A156T - - - + - - + 8 38 F G85D - - - - - - + 9 42 F A156T - + - - - - + 10 37 M A156T - - - - - - + 11 15 M N34S - - - - - - + 12 15 F A156T - - - - - - + 13 33 F A156T - - + - - - - 14 27 F G85D + - - - - - + 15 34 F N34S + - - - - - + 16 54 F A156T - - + - - - + 17 34 M G85D - - - - - - + 18 18 M N34S - - - - - - + 19 59 F G85D + - - + - - + 20 48 F N34S - - + - - - + 21 55 F G85D - - - + - - + 22 35 M G85D - - - - - D, KT + 23 24 M N34S - - - - - - + 24 58 F G85D + - + - - - + 25 58 F G85D - + - - - - + 26 26 M G85D - - - - - - + 27 39 F G85D - - - - - - - 28 33 F G85D - - - - - - + 29 18 F c.369 +3_c.547 - - - - - - + 30 50 F G10694 - - - - - - + 31 47 M G10694 - - - - - - + 32 40 F G85D - - - - - - + 33 66 F G271S + - + - - - + 34 29 M G85D - - - - D, KT - - 35 35 M N34S - - - - - - - 36 44 F D313Y - - - - - - - 37 39 F G271S + D313Y - - - - - - -* 38 40 F A156T - - - - - - - 39 64 F I232T - - - - - - - (Continued) Table 1. Clinical and genetic features of the Fabry patients. Patient no. Brain Imaging with PET and MRI in Fabry Disease Table 1. Clinical and genetic features of the Fabry patients. Patient no. Clinical and genetic features of the Fabry patients. PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 4 / 13 Significant regional deviation from the mean was expressed by a Z- score using a threshold value of Z >3.09 (p < 0.001, one sided). Progression of any pathology on the PET-scans were noted and described. A trained neuroradiologist (BB) evaluated all MRI scans. WMLs were defined as bright lesions of  5 mm on FLAIR images, and WML severity was graded on a 4-rate scale [17,18]. Images with no or a single punctate lesion were classified as 0, multiple punctate lesions were classified as 1, beginning confluence of lesions was classified as 2 and large confluent lesions were classified as 3. Lesion progression was noted and classified as minor if lesion-load had increased by one to four additional punctuate lesions on follow-up, and lesion progression was noted as marked if  five punctuate lesions had developed or if there was a transition to confluent lesions [19,20]. In addition, infarcts, hemorrhages and other vascular pathology were described. Brain Imaging with PET and MRI in Fabry Disease Table 1. (Continued) Patient no. Age at inclusion Sex Mutation Cerebro- vascular disease before inclusion Cerebro- vascular disease during study Cardio- vascular disease before inclusion Cardio- vascular disease during study Renal event before inclusion Renal event during study ERT 40 36 M I232T - - - - - - + “+” yes “-”no *Chaparone-therapy. ERT: Enzyme replacement treatment Cerebrovascular disease: Infarct or hemorrhage Cardiovascular disease: Arrhythmia, congestive heart failure or myocardial infarction Renal event: Dialysis (D) or kidney transplantation (KT) Unknown: U doi:10.1371/journal.pone.0143940.t001 Table 1. (Continued) atient no. Age at inclusion Sex Mutation Cerebro- vascular disease before inclusion Cerebro- vascular disease during study Cardio- vascular disease before inclusion Cardio- vascular disease during study Renal event before inclusion Renal event during study ERT 40 36 M I232T - - - - - - + using the “3D” program in the Siemens Multimodality Workplace by a specially trained nuclear medicine physician (IL). In addition to visual analysis, a quantitative 3-dimensional stereotactic surface projection analysis (Neurostat) was employed that allows direct visualiza- tion of the extent and topography of FDG uptake abnormalities [16]. This procedure involved reconstruction of the images with an 8 mm Hann filter, warping them to a standard stereotactic space and projecting regional cortical activity normalized to the cerebellum unto the cerebral surfaces. The surface projections were subsequently compared voxel-by-voxel to three cohorts of age-matched groups of healthy subjects depending on the patients’ age (19–34 years, 30–60 years, and 55–90 years). Significant regional deviation from the mean was expressed by a Z- score using a threshold value of Z >3.09 (p < 0.001, one sided). using the “3D” program in the Siemens Multimodality Workplace by a specially trained nuclear medicine physician (IL). In addition to visual analysis, a quantitative 3-dimensional stereotactic surface projection analysis (Neurostat) was employed that allows direct visualiza- tion of the extent and topography of FDG uptake abnormalities [16]. This procedure involved reconstruction of the images with an 8 mm Hann filter, warping them to a standard stereotactic space and projecting regional cortical activity normalized to the cerebellum unto the cerebral surfaces. The surface projections were subsequently compared voxel-by-voxel to three cohorts of age-matched groups of healthy subjects depending on the patients’ age (19–34 years, 30–60 years, and 55–90 years). Brain Imaging with PET and MRI in Fabry Disease Table 2. FDG-PET and MRI-features of the Fabry patients. Patient no. No of brain PET/brain MRI Brain PET pathology WML on any brain MRI Other brain MRI pathology incl. infarcts Progression of pathology on PET/MRI c 1 3/5 - - Minimal hyperintensity of both pulvinar on T1 - / - 2 4/4 - - - - / - 3 4/4 Hypometabolic areas corresponding to infarcts; thalamocortical diaschisis +crossed cerebello-cortical disachisis WML grade 2 Multiple infarcts in right frontal region, left basal ganglia and left cerebellar hemisphere Aggravation of crossed cerebello-cortical diaschisis / - 4 4/4 Hypometabolic areas corresponding to infarcts and in left mesial temporal region. WML grade 2 Multiple infarcts in both frontal regions and cerebellum Development of reduced metabolism in left mesial temporal region / - 5 4/2 Probably normal a WML grade 2 - - / - 6 4/3 Discretely reduced metabolism in frontal areas bilaterally WML grade 2 Infarct in right frontal region - / - 7 4/3 - WML grade 1 - - / - 8 2/3 - WML grade 1 - - / Minor progression (WML grade 0 to grade 1) 9 3/4 Hypometabolic area in right corona radiate corresponding to infarct WML grade 1 Infarct in right corona radiate Development of a hypometabolic area corresponding to infarct / Infarct in corona radiata. Marked progression (5 new focal WMLs) 10 4/4 - - - - / - 11 4/5 - - - - / - 12 4/5 - - - - / - 13 3/3 - - - - / - 14 4/5 Hypometabolic area in pons (infact), diffuse hypometabolism in both cerebellar hemispheres - Infarct in left side of pons - / - 15 3/2 Hypometabolic area in right insula (infact) including adjoining temporal cortex - Infarct in right insula with gliosis. Occluded right carotid artery - / - 16 3/3 - - - - / - 17 3/2 - - Small arachnoidal cyst - / - 18 3/3 - - - - / - 19 3/3 Hypometabolic areas corresponding to infarcts in frontal cortex and thalamus. WML grade 1 Multiple infarcts in frontal regions and left thalamus - / Minor progression in WML (one new focal WML) 20 3/3 - - - - / - 21 3/3 - WML grade 2 - - / - 22 1/2 Probably normal b - - n.a. Results Forty patients underwent a brain FDG-PET-scan at inclusion, and 31 patients were followed with brain FDG-PET ranging from two to four scans per patient for up to seven years (Table 2). Nine patients were only scanned once. Patients underwent brain MRI biannually performed around the time of the PET-scan, and were followed for up to nine years ranging from two to five scans per patient (five patients only had one MRI-scan during the study period, and one patient never had an MRI-scan per- formed) (Table 2) In 28 patients FDG-PET scan of the brain was normal both initially and during follow-up, that is, no areas of hypo- or hypermetabolism were detected compared to an age-matched con- trol-group (Table 2). MRI of the brain was normal in 23 of these 28 patients (apart from minor 5 / 13 PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 Brain Imaging with PET and MRI in Fabry Disease Table 2. (Continued) Table 2. (Continued) Patient no. No of brain PET/brain MRI Brain PET pathology WML on any brain MRI Other brain MRI pathology incl. infarcts Progression of pathology on PET/MRI c 28 2/2 - - - - / - 29 2/2 - - - - / - 30 1/1 - - - n.a. / n.a. 31 1/2 - WML grade 2 - n.a. / - 32 2/2 - - - - / - 33 2/2 Hypometabolic areas corresponding to infarcts. Reduced metabolism in right temporooccipital area WML grade 2 Infarcts in brainstem and right basal ganglia - / - 34 1/0 - n.a n.a n.a. / n.a. 35 1/2 - - - n.a. / - 36 1/1 - - - n.a. / n.a. 37 1/1 - - - n.a. / n.a. 38 2/2 - - - - / - 39 1/1 - - - n.a. / n.a. 40 1/1 - - - n.a. / n.a. n.a. not applicable - WML grade 0 or pathology not present - / - no changes in neither PET nor MRI a Patient no. 5: symmetrical mildly reduced activity parietotemporally bilaterally b Patient no. 22: symmetrical mildly reduced activity in both thalami Cont. c Progression of pathology on either PET or MRI was detected in the following patients: Patient no. 3: PET-study period: seven years Patient no. 4: PET-study period: six years. Patient no. 8: MRI study period: three years Patient no. 9: PET-study period: five years. MRI study period: seven years. Patient no. 19: MRI study period: five years Patient no. 25: PET/MRI study period: two years. doi:10 1371/journal pone 0143940 t002 n.a. not applicable findings in pts. no. 1 and 17 where hyperintensity of the pulvinar on T1-weighted images, respectively an arachnoidal cyst was detected). Of the remaining five patients in this group, one patient never had an MRI-scan, four patients had WMLs (grade 1 or 2), and progression of WMLs was seen in one of these patients (pt. no. 8) (Table 2). In two patients (no. 5 and 22) FDG-PET of the brain was probably normal (one patient showed symmetrically mildly reduced activity parieto-temporally bilaterally which remained stable over seven years (four PET-scans)), and one patient showed symmetrically mildly reduced activity in both thalami (only PET-scanned once). / - 23 3/3 - - - - / - 24 2/2 Hypometabolic area in left thalamus corresponding to infarct WML grade 3 Infarct in left thalamus - / - 25 2/2 Hypometabolic area in cerebellum corresponding to tissue loss. Cerebello-cortical diaschisis WML grade 1 Hemorrhage in left cerebellar hemisphere Development of hypometabolic area corresponding to tissue loss. Development of cerebello-cortical diaschisis / Cerebellar hemorrhage 26 2/2 - - - - / - 27 2/2 - - - - / - (Continued) Table 2. FDG-PET and MRI-features of the Fabry patients. (Continued) PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 6 / 13 PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 Brain Imaging with PET and MRI in Fabry Disease 19) showed progression on MRI (minor progression in WML, one new focal WML). The remaining patients in this group developed no new structural changes detectable with MRI. 19) showed progression on MRI (minor progression in WML, one new focal WML). The remaining patients in this group developed no new structural changes detectable with MRI. In four patients progression of pathology of glucose metabolism was seen on FDG-PET dur- ing follow-up: One patient (no. 25) suffered from a cerebellar hemorrhage and developed a hypometabolic area corresponding to tissue loss in the cerebellum (visualized by MRI) in addi- tion to a cerebello-cortical diaschisis (see Figs 1 and 2). Another patient (no. 9) suffered from an infarct in the right corona radiata with tissue loss seen on MRI and corresponding hypometabolism on FDG-PET. In addition, marked progres- sion with five new focal WMLs was seen in this patient. Both of these patients had a normal FDG-PET scan initially. A third patient (no. 3) developed a more pronounced crossed cerebellar diaschisis without any new structural changes on MRI, and the fourth patient (no. 4) developed slightly reduced glucose metabolism in the left temporal region mesially also without any evidence of new struc- tural changes on MRI. In addition to the hypometabolic areas corresponding to infarcts, minor pathology was seen on FDG-PET in a few patients e.g. diffuse hypometabolism in both cerebellar hemispheres (patient no. 14 (infarct in pons)) and a hypometabolic area in adjoining temporal cortex (patient no.15 (infarct in insula)) (see Table 2). In total, WMLs grade 1–3 were seen in 13 of 40 patients (32.5%) (median age 58 years at inclusion, range 37–66). Lesions were classified as grade 1 in five patients, as grade 2 in seven patients, and as grade 3 in one patient (Table 2). Progression of WMLs was seen in three patients (follow-up: range 3–7 years), and one of these patients (no. 9) also developed a new infarct. Cerebral infarcts were detected on MRI in nine of 40 patients (22.5%), and cerebral hemor- rhage in one patient (2.5%). Eight of the 10 patients with infarcts/hemorrhages had WMLs. Abnormal bright signal in the pulvinar on T1-weighted images was seen in one patient (patient no 1, male). Nine of the female patients (34.6%) had cerebrovascular disease, and one (7.1%) of the male patients (Table 1). Seven patients had a history of cardiovascular disease (arrhythmia, congestive heart failure or myocardial infarction) prior to inclusion, and six patients developed cardiovascular disease during the study. In the group of patients with cerebral infarcts/hemorrhages (10 patients), six patients also had cardiovascular disease. Two patients had renal disease prior to inclusion (one patient was in renal dialysis; one patient had undergone kidney transplantation). The patient in renal dialysis was transplanted during the study. A third patient begun renal dialysis during the study and was later trans- planted (see Table 1). Only one of the patients with infarcts also had renal disease. The patient with mildly reduced activity parieto-temporally bilaterally had WMLs grade 2 on MRI which did not progress dur- ing follow-up (short follow-up: 2 years). The patient with mildly reduced activity in both thal- ami had two normal MRI of the brain. In 10 patients hypometabolic areas were observed on FDG-PET of the brain either at inclu- sion or at follow-up. All of these patients had cerebral infarcts/hemorrhages visualized on MRI corresponding to the main hypometabolic areas. In six of these patients the metabolic changes were stationary over time. One of the patients with stationary metabolic defects (patient no. 7 / 13 PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 Discussion Cerebrovascular events are common in patients with Fabry disease and in order to monitor long-time brain function and structure, our cohort of patients with Fabry disease underwent FDG-PET- and MRI-scans of the brain biannually in a prospective follow-up study. Overall, we found that both modalities detect infarcts, however, more detailed information about brain structure and pathology are discovered with MRI including WMLs. Cerebral glucose metabolism in patients with Fabry disease has previously been investigated by Moore et al [21] who found that the mean global cerebral glucose metabolism (CMRGlu) was not significantly different between patients with Fabry disease and controls. This was in 8 / 13 PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 Brain Imaging with PET and MRI in Fabry Disease Fig 1. PET and MRI of the brain of patient no 25. Patient no. 25 suffered from a cerebellar hemorrhage and developed a hypometabolic area corresponding to tissue loss in the left cerebellar hemisphere (b + d) in addition to a cerebello-cortical diaschisis (a + b). a: Cortex (MRI fusioned with PET)–decreased activity in the right hemisphere. b: Cerebellum (MRI fusioned with PET)–decreased activity in the left cerebellar hemisphere. c: Cortex (MRI)–no structural changes. d: Cerebellum (MRI)–sequelae after hemorrhage. doi:10 1371/journal pone 0143940 g001 Fig 1. PET and MRI of the brain of patient no 25. Patient no. 25 suffered from a cerebellar hemorrhage and developed a hypometabolic area corresponding to tissue loss in the left cerebellar hemisphere (b + d) in addition to a cerebello-cortical diaschisis (a + b). a: Cortex (MRI fusioned with PET)–decreased activity in the right hemisphere. b: Cerebellum (MRI fusioned with PET)–decreased activity in the left cerebellar hemisphere. c: Cortex (MRI)–no structural changes. d: Cerebellum (MRI)–sequelae after hemorrhage. doi:10.1371/journal.pone.0143940.g001 doi:10.1371/journal.pone.0143940.g001 Fig 2. Quantitative 3-dimensional surface projection-analysis of FDG-uptake in patient no 25. PET-images of patient no. 25 are normalized to whole brain using a database of normal subjects and scaled to Z values from - 4.0 to 4.0 (Neurostat). Projections of Z values are shown onto, respectively, the right and left lateral hemispheric surfaces, the superior and inferior surfaces, and the anterior and posterior surfaces. The uptake in the left cerebellar hemisphere is reduced in addition to uptake in the right frontal cortex. MRI surface projections are presented for anatomical reference in a standard stereotactic space Fig 2. Quantitative 3-dimensional surface projection-analysis of FDG-uptake in patient no 25. PET-images of patient no. Brain Imaging with PET and MRI in Fabry Disease accordance with our results, where we find a normal cerebral glucose metabolism distribution in the group of patients without infarcts/hemorrhages. However, Moore et al [21] also reported that glucose metabolism was decreased in some regions of white matter including areas with WMLs seen on the FLAIR-sequence on MRI. In our study WMLs were detected in 13 patients, however, we did not see any clear or obvious changes in glucose-metabolism corresponding to the WMLs. This could in part be explained by a small size of the WMLs and resolution limitations. In the analysis of glucose metabolism we used Neurostat as it has been shown that aid from this program increases sensitivity and reduces intra-observer variability in the identification of hypo- or hypermetabolic areas [22]. In another study, also by Moore et al [23], it was demonstrated that resting global cerebral blood flow (CBF) was not different between patients and controls; however, in specific brain regions (the brain stem, cerebellum, bilateral temporal, posterior occipital and inferior frontal regions) an increased regional CBF was seen in patients. After ERT the resting global CBF fell significantly in the treated group, whereas in the placebo group it increased together with an increase in regional CBF. Therefore, it was speculated if the elevated CBF in the placebo group could contribute to endothelial dysfunction and vessel wall dilation and result in an abnormal flow state together with an increased risk of emboli and thrombosis. As Fellgiebel states [5] it is unknown if ERT has long term protective effects against cere- brovascular events and only longitudinal studies including patients under ERT as well as untreated patients can answer this question. Unfortunately in our study, only six patients were FDG-PET-scanned both before and after commencement of treatment with ERT, ren- dering a comparison of scans before and after treatment with ERT impossible; in addition development of pathology detected with PET was only seen in four patients (follow-up 2–7 years); with MRI development of pathology was also detected in four patients, however not the same (follow-up 2–7 years). A third study—also by Moore et al [24] did not report any abnormalities in visual regional CBF reactivity suggesting normal neurovascular coupling mechanisms in patients with Fabry. However, a prolonged cerebral vascular response time after acetazolamid challenge was seen in patients with Fabry, suggesting an abnormality on the luminal side of the vessels. Discussion 25 are normalized to whole brain using a database of normal subjects and scaled to Z values from - 4.0 to 4.0 (Neurostat). Projections of Z values are shown onto, respectively, the right and left lateral hemispheric surfaces, the superior and inferior surfaces, and the anterior and posterior surfaces. The uptake in the left cerebellar hemisphere is reduced in addition to uptake in the right frontal cortex. MRI surface projections are presented for anatomical reference in a standard stereotactic space Fig 2. Quantitative 3-dimensional surface projection-analysis of FDG-uptake in patient no 25. PET-images of patient no. 25 are normalized to whole brain using a database of normal subjects and scaled to Z values from - 4.0 to 4.0 (Neurostat). Projections of Z values are shown onto, respectively, the right and left lateral hemispheric surfaces, the superior and inferior surfaces, and the anterior and posterior surfaces. The uptake in the left cerebellar hemisphere is reduced in addition to uptake in the right frontal cortex. MRI surface projections are presented for anatomical reference in a standard stereotactic space doi:10.1371/journal.pone.0143940.g002 doi:10.1371/journal.pone.0143940.g002 9 / 13 PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 Ethical Standards The study was approved by the Regional Ethics Committee (02-038/02, H-3-2014-FSP8), and informed consent was obtained from all participating subjects. There were few and minor findings on FDG-PET not detectable on MRI (diaschisis, discrete hypometabolism), however, In conclusion, we found that the majority of hypometabolic areas on FDG-PET corre- sponded to cerebral infarcts or hemorrhages detectable on conventional MRI of the brain. No areas with hypermetabolism on FDG-PET were detected. There were few and minor findings on FDG-PET not detectable on MRI (diaschisis, discrete hypometabolism), however, FDG-PET provided no additional clinical relevant information. As MRI can diagnose brain pathology and is without radiation in contrast to FDG-PET, where the patient is given a con- siderable dose of radiation, we suggest MRI as the preferable clinical modality when monitor- ing cerebral status in patients with Fabry disease FDG-PET provided no additional clinical relevant information. As MRI can diagnose brain pathology and is without radiation in contrast to FDG-PET, where the patient is given a con- siderable dose of radiation, we suggest MRI as the preferable clinical modality when monitor- ing cerebral status in patients with Fabry disease. However, as more and more patients with Fabry disease have implantable cardioverter defi- brillators or pacemakers (ICDs) due to heart disease, making MRI difficult to apply, FDG- PET/CT might be used in these patients, when monitoring brain function. To our knowledge a prospective brain FDG-PET-study spanning over several years has not yet been published, and we find it of great importance to point out that FDG-PET is not the clinical modality to choose when monitoring brain status in patients with Fabry disease unless MRI of the brain is not applicable. Brain Imaging with PET and MRI in Fabry Disease Fellgiebel et al [5] reported that seven of 25 patients (28%) with Fabry had markedly elevated WML-volumes, however, mean volume of WMLs between the rest of the patients was not sig- nificantly different from the control group. In addition they also studied mean diffusivity with MRI and mean diameter of the basilar artery, and found that basilar artery diameter was signifi- cantly larger in patients with Fabry than in controls. Global mean diffusivity was significantly elevated in Fabry patients compared to controls, and this has also previously been reported even in patients without WMLs, suggesting a sensitive marker of early brain involvement [27]. The pulvinar sign (a bilateral neuroradiological sign detected in T1-weighted MRI of the lat- eral part of the pulvinar—mostly seen in male patients—and considered expression of hyper- perfusion with subsequent dystrophic calcification of the pulvinar [28]) was seen in one of our patients (patient no 9). In a study by Burlina et al [29] the pulvinar sign was found in five male patients (out of 36 patients), and the sign was associated with cardiac disease and severe kidney involvement; however, in our study the patient with the pulvinar sign had no cardiovascular or renal disease. Reisin et al. [26] reported that the pulvinar sign was seen in one patient among 36 adult patients, which is in accordance with our results. Nine of the patients with cerebrovascular disease were female (of a total of 26 female patients), one was male (of a total of 14 male patients) (Table 1). In the Fabry Outcome Survey (FOS) the prevalence of stroke or transitory cerebral ischemia was also higher in female (15.7%) than in male (11.1%) patients [30]. In the group of patients with pathology detected with MRI, however with normal or proba- bly normal FDG-PET, one patient—with progression of WMLs during the study period—suf- fered from a cerebral infarct years later—this corresponds well to the fact that there is a known association between WMLs and stroke [31]. Another patient with WMLs and normal FDG-PET died a year after the study period—however, the cause of death was unknown. In conclusion, we found that the majority of hypometabolic areas on FDG-PET corre- sponded to cerebral infarcts or hemorrhages detectable on conventional MRI of the brain. No areas with hypermetabolism on FDG-PET were detected. As we did not investigate cerebral blood flow in our group of patients, we cannot collate the results. In our cohort of patients cerebral infarcts were detected on MRI in nine of 40 patients (22.5%), cerebral hemorrhage in one patient (2.5%), and WMLs were seen in 13 of 40 patients (32.5%). Median age of patients with WMLs was 58 years in comparison to 39 years (whole group). Lesions were punctate (grade 1) in five patients (12.5%) and confluent (grade 2 or 3) in eight patients (20.0%). In other studies of patients with Fabry disease focal WMLs were detected in respectively 22% and 28% of patients, and confluent WMLs were detected in respectively 33% and 17% of the patients [25,26]. These numbers corresponded fairly well to our result. Twelve patients in the latter study had two brain MRIs during the study period, and in eight patients no changes occurred during a follow-up of approximately 2 years, whereas new WMLs were identified in four patients [26]. This was in reasonable accordance with our results, where new lesions were identified in 3 of 40 patients during follow-up (range 3–7 years). Crutchfield [8] et al found that lesion burden detectable by MRI increased with age, and in addition they found that 37.5% of patients with WMLs had had a symptomatic stroke and 21.9% had had a transient ischemic attack (TIA), whereas only 6.2% of patients without WMLs on MRI had had a TIA and none had had a symptomatic stroke. In our cohort 13 patients had WMLs and 8 of these (69%) had suffered from a cerebrovascular event. Twenty-seven patients had no WMLs and two of these (7%) had suffered from a cerebrovascular event. 10 / 13 PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 References 1. Brady RO, Gal AE, Bradley RM, Martensson E, Warshaw AL, Laster L. Enzymatic defect in Fabry's dis- ease. Ceramidetrihexosidase deficiency. N Engl J Med. 1967; 276[21]:1163–1167. PMID: 6023233 2. Germain DP. Fabry disease. Orphanet J Rare Dis. 2010; 5:30. doi: 10.1186/1750-1172-5-30 PMID: 21092187 3. Eng CM, Guffon N, Wilcox WR, Germain DP, Lee P, Waldek S, et al. Safety and efficacy of recombinant human alpha-galactosidase A—replacement therapy in Fabry's disease. N Engl J Med. 2001; 345 [1]:9–16. PMID: 11439963 4. Schiffmann R, Kopp JB, Austin HA III, Sabnis S, Moore DF, Weibel T, et al. Enzyme replacement ther- apy in Fabry disease: a randomized controlled trial. JAMA 2001; 285[21]: 2743–2749. PMID: 11386930 5. Fellgiebel AM, Keller IM, Marin D, Muller MJM, Schermuly IP, Yakushev I, et al. Diagnostic utility of dif- ferent MRI and MR angiography measures in Fabry disease. Neurology. 2009; 72[1]:63–68. doi: 10. 1212/01.wnl.0000338566.54190.8a PMID: 19122032 6. Feldt-Rasmussen U. Fabry disease and early stroke. Stroke Res Treat. 2011;615218. 7. Mitsias P, Levine SR. Cerebrovascular complications of Fabry's disease. Ann Neurol. 1996; 40[1]:8– 17. PMID: 8687196 8. Crutchfield KE, Patronas NJ, Dambrosia JM, Frei KP, Banerjee TK, Barton NW, et al. Quantitative anal- ysis of cerebral vasculopathy in patients with Fabry disease. Neurology. 1998; 50[6]:1746–1749. PMID: 9633721 9. Moore DF, Kaneski CR, Askari H, Schiffmann R. The cerebral vasculopathy of Fabry disease. J Neurol Sci. 2007; 257[1–2]:258–263. PMID: 17362993 10. DeGraba T, Azhar S, gnat-George F, Brown E, Boutiere B, Altarescu G, et al. Profile of endothelial and leukocyte activation in Fabry patients. Ann Neurol. 2000; 47[2]:229–233. PMID: 10665494 11. Schiffmann R. Fabry disease. Pharmacol Ther. 2009; 122[1]:65–77. doi: 10.1016/j.pharmthera.2009. 01.003 PMID: 19318041 12. Moller AT, Feldt-Rasmussen U, Rasmussen AK, Sommer C, Hasholt L, Bach FW, et al. Small-fibre neuropathy in female Fabry patients: reduced allodynia and skin blood flow after topical capsaicin. J Peripher Nerv Syst. 2006; 11[2]:119–125. PMID: 16787509 13. Mersebach H, Johansson JO, Rasmussen AK, Bengtsson BA, Rosenberg K, Hasholt L, et al. Osteope- nia: a common aspect of Fabry disease. Predictors of bone mineral density. Genet Med. 2007; 9 [12]:812–818. PMID: 18091430 14. Prabakaran T, Birn H, Bibby BM, Regeniter A, Sorensen SS, Feldt-Rasmussen U, et al. Long-term enzyme replacement therapy is associated with reduced proteinuria and preserved proximal tubular function in women with Fabry disease. Nephrol Dial Transplant. 2014; 29[3]:619–625. doi: 10.1093/ndt/ gft452 PMID: 24215016 15. Author Contributions Conceived and designed the experiments: UFR LH IL. Performed the experiments: UFR HG BB AKR. Analyzed the data: KK IL. Wrote the paper: KK. Manuscript editing and final approval: KK UFR HG LH BB AKR IL. 11 / 13 PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 Brain Imaging with PET and MRI in Fabry Disease PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 References Lewellen TK, Kohlmyer SG, Miyaoka RS, Kaplan MS, Stearns CW, Schubert SF. Investigation of the performance of the General Electric ADVANCE positron emission tomograph in 3D mode. Nuclear Sci- ence, IEEE Transactions on. 1996; 43[4]:2199–2206. 16. 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PMID: 17321549 21. Moore DF, Altarescu G, Barker WC, Patronas NJ, Herscovitch P, Schiffmann R. White matter lesions in Fabry disease occur in 'prior' selectively hypometabolic and hyperperfused brain regions. Brain Res Bull. 2003; 62[3]:231–240. PMID: 14698356 22. Drzezga A, Arnold S, Minoshima S, Noachtar S, Szecsi J, Winkler P, et al. 18F-FDG PET studies in patients with extratemporal and temporal epilepsy: evaluation of an observer-independent analysis. J Nucl Med. 1999; 40[5]:737–746. PMID: 10319744 12 / 13 PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 Brain Imaging with PET and MRI in Fabry Disease 23. Moore DF, Scott LT, Gladwin MT, Altarescu G, Kaneski C, Suzuki K, et al. Regional cerebral hyperper- fusion and nitric oxide pathway dysregulation in Fabry disease: reversal by enzyme replacement ther- apy. Circulation. 2001; 104[13]:1506–1512. PMID: 11571244 24. Moore DF, Altarescu G, Herscovitch P, Schiffmann R. Enzyme replacement reverses abnormal cere- brovascular responses in Fabry disease. BMC Neurol. 2002; 2:4. PMID: 12079501 25. Fellgiebel AM, Muller MJM, Mazanek MM, Baron K, Beck MM, Stoeter PM. White matter lesion severity in male and female patients with Fabry disease. Neurology. 2005; 65[4]:600–602. PMID: 16116124 26. Reisin RC, Romero C, Marchesoni C, Napoli G, Kisinovsky I, Caceres G, et al. Brain MRI findings in patients with Fabry disease. J Neurol Sci. 2011; 305[1–2]:41–44. PLOS ONE | DOI:10.1371/journal.pone.0143940 December 2, 2015 References doi: 10.1016/j.jns.2011.03.020 PMID: 21463870 27. Fellgiebel A, Mazanek M, Whybra C, Beck M, Hartung R, Muller KM, et al. Pattern of microstructural brain tissue alterations in Fabry disease: a diffusion-tensor imaging study. J Neurol. 2006; 253[6]:780– 787. PMID: 16511647 28. Moore DF, Ye F, Schiffmann R, Butman JA. Increased signal intensity in the pulvinar on T1-weighted images: a pathognomonic MR imaging sign of Fabry disease. Am J Neuroradiol. 2003; 24[6]:1096– 1101. PMID: 12812932 29. Burlina A, Manara R, Caillaud C, Laissy JP, Severino M, Klein I, et al. The pulvinar sign: frequency and clinical correlations in Fabry disease. Journal of Neurology. 2008; 255[5]:738–744. doi: 10.1007/ s00415-008-0786-x PMID: 18297328 30. Mehta A, Ginsberg L. Natural history of the cerebrovascular complications of Fabry disease. Acta Pae- diatr Suppl. 2005; 94[447]:24–27. PMID: 15895708 31. Buyck JF, Dufouil C, Mazoyer B, Maillard P, Ducimetiere P, Alperovitch A, et al. Cerebral white matter lesions are associated with the risk of stroke but not with other vascular events: the 3-City Dijon Study. Stroke. 2009; 40[7]:2327–2331. doi: 10.1161/STROKEAHA.109.548222 PMID: 19443799 13 / 13
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Penentuan Karakteristik Aspal Porus Menggunakan Agregat Kasar Batu Pecah Parengan Tuban Dengan Metode Uji Marshall
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ISSN: 2502-3152 ISSN: 2502-3152 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 Erik Dwi Setiawan1, Alfia Nur Rahmawati2, Soegyarto2 1Mahasiswa Program Studi Teknik Sipil, Fakultas Sain dan Tenik, Universitas Bojonegoro 2Dosen Program Studi Teknik Sipil, Fakultas Sain dan Tenik, Universitas Bojonegoro ABSTRAK Aspal porus merupakan teknologi perkerasan jalan dengan beberapa kelebihan seperti mengurangi aquaplaning dan meredam kebisingan. Aspal porus saat ini sedang terus dikembangkan oleh beberapa negara, tak terkecuali di Indonesia. Penelitian ini secara umum bertujuan untuk mengetahui kinerja perkerasan campuran aspal porus menggunakan agregat kasar batu pecah Parengan dan dengan aspal penetrasi 60/70. Analisis kinerja fungsi kekuatan campuran aspal porus dilakukan dengan Marshall test dengan melihat hasil pengujian parameter seperti Stabilitas, VMA, VFB, VIM, kelelehan (flow) dan MQ (Marshall quotient). Untuk analisis resapan campuran aspal porus dilakukan dengan metode Falling Head Permeability (FHP), dimana air di dalam tabung jatuh bebas dengan ketinggian tertentu sampai melewati rongga pada campuran aspal berpori menggunakan rentang kadar aspal varisai rencana antara lain 4%, 5%, 6% dan 7%. Hasil penelitian didapatkan Stabilitas aspal porus dari empat variasi kadar aspal rata-rata yaitu 248,3 Kg. Untuk nilai rata-rata VIM pada empat variasi kadar aspal yaitu 10,78%. Nilai rata-rata flow pada empat variasi kadar aspal 1,31 mm. Untuk nilai rata-rata VMA pada empat variasi kadar aspal yaitu 19,33%. Nilai rata-rata VFB pada empat variasi kadar aspal yaitu 44,3 gr/cc. Dan nilai Marshall Quetient 237,43 Kg/mm. Hasil penelitian permeabilitas aspal porus pada empat variasi kadar aspal yaitu 0,366 cm/s. p y Kata Kunci : Aspal Porus, Karakteristik, Marshall, permeabilitas Kata Kunci : Aspal Porus, Karakteristik, Marshall, permeabilitas Abstract Abstract 34 Porous asphalt is a road pavement technology with several advantages such as reducing aquaplaning and reducing noise. Porous asphalt is currently being developed by several countries, including Indonesia. In general, this study aims to determine the performance of a porous asphalt mixture pavement using coarse aggregate of Parengan crushed stone and with 60/70 penetration asphalt. Performance analysis of the strength function of the porous asphalt mixture was carried out using the Marshall test by looking at the results of testing parameters such as stability, VMA, VFB, VIM, flow and MQ (Marshall quotient). For the absorption analysis of the porous asphalt mixture, the Falling Head Permeability (FHP) method is carried out, where the water in the tube falls freely with a certain height until it passes through the cavity in the porous asphalt mixture using the design variation of asphalt content, including 4%, 5%, 6% and 7%. The results showed that the porous asphalt stability of the four variations of the average asphalt content was 248.3 Kg. For the average value of VIM on the four variations of asphalt content is 10.78%. The average value of flow in the four variations of asphalt content is 1.31 mm. For the average value of VMA on the four variations of asphalt content is 19.33%. The average value of VFB in the four variations of asphalt content is 44.3 gr/cc. And the Marshall 34 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 ISSN: 2502-3152 ISSN: 2502-3152 Quetient value is 237.43 Kg/mm. The results of the research on the permeability of porous asphalt in four variations of asphalt content are 0.366 cm/s. Keywords: Porous Asphalt, Characteristics, Marshall, permeability 1. Pendahuluan . e da u ua Aspal porus merupakan salah satu inovasi untuk meningkatkan resapan air hujan menuju saluran drainase untuk mengurangi adanya genangan di atas jalan. Fungsi aspal porus biasanya di gunakan pada jalan kecil dan fasilitas parkir kendaraan ringan. Dikutip dari Sofyan (2014) yang di sebutkan pada Australian Asphalt Pavement Association / AAPA, (2004) aspal porus harus diposisikan pada lapisan permukaan jalan sebagai lapisan yang bersifat non struktural dan diatas lapisan pondasi yang kedap air (Asphalt Treated Base). Aspal porus juga salah satu alternatif untuk meningkatkan keselamatan di jalan dan dapat mengurangi kebisingan (noise). Aspal porus dibuat untuk mendapatkan kadar rongga yang lebih besar untuk meneruskan aliran air kesaluran drainase samping dan lapisan dasar yang kedap air untuk mencegah air meresap ke badan jalan. Sehingga genangan air yang berada di atas jalan dapat diatasi. Kondisi ini bisa terjadi karena gradasi yang digunakan merupakan (open graded) gradasi terbuka yang mempunyai fraksi agregat kasar 85%-95% dari berat total campuran sehingga hasil strukturnya memiliki rongga antar butir yang lebih tinggi dan memiliki permeabilitas yang tinggi juga. Aspal porus juga dapat digunakan sebagai anti slip sehingga mampu mengurangi angka kecelakaan lalu lintas yang terjadi. Porositas yang cukup tinggi pada aspal porus akan berdampak pada umur aspal porus sendiri. Sehingga umur pelayanan aspal porus sendiri lebih singkat daripada kontruksi perkerasan konvensional. Ini dikarenakan porositas yang tinggi sehingga stabilitasnya kecil (Takahashi dkk, 1999). Dalam penelitian ini menggunakan pengikat aspal penetrasi 60/70 yang kebanyakan sering digunakan di Indonesia. Aspal dengan penetrasi 60/70 sesuai bila digunakan di Indonesia karena memiliki iklim dan cuaca yang panas serta volume lalu lintas yang sedang dan tinggi. Dalam pengerjaan Aspal Porus, aspal merupakan faktor utama dalam pembuatan pekerjaan perkerasan lentur. Agregat dengan volume terbesar turut berperan dalam kekuatan perkerasan lentur. Untuk mencapai umur yang di rencanakan maka diperlukan mutu dan kualitas material yang memenuhi syarat. Maka tingkat ketahanan dan keawetan konstruksi jalan akan lebih baik. Pada penelitian ini memanfaatkan material dari batu pecah sebagai agregat kasar penyusun perkerasan lentur. Batu pecah yang akan di gunakan dalam penelitian ini berasal dari Kec. Parengan. 35 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 ISSN: 2502-3152 ISSN: 2502-3152 Sejauh ini karena terbatasnya pengalaman penelitian mengenai pembuatan aspal porus menggunakan agregat kasar batu pecah pada daerah parengan tuban. Maka dari itu penelitian mengenai sifat-sifat aspal porus ini perlu dilakukan. 2.1 Aspal Porus Aspal porus merupakan campuran beraspal yang memiliki banyak rongga-rongga udara sehingga memungkinkan untuk mengalirkan air secara vertikal maupun horizontal melalui rongga- rongga udara yang ada. Rongga-rongga udara yang terbentuk dalam campuran aspal porus sengaja direncanakan dengan penggunaan agregat bergradasi terbuka dan didominasi dengan agregat kasar sebanyak 80%-95%, sehingga dengan dominasi ukuran agregat yang relatif sama maka akan ada bagian rongga yang tidak terisi oleh agregat dan aspal sehingga akan membentuk rongga-rongga udara yang saling berkesinambungan. Rongga udara inilah yang akan membuat campuran aspal porus dapat bersifat porus atau mengalirkan air. Adapun porositas untuk campuran aspal porus berkisar antara 18%-25%. Menurut Sanusi dan Setyawan (2008), campuran aspal porus merupakan generasi baru dalam perkerasan lentur, yang membolehkan air meresap ke dalam lapisan atas wearing course secara vertikal dan horizontal . Lapisan ini menggunakan gradasi terbuka open graded yang dihamparkan diatas lapisan aspal yang kedap air. Lapisan aspal porus ini secara efektif dapat memberikan tingkat keselamatan yang lebih, terutama diwaktu hujan agar tidak terjadi aquaplaning sehingga menghasilkan kekesatan permukaan yang lebih kasar, dan dapat mengurangi kebisingan noise reduction. 3. Metode Penelitian Dalam penelitian ini menggunakan metode eksperimental, agar dapat menentukan apakah batu pecah parengan bisa sebagai bahan aspal porus. 3.2 Teknik Analisis Data Metode analisis yang digunakan dalam penelitian ini adalah metode analisis kuantitatif yaitu metode dengan menggunakan perhitungan secara matematis dengan Uji Marshall mengacu pada SNI 06-2489-1991. Dengan standart gradasi Australian Asphalt Pavement Assosiation (AAPA) dengan 24 benda uji dengan variasi aspal 4%-7%. 3.1 Teknik Pengumpulan Data Dalam penelitian ini semua data yang digunakan adalah data primer yang merupakan hasil uji dari laboratorium. Mulai dari hasil uji agregat samapai data dari hasil uji marshal. 2.2 Spesifikasi Aspal Porus Dengan Metode Marshall Pengujian kinerja aspal padat dilakukan melalui pengujian Marshall, yang dikembangkan pertama kali oleh Bruce Marshall dan dilanjutkan oleh U.S. Corps Engineer. Kinerja aspal padat ditentukan melalui pengujian benda uji yang meliputi: enentuan berat volume benda uji 1. Penentuan berat volume benda uji 1. Penentuan berat volume benda uji 2. Pengujian nilai stabilitas, adalah kemampuan maksimum beton aspal menerima beban sampai terjadi kelelahan plastis. 3. Pengujian kelelahan (flow), adalah besarnya perubahan bentuk plastis dari beton aspal padat, akibat adanya beban sampai batas keruntuhan. 4. Perhitungan kofisien marshall, adalah perbandingan antara nilai stabilitas dan flow. 5. Perhitungan berbagai jenis volume pori dalam beton aspal padat (VIM, VMA dan VFB). 36 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 ISSN: 2502-3152 ISSN: 2502-3152 Berdasarkan data diatas ketententuan spesifikasi aspal porus adalah sebagai berikut : Tabel. 1 Ketentuan Spesifikasi Aspal Porus No. Spesifikasi Ashpalt Porus Nilai 1 Koefisien Permeabilitas (cm/s) 0,1 - 0,5 2 Kadar Rongga di Dalam Campuran (VIM%) 18 - 25 3 Void In Mineal Agregate (VMA%) - 4 Void Filled with Bitumen(VFB%) - 5 Stabilitas Marshall (Kg) min. 500 6 Kelelehan Marshal/Flow (mm) 2 - 6 7 Marshall Question (Kg/mm) Min. 250 8 Jumlah Tumbukan Perbidang 75 Sumber (AAPA,2004) Tabel. 1 Ketentuan Spesifikasi Aspal Porus 4.1 Pengujian Gradasi Agregat Halus Pengujian gradasi agregat halus dilakukan untuk mengetahui nilai analisis saringan yang akan di gunakan untuk pembuatan gradasi gabungan sehingga dapat di gunakan dalam pembuatan benda uji. Uji agregat halus menggunakan 2 sampel, dengan tujuan untuk mencari nilai rata-rata agregat halus. Adapun hasil pengujian gradasi agregat halus dengan mengacu pada SNI 03-1968-1990 dapat dilihat pada tabel 2 sebagai mana berikut. 37 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 ISSN: 2502-3152 Tabel 2. Hasil uji ayakan agregat halus UKURAN AYAKAN SAMPEL – 1 SAMPEL – 2 AVER AGE Berat terting gal (gr) komula tif tertahan (gr) komula tif tertaha n % lolos (gr) % Berat tertin ggal (gr) Komul atif Tertaha n (gr) Komul atif Tertaha n % Lolos (gr) % (mm) No. 19,1 3/4" 0 0 0 100 0 0 0 100 100 13,2 1/2" 0 0 0 100 0 0 0 100 100 9,60 3/8" 0 0 0 100 0 0 0 100 100 6,7 1/4" 0 0 0 100 0 0 0 0 100 4,80 4 6 6 0.24 99.76 10 10 0.4 99.6 99.68 2,40 8 30 36 1.44 98.56 34 44 1.76 98.24 98.4 1,20 16 43 79 3.16 96.84 69 113 4.52 95.48 96.16 0,60 30 1917 1996 79.84 20.16 1682 1795 71.8 28.2 24.18 0,30 50 293 2289 91.56 8.44 453 2248 89.92 10.08 9.26 0,15 100 77 2366 94.64 5.36 111 2359 94.36 5.64 5.5 0,075 200 121 2487 99.48 0.52 128 2487 99.48 0.52 0.52 PAN PAN 13 2500 100 0 13 2500 100 0 0 JUMLAH 2500 2500 (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2022) (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2022) 4.2. Pengujian Agregat Kasar Agregat kasar yang berasal dari parengan dilakukan pengujian analisis saringan dengan acuan SNI 03-1968-1990. Analisis saringan pada agregat kasar batu pecah Parengan Tuban dengan ukuran 0,5 dilakukan 2 kali pada ukuran saringan 3/4 “ dan 1/2 “ agregat tertinggal 0 gr. Pada analisis 1 ukuran saringan 3/8 “ berat tertinggal 14 gr, 1/4 “ tertinggal 892 gr, 4 “ tertinggal 1251 gr, 8 “ tertinggal 176 gr dan di Pan tertinggal 167 gr. Pada analisis 2 ukuran saringan 3/8 “ berat tertinggal 11 gr, 1/4 “ tertinggal 890 gr, 4 “ tertinggal 1253 gr, 8 “ tertinggal 170 gr dan di Pan tertinggal 176 gr. Begitu juga ukuran 1 : 2 dapat dilihat pada tabel.Tabel 4.2 Hasil uji ayakan agregat kasar ukuran 0,5 38 Tabel 3. Hasil uji ayakan agregat kasar UKURAN AYAKAN SAMPEL – 1 SAMPEL – 2 AVER AGE Berat tertin ggal (gr) komul atif tertaha n (gr) komul atif tertaha n % lolos (gr) % Berat terting gal (gr) Komul atif Tertaha n (gr) Komul atif Tertah an % Lolos (gr) % (mm) No. 19,1 3/4" 0 0 0 100 0 0 0 100 100 13,2 1/2" 0 0 0 100 0 0 0 100 100 9,60 3/8" 14 14 0.56 99.44 11 11 0.44 99.56 99.5 6,7 1/4" 892 906 36.24 63.76 890 901 36.04 63.96 63.86 4,80 4 1251 2157 86.28 13.72 1253 2154 86.16 13.84 13.78 Tabel 3. Hasil uji ayakan agregat kasar 38 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 ISSN: 2502-3152 UKURAN AYAKAN SAMPEL – 1 SAMPEL – 2 AVER AGE Berat tertin ggal (gr) komul atif tertaha n (gr) komul atif tertaha n % lolos (gr) % Berat terting gal (gr) Komul atif Tertaha n (gr) Komul atif Tertah an % Lolos (gr) % (mm) No. 2,40 8 176 2333 93.32 6.68 170 2324 92.96 7.04 6.86 PAN 167 2500 100 0 176 2500 100 0 0 JUMLAH 2500 2500 (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2020) Tabel 4. Hasil uji ayakan agregat kasar ukuran 1 : 2 UKURAN AYAKAN SAMPEL – 1 SAMPEL – 2 AVER AGE Berat terting gal (gr) komul atif tertaha n (gr) komul atif tertaha n % lolos (gr) % Berat terting gal (gr) Komul atif Tertah an (gr) Komulat if Tertaha n % Lolos (gr) % (mm) No. 4.2. Pengujian Agregat Kasar 19,1 3/4" 0 0 0 100 0 0 0 100 100 13,2 1/2" 485 485 19.4 80.6 487 487 19.48 80.52 80.56 9,60 3/8" 1344 1829 73.16 26.84 1340 1827 73.08 26.92 26.88 6,7 1/4" 301 2130 85.2 14.8 305 2132 85.28 14.72 14.76 4,80 4 230 2360 94.4 5.6 232 2364 94.56 5.44 5.52 2,40 8 115 2475 99 1 113 2477 99.08 0.92 0.96 PAN 25 2500 100 0 23 2500 100 0 0 JUMLAH 2500 2500 (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2020) Tabel 4. Hasil uji ayakan agregat kasar ukuran 1 : 2 (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2020) 4.3. Pengujian Filler Pengujian filler semen Portland ini dilakukan dengan spesifikasi SNI 03-1968-1990. Dengan hasil pengujian sebagai berikut Pengujian filler semen Portland ini dilakukan dengan spesifikasi SNI 03-1968-1990. Dengan hasil pengujian sebagai berikut Tabel 5. Hasil uji ayakan filler UKURAN AYAKAN SAMPEL – 1 SAMPEL – 2 AVER AGE Berat tertin ggal (gr) kom ulatif terta han (gr) komul atif tertaha n % lolos (gr) % Berat tertin ggal (gr) Komul atif Tertah an (gr) Kom ulatif Terta han % Lolos (gr) % (mm) No. 0,30 50 0 0 0 0 0 0 0 0 0 0,15 100 0 0 0 100 0 0 0 100 100 0,075 200 4 4 0.8 99.2 3 3 0.6 99.4 99.3 PAN 496 500 100 0 497 500 100 0 0 JUMLAH 500 500 (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2020) Tabel 5. Hasil uji ayakan filler 39 ISSN: 2502-3152 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 4.4 Pengujian Berat Jenis Dan Penyerapan Agregat Agregat kasar yang berasal dari parengan dan pamotan di lakukan pengujian berat jenis dan penyerapan dengan mengacu pada SNI 03-1969-1990, sedangkan agregat halus yang berasal dari sungai bengawan solo yaitu daerah Gelagahsari, dilakukan pengujian berat jenis dan penyerapan dengan mengacu pada SNI 03-1970-1990. Berikut adalah hasil pengujian berat jenis dan penyerapan agregat. Tabel 6. Berat Jenis Agregat No. Agregat Jenis Persyaratan Hasil Satuan Pengujian Min. Maks. Agregat Berat jenis bulk 2,5 - 2,50 gr/cc 1. kasar Berat jenis SSD 2,5 - 2,56 gr/cc Size 0,5 Berat jenis semu 2,5 - 2,55 gr/cc Penyerapan - 3 2,43 % Agregat Berat jenis bulk 2,5 - 2,51 gr/cc 2. kasar Berat jenis SSD 2,5 - 2,56 gr/cc Size 1 : 2 Berat jenis semu 2,5 - 2,64 gr/cc Penyerapan - 3 1,94 % 3. Agregat Halus Berat jenis bulk 2,5 - 2,61 gr/cc Berat jenis SSD 2,5 - 2,69 gr/cc Berat jenis semu 2,5 - 2,83 gr/cc Penyerapan - 3 2,88 % (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2020) Tabel 6. Berat Jenis Agregat 4.5 Gradasi Gabungan Pada perencanaan gradasi agregat gabungan dilakukan berdasarkan hasil pengujian dari agregat yang akan digunakan untuk campuran dengan spesifikasi AAPA, 2004. Pada perencanaan gradasi agregat gabungan dilakukan berdasarkan hasil pengujian dari agregat yang akan digunakan untuk campuran dengan spesifikasi AAPA, 2004. Tabel 7. Gradasi Gabungan Aspal Porus Tabel 7. Gradasi Gabungan Aspal Porus SIEVE SIZE HOT BIN I HOT BIN II HOT BIN III FILLER jumlah Spesifikasi BB BA 3/4" 19,1 100.00 100.00 100.00 100.00 100 100 100 1/2" 13,2 80.56 100.00 100.00 100.00 89.11 85 100 3/8" 9,60 26.88 99.50 100.00 100.00 58.87 45 70 1/4" 6,7 14.76 63.86 100.00 100.00 39.61 25 45 NO 4 4,80 5.52 13.78 99.68 100.00 16.89 10 25 NO 8 2,40 0.96 6.68 98.40 100.00 11.79 7 15 NO 16 1,20 0.00 0.00 96.16 100.00 8.80 6 12 NO 30 0,60 0.00 0.00 24.18 100.00 5.20 5 10 NO 50 0,30 0.00 0.00 9.26 100.00 4.46 4 8 NO 100 0,15 0.00 0.00 5.50 100.00 4.27 3 7 NO 200 0,075 0.00 0.00 0.52 99.30 3.99 2 5 40 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 ISSN: 2502-3152 (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2020) (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2020) Gambar 1. Gradasi Agregat gabungan 0 20 40 60 80 100 19,1 13,2 9,60 6,7 4,80 2,40 1,20 0,60 0,30 0,15 0,075 3/4" 1/2" 3/8" 1/4" NO 4 NO 8 NO 16 NO 30 NO 50 NO 100 NO 200 gradasi Bts bwh bts ats (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2020) (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2020) Gambar 1. Gradasi Agregat gabungan 4.6 Job Mix Formula Pada penelitian ini digunakan metode trial and error yang mengacu pada persyaratan Bina Marga 2018 dan AAPA, 2004.Berikut adalah hasil Job mix formula : Pada penelitian ini digunakan metode trial and error yang mengacu pada persyaratan Bina Marga 2018 dan AAPA, 2004.Berikut adalah hasil Job mix formula : Tabel 8. Job Mix Formula No. Kadar Aspal Agregat Kapasitas Hot Bin I Hot Bin II Hot Bin III Filler 1 49.2 661.25 413.28 59.04 47.23 1230 2 61.5 654.36 408.97 59.04 46.74 1230 3 73.8 647.47 413.28 57.81 46.25 1230 4 86.1 640.58 413.28 57.2 45.76 1230 (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2020) Gambar 2. Grafik Asphalt Flow Down Gambar 2. Grafik Asphalt Flow Down p Dari Gambar 2 nilai AFD tertinggi untuk aspal dengan agregat kasar Parengan berada pada kadar aspal 7% dengan nilai 0,29% sedangkan terendah dengan kadar aspal 4% dengan nilai 0,18% (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2020) 4.7. Aspal Flow Down 4.7. Aspal Flow Down 41 Besarnya asphalt flow down dapat dihitung dengan persamaan : AFD = [ (m3-m1) / (m2-m1) ] x 100 0,18 0,22 0,25 0,29 y = 1E-16x2 + 0,036x + 0,037 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 3 4 5 6 7 8 AFD % Kadar Aspal AFD Poly. (AFD) Besarnya asphalt flow down dapat dihitung dengan persamaan : AFD = [ (m3-m1) / (m2-m1) ] 100 Besarnya asphalt flow down dapat dihitung dengan persamaan : AFD = [ (m3-m1) / (m2-m1) ] x 100 41 ISSN: 2502-3152 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 4.8 Pengukuran Dimensi Dan Berat Benda Uji Pengujian benda uji bertujuan untuk mencari tinggi dan diameter benda uji dari tiga sisi permukaan dan menimbang berat benda uji baik itu berat di udara, berat dalam air, dan berat SSD, sehingga dapat di gunakan untuk menghitung hasil marshall Tabel 9. Pengukuran Dimensi Benda Uji Aspal Porus PRES ENTA SE ASPA L KOD E BEN DA UJI TINGGI DIAMETER RATA-RATA TOTAL sisi 1 sisi 2 sisi 3 sisi 1 sisi 2 sisi 3 Ting gi Dmt ting gi dm t AP 4% AP1 79.8 77.1 77.9 103.7 102.4 104.7 78.2 103.6 77.22 102.90 AP2 77.2 77.9 75.6 102.4 102.5 102.1 76.9 102.3 AP3 76.8 76.1 74.8 102.2 102.2 102.2 75.9 102.2 AP4 76.7 77.0 76.9 102 102.2 102.2 76.9 102.1 AP5 75.8 76.6 76.1 102.6 102.8 103.0 76.2 102.8 AP6 78.9 79.8 78.3 103.6 104.7 103.1 79.0 104.1 AP 5% AP1 75.5 76.1 76.4 101.9 102.1 102.7 76.0 102.2 76.47 102.52 AP2 75.7 76.5 77.4 103.0 102.8 103.5 76.5 103.6 AP3 77.7 76.9 78.1 102.4 102.1 102.8 77.6 102.4 AP4 75.1 75.6 72.4 102.1 101.9 102.2 74.4 102.1 AP5 77.9 76.0 75.7 102.5 102.4 102.1 76.5 102.3 AP6 78.1 78.0 76.7 102.1 103.0 102.9 77.6 102.7 AP 6% AP1 76.5 75.4 75.6 103.3 103.3 103.5 75.8 103.4 76.40 103.43 AP2 74.3 72.5 74.5 102.3 102.3 102.2 73.8 102.3 AP3 77.2 79.3 77.5 102.7 102.7 103.8 78.0 103.1 AP4 77.0 76.5 77.5 102.3 102.3 102.4 77.0 102.4 AP5 77.0 76.3 76.6 106.9 106.9 102.5 76.6 105.4 AP6 77.5 76.9 76.2 103.9 103.6 103.9 76.9 103.7 AP 7% AP1 77.5 76.1 76.3 102.0 102.8 102.2 76.6 102.3 75.86 102.81 AP2 75.6 76.8 74.3 102.3 102.3 102.3 75.6 102.3 AP3 76.7 77.8 76.2 102.9 103.3 103.3 76.9 103.1 AP4 75.9 73.4 74.6 103.0 102.7 102.7 74.6 102.8 AP5 74.5 74.5 74.0 103.6 103.8 103.8 74.3 103.7 AP6 76.7 75.8 77.9 102.4 102.3 102.3 76.8 102.3 (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2020) 42 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 ISSN: 2502-3152 Tabel 10. 4.9.1 Kepadatan (Density) Nilai density tertinggi untuk aspal dengan agregat kasar Parengan berada pada kadar aspal 4% dengan nilai 2,21 gr/cc sedangkan terendah dengan kadar aspal 6% dengan nilai 2,11 gr/cc. 4.8 Pengukuran Dimensi Dan Berat Benda Uji Data Berat Benda Uji PRESENT ASE ASPAL KODE BENDA UJI BERAT KERIN G BERAT SSD BERAT DLM AIR RATA-RATA KERING SSD DLM AIR AP 4% AP1 1183 1212 671 1180.66 1206.17 670.83 AP2 1201 1231 678 AP3 1174 1200 670 AP4 1167 1178 667 AP5 1180 1202 664 AP6 1179 1214 675 AP 5% AP1 1198 1219 669 1204.17 1225.83 670.50 AP2 1211 1230 671 AP3 1191 1217 673 AP4 1176 1203 666 AP5 1228 1248 673 AP6 1221 1238 671 AP 6% AP1 1228 1240 664 1219.83 1234.50 657.50 AP2 1206 1220 657 AP3 1224 1242 659 AP4 1209 1231 658 AP5 1236 1240 652 AP6 1216 1234 655 AP 7% AP1 1207 1222 650 1209.33 1223.33 652.83 AP2 1194 1211 646 AP3 1201 1219 649 AP4 1224 1235 665 AP5 1205 1218 650 AP6 1225 1235 657 (Sumber : Hasil pengujian Laboratorium Teknik Sipil Unigoro,2020) Tabel 10. Data Berat Benda Uji 4.9 Pengujian Marshall ( ) Nilai VIM tertinggi dengan kadar campuran aspal 6% sebesar 11.4%, nilai VIM terendah dengan kadar campuran limbah 4% dengan nilai 10,1% . 4.9.5. Pelelehan (flow) Nilai flow tertinggi dengan kadar campuran aspal 4% sebesar 1,71 mm dan nilai flow terendah dengan kadar campuran aspal 5% sebesar 0,91 mm. Berdasarkan Spesifikasi AAPA ,2004, nilai persyaratan flow adalah 2-6 mm. 4.9.6. Stabilitas Nilai stabilitas tertinggi terdapat pada benda uji dengan kadar campuran aspal 7% dengan nilai 423,41 kg, nilai stabilitas terendah berada pada benda uji dengan kadar campuran aspal 4% dengan nilai 187,24 kg. 4.9.2. VIM (Void In Mix) Nilai VIM tertinggi dengan kadar campuran aspal 6% sebesar 11.4%, nilai VIM terendah dengan kadar campuran limbah 4% dengan nilai 10,1% . 4.9.3. VMA (Void In Mineral Agregate) 43 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 ISSN: 2502-3152 Nilai VMA tertinggi dengan kadar campuran aspal 6% sebesar 21,5%, nilai VMA terendah dengan kadar campuran aspal 4% dengan nilai 16,3% 4.9.7. Marshall quantity Nilai MQ tertinggi dengan kadar campuran aspal 7% sebesar 358,82 kg/mm, nilai MQ terendah dengan kadar campuran limbah plastik 4% sebesar 109,50 kg/mm. 4.9.8. Pengujian Koefisien Permebilitas Nilai rata-rata dari pengujian permeabilitas adalah 0,366. Dang angka tersebut masih bisa di kategorikan aspal porus. Nilai rata-rata dari pengujian permeabilitas adalah 0,366. Dang angka tersebut masih bisa di kategorikan aspal porus. 4.9.4. VFB (Void Filled With Bitumen) Nilai VFB tertinggi dengan kadar campuran aspal 7% sebesar 47,50%, nilai VFB terendah dengan kadar campuran aspal 4% sebesar 38,50% 6.Saran Pada penelitian selanjutnya disarankan bisa menggunakan bahan tambah atau bahan subtitusi sebagai bahan campuran aspal porus sehingga bisa memenuhi spesifikasi yang ditentukan. 5. Kesimpulan Hasil pengujian Marshall test ada lah sebagai berikut : Hasil pengujian Marshall test ada lah sebagai berikut : ➢ Berdasarlan teori agregat bergradasi senggang memberikan rongga dalam campuran (VIM) yang sedang maka menghasilkan nilai stabilitas yang sedang. Dibuktikan dengan hasil pada kadar aspal 7% memiliki nilai VIM yang sedang 11,2% sehingga nilai stabilitas yang dihasilkan tidak begitu tinggi yaitu sebesar 423,41 Kg, Nilai VIM dari empat variasi kadar aspal memiliki rata-rata 10,78% ➢ Nilai Flow yang rendah mengindikasikan campuran tidak bersifat plastis dan belum mampu mengikuti deformasi akibat beban. Dari hasil pengujian didapatkan pada kadar 4% nilai Flow diketahui paling tinggi yaitu sebesar 1.71mm. Nilai Flow dari empat variasi kadar aspal memiliki rata-rata 1,30mm. ➢ Kenaikan dan penurunan nilai MQ dipengaruhi oleh hasil bagi antara stabilitas dan flow, mengindikasikan pendekatan kekakuan dan fleksibilitas dari suatu campuran aspal. Pada 44 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 ISSN: 2502-3152 ISSN: 2502-3152 kadar 7% menghasilkan nilai MQ tertinggi sebesar 358.82 Kg/mm. Nilai MQ dari empat variasi kadar aspal memiliki rata-rata 237,43 Kg/mm kadar 7% menghasilkan nilai MQ tertinggi sebesar 358.82 Kg/mm. Nilai MQ dari empat variasi kadar aspal memiliki rata-rata 237,43 Kg/mm kadar 7% menghasilkan nilai MQ tertinggi sebesar 358.82 Kg/mm. Nilai MQ dari empat variasi kadar aspal memiliki rata-rata 237,43 Kg/mm kadar 7% menghasilkan nilai MQ tertinggi sebesar 358.82 Kg/mm. Nilai MQ dari empat variasi kadar aspal memiliki rata-rata 237,43 Kg/mm ➢ Stabilitas merupakan kemampuan maksimum aspal menerima beban sampai terjadi kelelehan plastis. Pada kadar aspal 7% menghasilkan nilai Stabilitas tertinggi sebesar 423,41 Kg. Nilai Stabilitas dari empat variasi kadar aspal memiliki rata-rata 248,3 Kg. ➢ Nilai Permeabilitas rata-rata aspal porus sebesar 0,366 cm/s dan memenuhi standar spesifikasi aspal porus yaitu antara 0,1 – 0,5 cm/s. Dari Hasil diatas didapatkan bahwa Aspal Porus dengan menggunakan agregat kasar batu pecah parengan Tuban belum mampu mencapai standart spesifikasi yang ditentukan AAPA, 2004 Dari Hasil diatas didapatkan bahwa Aspal Porus dengan menggunakan agregat kasar batu pecah parengan Tuban belum mampu mencapai standart spesifikasi yang ditentukan AAPA, 2004 7.Daftar Pustaka AAPA, Australia Asphalt Pavement Association, (1997). Open Graded Asphalt Design Guide, Australia. Australia Asphalt Pavement Association (AAPA), (2004). National Asphalt Specification. Australian Asphalt pavement Association (AAPA). Road Engineering Association of Australian Joint with Jabatan Kerja Raya Malaysia Diana. I W., Siswosoebrotho. B. I, Karsaman. R. B. (2009). Sifat-sifat Teknik dan permeabilitas pada aspal porus. Symposium III FSTPT, ISBN No. 979-96241-0-X. Direktorat Jenderal Bina Marga, Spesifikasi Umum Bina Marga 2018 Djumari, S. D (2009). Perencanaan Gradasi Aspal Porus Menggunakan Material Lokal dengan Metode Pemampatan Kering. Media Teknik Sipil. Vol XI. 9-14. Hamirhan Saodang (2005). Perancangan Perkerasan Jalan Raya, Nova: Bandung. Hardimana, (2008). Tinjauan Aspal Porus Dwilapis Permukaan Jalan yang Ramah dengan Lingkungan Perkotaan. ISBN. No 978-979. 140-145 Jauhari, S. N; (2013), Karakteristik Marshall test Pada Lapisan Perkerasan Aspal Berongga menggunakan Batu Karang dan Buton Natural Asphalt, Makasar : Sripsi Teknik Sipil Universitas Hasanudin. AAPA, Australia Asphalt Pavement Association, (1997). Open Graded Asphalt Design Guide, Australia. ustralia Asphalt Pavement Association (AAPA), (2004). National Asphalt Specification. Australian Asphalt pavement Association (AAPA). Road Engineering Association of Australian Joint with Jabatan Kerja Raya Malaysia Diana. I W., Siswosoebrotho. B. I, Karsaman. R. B. (2009). Sifat-sifat Teknik dan permeabilitas pada aspal porus. Symposium III FSTPT, ISBN No. 979-96241-0-X. Diana. I W., Siswosoebrotho. B. I, Karsaman. R. B. (2009). Sifat-sifat Teknik dan permeabilitas pada aspal porus. Symposium III FSTPT, ISBN No. 979-96241-0-X. Direktorat Jenderal Bina Marga, Spesifikasi Umum Bina Marga 2018 Djumari, S. D (2009). Perencanaan Gradasi Aspal Porus Menggunakan Material Lokal dengan Metode Pemampatan Kering. Media Teknik Sipil. Vol XI. 9-14. Djumari, S. D (2009). Perencanaan Gradasi Aspal Porus Menggunakan Material Lokal dengan Metode Pemampatan Kering. Media Teknik Sipil. Vol XI. 9-14. Hamirhan Saodang (2005). Perancangan Perkerasan Jalan Raya, Nova: Bandung. Hardimana, (2008). Tinjauan Aspal Porus Dwilapis Permukaan Jalan yang Ramah dengan Lingkungan Perkotaan. ISBN. No 978-979. 140-145 Jauhari, S. N; (2013), Karakteristik Marshall test Pada Lapisan Perkerasan Aspal Berongga menggunakan Batu Karang dan Buton Natural Asphalt, Makasar : Sripsi Teknik Sipil Universitas Hasanudin. 45 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 De’Teksi Jurnal Teknik Sipil Unigoro Vol. 7 No. 2, Juli 2022 ISSN: 2502-3152 Nurcahya,A. (2013), Analisis Kinerja Campuran Aspal Porus Menggunakan Aspal Pen 60/70 Dan Aspal Modifikasi Polimer Evaloy. Program Magister Sistem Teknik dan Jalan Raya Institut Teknologi Bandung Nurcahya,A. (2013), Analisis Kinerja Campuran Aspal Porus Menggunakan Aspal Pen 60/70 Dan Aspal Modifikasi Polimer Evaloy. 7.Daftar Pustaka Program Magister Sistem Teknik dan Jalan Raya Institut Teknologi Bandung Sanusi, S. A(2008). Observasi Properties Porus Berbagai Gradasi dengan Material local. Media Teknik Sipil. 15-20. Sofyan, M. S(2014), Karakteristik Campuran Aspal Porus Dengan Subtitusi Styrofoam Pada Aspal Penetrasi 60/70. Jurnal Teknik Sipil Vol 21 Standar Nasional Indonesia (SNI). 03-1737-1989, Tata Cara Pelaksanaan Lapis Aspal Beton (LASTON) Untuk Jalan Raya Jakarta. Standar Nasional Indonesia (SNI). 06-2489-1991, Metode Pengujian Campuran Aspal Dengan Alat Marshall. Jakarta. Sukirman,S. (1999), Perkerasan Lentur Jalan Raya, Penerbit Nova: Bandung. Sukirman,S. (2003), Beton Aspal Campuran Panas, Penerbit Granit: Jakarta Takahashi, K.,1999. Body Temperature correlates with functional outcome and the lesion size of cerebral infarction. Acta Neurol Scan, Volume 100, pp. 385-390. Tenriajeng, A.T.,2002. Seri Diktat Kuliah, Rekayasa Jalan Raya-2. Gunadarma. Jakarta. 207 hlm. Sanusi, S. A(2008). Observasi Properties Porus Berbagai Gradasi dengan Material local. Media Teknik Sipil. 15-20. Sanusi, S. A(2008). Observasi Properties Porus Berbagai Gradasi dengan Material local. Media Teknik Sipil. 15-20. Media Teknik Sipil. 15-20. Sofyan, M. S(2014), Karakteristik Campuran Aspal Porus Dengan Subtitusi Styrofoam Pada Aspal Penetrasi 60/70. Jurnal Teknik Sipil Vol 21 Standar Nasional Indonesia (SNI). 03-1737-1989, Tata Cara Pelaksanaan Lapis Aspal Beton Sofyan, M. S(2014), Karakteristik Campuran Aspal Porus Dengan Subtitusi Styrofoam Pada Aspal Penetrasi 60/70. Jurnal Teknik Sipil Vol 21 Sofyan, M. S(2014), Karakteristik Campuran Aspal Porus Dengan Subtitusi Styrofoam Pada Aspal Penetrasi 60/70. Jurnal Teknik Sipil Vol 21 Standar Nasional Indonesia (SNI). 03-1737-1989, Tata Cara Pelaksanaan Lapis Aspal Beton (LASTON) Untuk Jalan Raya Jakarta. Standar Nasional Indonesia (SNI). 03-1737-1989, Tata Cara Pelaksanaan Lapis Aspal Beton (LASTON) Untuk Jalan Raya Jakarta. Standar Nasional Indonesia (SNI). 06-2489-1991, Metode Pengujian Campuran Aspal Dengan Alat Marshall. Jakarta. Standar Nasional Indonesia (SNI). 06-2489-1991, Metode Pengujian Campuran Aspal Dengan Alat Marshall. Jakarta. Sukirman,S. (1999), Perkerasan Lentur Jalan Raya, Penerbit Nova: Bandung. Sukirman,S. (1999), Perkerasan Lentur Jalan Raya, Penerbit Nova: Bandung. Takahashi, K.,1999. Body Temperature correlates with functional outcome and the lesion size of cerebral infarction. Acta Neurol Scan, Volume 100, pp. 385-390. Takahashi, K.,1999. Body Temperature correlates with functional outcome and the lesion size of cerebral infarction. Acta Neurol Scan, Volume 100, pp. 385-390. Tenriajeng, A.T.,2002. Seri Diktat Kuliah, Rekayasa Jalan Raya-2. Gunadarma. Jakarta. 207 hlm. Tenriajeng, A.T.,2002. Seri Diktat Kuliah, Rekayasa Jalan Raya-2. Gunadarma. Jakarta. 207 hlm. 46
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Intercomparison of Cosmic-Ray Neutron Sensors andWater Balance Monitoring in an Urban Environment
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Correspondence: Martin Schrön (martin.schroen@ufz.de) Correspondence: Martin Schrön (martin.schroen@ufz.de) Received: 13 May 2017 – Discussion started: 7 July 2017 Received: 13 May 2017 – Discussion started: 7 July 2017 Received: 13 May 2017 – Discussion started: 7 July 2017 Revised: 27 January 2018 – Accepted: 3 February 2018 – Published: 9 March 2018 Revised: 27 January 2018 – Accepted: 3 February 2018 – Published: 9 March 2018 Abstract. Sensor-to-sensor variability is a source of error common to all geoscientific instruments that needs to be assessed before comparative and applied research can be performed with multiple sensors. Consistency among sen- sor systems is especially critical when subtle features of the surrounding terrain are to be identified. Cosmic-ray neutron sensors (CRNSs) are a recent technology used to monitor hectometre-scale environmental water storages, for which a rigorous comparison study of numerous co-located sensors has not yet been performed. In this work, nine stationary CRNS probes of type “CRS1000” were installed in rela- tive proximity on a grass patch surrounded by trees, build- ings, and sealed areas. While the dynamics of the neutron count rates were found to be similar, offsets of a few percent from the absolute average neutron count rates were found. Technical adjustments of the individual detection parameters brought all instruments into good agreement. Furthermore, we found a critical integration time of 6 h above which all sensors showed consistent dynamics in the data and their RMSE fell below 1 % of gravimetric water content. The residual differences between the nine signals indicated local effects of the complex urban terrain on the scale of several metres. Mobile CRNS measurements and spatial simulations with the URANOS neutron transport code in the surrounding area (25 ha) have revealed substantial sub-footprint hetero- geneity to which CRNS detectors are sensitive despite their large averaging volume. The sealed and constantly dry struc- tures in the footprint furthermore damped the dynamics of the CRNS-derived soil moisture. We developed strategies to correct for the sealed-area effect based on theoretical insights about the spatial sensitivity of the sensor. This procedure not only led to reliable soil moisture estimation during dry-out periods, it further revealed a strong signal of intercepted wa- ter that emerged over the sealed surfaces during rain events. The presented arrangement offered a unique opportunity to demonstrate the CRNS performance in complex terrain, and the results indicated great potential for further applications in urban climate research. 1 Introduction The monitoring of water states and fluxes is important to understand processes of the hydrological cycle, to facilitate weather predictions, and to make timely decisions (Wood et al., 2011; Beven and Cloke, 2012). Soil moisture and air humidity are interlinked key quantities that can control plant water availability, groundwater recharge, air temper- Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 https://doi.org/10.5194/gi-7-83-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 3.0 License. Intercomparison of cosmic-ray neutron sensors and water balance monitoring in an urban environment Martin Schrön1, Steffen Zacharias1, Gary Womack2, Markus Köhli1,3,4, Darin Desilets2, Sascha E. Oswald5, Jan Bumberger1, Hannes Mollenhauer1, Simon Kögler1, Paul Remmler1, Mandy Kasner1,6, Astrid Denk1,7, and Peter Dietrich1 toring and Exploration Technologies, Helmholtz Centre for Environmental Research GmbH – UFZ 1Dep. Monitoring and Exploration Technologies, Helmholtz Centre for Environmental Research G Leipzig, Germany 2Hydroinnova LLC, Albuquerque, USA 3Physikalisches Institut, Heidelberg University, Heidelberg, Germany 4Physikalisches Institut, University of Bonn, Bonn, Germany 5Institute of Earth and Environmental Science, University of Potsdam, Potsdam, Germany 6Institute of Geosciences and Geography, University of Halle-Wittenberg, Halle, Germany 7Dep. of Geosciences, University of Tübingen, Tübingen, Germany M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 84 ple, Chiba et al. (1975) and Oh et al. (2013) revealed clear discrepancies between high-energy neutron monitors which were related to device-specific configurations. Intercalibra- tion may be employed to normalize such differences from unit to unit and also to account for any residual instrumental configuration inconsistencies (Bachelet et al., 1965; Krüger et al., 2008). ature, and regional weather phenomena (Seneviratne et al., 2010). In urban environments, sealed surfaces reduce water infiltration and promote high evaporation from ponded wa- ter. These effects are linked with the formation and impact of urban heat islands and can be a major threat for society (Arnfield, 2003; Starke et al., 2010; UN, 2015). Conventional measurement methods for soil and evapora- tion water operate on a point scale and are not representa- tive of complex areas (Famiglietti et al., 2008; Schelle et al., 2013), while remote-sensing products are often limited to low resolution and shallow penetration depth (Nouri et al., 2013; Fang and Lakshmi, 2014). Up to now, few methods have been available to assess the components of the hydro- logical cycle non-invasively and on relevant scales (Robinson et al., 2008). When it comes to data analysis and interpretation, spatial heterogeneity could have a biasing effect on neutron detec- tors. Despite the large footprint, the sensor is not equally sensitive to every part. Its radial sensitivity decreases non- linearly with distance, showing pronounced sensitivity to the nearest few metres around the probe (Köhli et al., 2015). This might become a particular issue for co-located sensors dur- ing an intercomparison study and for the reliability of soil moisture estimations in complex terrain (Franz et al., 2013; Schrön et al., 2017a). Nonetheless, researchers have chal- lenged the task to interpret CRNS data in complex environ- ments (Bogena et al., 2013; Franz et al., 2016; Schattan et al., 2017; Schrön et al., 2017b), but open questions remain of how and to what degree spatial heterogeneity should be ac- counted for. The method of cosmic-ray neutron sensing (CRNS) com- bines the geoscientific research fields of cosmic-ray neutron detection and environmental hydrology (Desilets et al., 2010; Zreda et al., 2012). The instrument is an epithermal neutron detector that measures the natural cosmic-ray-induced radi- ation 1–2 m above the ground and is highly sensitive to the abundance of hydrogen atoms in the surrounding area. M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment As neutrons can penetrate the soil up to depths of approximately 80 cm and are then able to travel several hundreds of me- tres in air, the unique feature of the technology is the large averaging volume (Köhli et al., 2015). The CRNS research field aims to fill the gap between point-scale and large-scale measurements by using the sensor in a stationary and mobile mode. The main advantages of the method are its capabil- ities to capture different components of the water cycle in air, soil, and vegetation non-invasively (e.g. Baroni and Os- wald, 2015) and to provide a representative spatial average of the environmental water content. Therefore, the method is a promising candidate to support hydrogeophysical and cli- mate research in complex terrain (e.g. urban environments). The CRNS measurements come with an intrinsic statis- tical uncertainty which is higher for lower count rates and decreases with longer integration time. Among others, Evans et al. (2016) and Hawdon et al. (2014) reported issues with low hourly count rates in wet regions and at low altitude. Bogena et al. (2013) found that the error in volumetric soil moisture estimates can be 10–20 % for hourly CRNS data in a forested environment. This statistical noise might become a problem for the reliability and consistency of CRNS mea- surements. The main objective of this paper is to advance the gener- ation of reliable CRNS products. To achieve this, we aim to explore the potential sensor-to-sensor variability of cosmic- ray neutron sensors in a systematic way and to provide so- lutions to improve the consistency of neutron measurements. With regards to the potential sources of variability in the neu- tron signal, we can formulate the following hypotheses: Consistency among the neutron signals is an important prerequisite towards joint usage of multiple sensors for scientific applications. Many studies relied on the consis- tent performance of a set of CRNS probes for monitor- ing (Rivera Villarreyes et al., 2011; Dong et al., 2014; Franz et al., 2015; Evans et al., 2016), modelling (Rosolem et al., 2014; Baatz et al., 2017; Andreasen et al., 2016), or remote-sensing validation purposes (Holgate et al., 2016; Montzka et al., 2017). Intercomparison studies are a prefer- able way to find sensor-to-sensor inconsistencies. M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment Geoscien- tific instruments such as point-scale soil moisture probes and remote-sensing instruments typically undergo intercompari- son (Walker et al., 2004; Kögler et al., 2013; Su et al., 2013), and the CRNS technology should not be an exception. A. The integrated neutron measurements may be sensitive to sensor location within a few metres. B. Device-specific differences may cause systematic vari- ations between the sensors. C. Statistical noise contributes to the count rate variabil- ity and determines the degree of comparability between sensors. The main application of intercomparison studies is inter- calibration, the determination of efficiency (or scaling) fac- tors for individual devices (see e.g. Baatz et al., 2015; Franz et al., 2015). Neutron detector signals often exhibit system- atic biases due to limitations in manufacturing and small differences in geometry and materials utilized. For exam- The hypotheses are tested using nine co-located CRNS probes within a maximum distance of 15 m in an urban en- vironment. Hypothesis A was addressed by investigating the effect of sensor permutation on the neutron counts. Hypoth- esis B was tested by changing detection parameters of the Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 2.1 Cosmic-ray neutron sensors Neutrons in the energy range of 10 to 104 eV are highly sen- sitive to hydrogen, which turns neutron detectors to highly efficient proxies for changes of environmental water con- tent. Zreda et al. (2012) presented the established method of cosmic-ray neutron sensing as a non-invasive and promising tool for hydrology applications. Köhli et al. (2015) provided more details of the underlying physics, the lateral footprint of several tens of hectares, and the sample depth of up to 80 cm (see also Franz et al., 2012; Desilets and Zreda, 2013). Connection to external sensors (T, h), rain gauge, solar panel N 100 90 Lower discriminator 30 Counts Pulse height spectrum Deposited energy in keV (b) 20 800 700 200 Bin number (arb. scale) Upper discrim. Wall effect (b) Neutron sensors of type “CRS1000” (Hydroinnova LLC, US) have been the standard in CRNS research and are com- mercially available in several configurations. The main com- ponents and configurations have been described by Zreda et al. (2012) and are summarized in Fig. 1a (see also the man- ufacturer’s web page: http://hydroinnova.com/ps_soil.html\ T1\textbackslash#stationary). Each system comprises a bare and a moderated neutron detector, two advanced neutron pulse detecting modules (NPM), and a data logger with in- tegrated telemetry. The mentioned components are housed in a sealed metal enclosure. The logger retrieves neutron counts and diagnostic pulse height information periodically from each NPM, which generate the high voltage required by the detector tubes. The data logger also samples from barometric pressure sensors and, in this work, from an external temper- ature and air humidity sensor (Campbell CS215, Campbell Scientific Inc., Logan, Utah, US). The data logger has fur- ther been configured to record signals from a tipping bucket rain gauge. Figure 1. (a) Inside view of the cosmic-ray neutron sensor (CRNS) of type “CRS1000”. The moderated tube (surrounded by a white polyethylene block) mainly detects epithermal neutrons and is thus sensitive to water in the environment. (b) A typical, measured pulse height spectrum (PHS) shows the deposited energy in the gas tube. Upper and lower discriminators (orange) delimit the region (grey) in which events are interpreted as neutron counts N. Illustrated dis- criminator positions are examples. The internal representation of released energy as bin numbers is a specific feature of the sensor. 2.1 Cosmic-ray neutron sensors mainly to survey soil moisture on the regional scale (Mc- Jannet et al., 2014; Dong et al., 2014; Chrisman and Zreda, 2013; Wolf et al., 2016), in agricultural fields (Franz et al., 2015; Schrön et al., 2017b), and also in urban areas (Chris- man and Zreda, 2013). In this study we aim to determine the spatial distribution of neutrons within parts of the stationary CRNS footprint. M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 85 sensors. Correlation and consistency tests of the sensor en- semble were performed with regards to temporal aggregation to address hypothesis C. GSM antenna Neutron pulse module (NPM) Data logger and cell modem 3 Bare He tube (thermal detector) 3 Moderated He tube (epithermal detector) Charge controller Maintenance-free battery (12 V) Connection to external sensors (T, h), rain gauge, solar panel N 100 90 Lower discriminator 30 Counts Pulse height spectrum Deposited energy in keV (b) (a) 20 800 700 200 Bin number (arb. scale) Upper discrim. Wall effect Figure 1. (a) Inside view of the cosmic-ray neutron sensor (CRNS) of type “CRS1000”. The moderated tube (surrounded by a white polyethylene block) mainly detects epithermal neutrons and is thus sensitive to water in the environment. (b) A typical, measured pulse height spectrum (PHS) shows the deposited energy in the gas tube. Upper and lower discriminators (orange) delimit the region (grey) in which events are interpreted as neutron counts N. Illustrated dis- criminator positions are examples. The internal representation of released energy as bin numbers is a specific feature of the sensor. GSM antenna Neutron pulse module (NPM) Data logger and cell modem 3 Bare He tube (thermal detector) 3 Moderated He tube (epithermal detector) Charge controller Maintenance-free battery (12 V) Connection to external sensors (T, h), rain gauge, solar panel (a) (a) To further understand the influence of sensor location, simulations and mobile measurements were consulted to re- veal the spatial heterogeneity of neutrons within the foot- print. We finally evaluated the sensor performance against in- dependent soil moisture observations, using a new approach to correct the neutron signal for the unwanted contribution of sealed areas in the footprint. 2.2 The mobile CRNS rover The cosmic-ray neutron rover is technically similar to the sta- tionary CRNS probes. The main differences are the added GPS functionality and much larger counting gas tubes. The larger size of the detector increases the probability to capture a neutron and consequently leads to higher count rates by factors of ≈11 compared to the stationary probes. This also allows for shorter integration periods of 1 min. The length of the track passed in that minute determines the spatial reso- lution of the measurement. Previous studies used the rover 2.3 Neutron detection A polyethylene shielded detector is employed to provide a moderated detector channel. The shielding material is de- signed to reduce the number of incoming thermal neutrons Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 www.geosci-instrum-method-data-syst.net/7/83/2018/ Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 www.geosci-instrum-method-data-syst.net/7/83/2018/ M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 2.3.2 Pulse height spectrum recordings As the energy of the reaction products in the gas is well known, a characteristic electronic pulse can be expected and translates to a prominent peak in a so-called pulse height spectrum (see Fig. 1b). However, sometimes the elements of the reaction product reach the wall of the tube before com- pletely depositing their energy into the proportional gas. The so-called “wall effect” is then visible in the PHS as a distri- bution of pulses of lower pulse height than the peak. As such, the typical shape of the PHS is independent of the absorbed neutron energy. It is rather a function of the reaction kine- matics and detector-specific details, including the geometry (Crane and Baker, 1991). M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 86 2.3.3 The lower discriminator and to slow incoming epithermal neutrons down to thermal (i.e. detectable) energies. An additional bare detector in the CRNS probe directly records incoming thermal neutrons, while it is less sensitive to epithermal energies (Andreasen et al., 2016; Köhli et al., 2018). The detector recognizes a neutron capture event if the re- leased electronic pulse lies between the lower discrimina- tor at the lower end and the upper discriminator at the upper end of the PHS (beyond the prominent peak). The lower dis- criminator is an important detection parameter that is often set up on the “wall-effect shelf” (the flat plateau in Fig. 1b), slightly to the right of the lower shelf edge (bins 30–35). This ensures maximum immunity to lower amplitude electronic noise which could otherwise be counted as neutron events. In addition, a high discriminator excludes signals from gamma pileups which could otherwise produce spurious counts when in the presence of significant gamma radiation. However, the discriminator position above the shelf results in some loss of the theoretical maximum sensitivity of the neutron detector and can cause some variation in sensitivity if the location of the lower discriminator relative to the peak location is not set consistently across multiple sensors. 2.3.1 Interactions with the detector gas Only thermal neutrons can be efficiently detected with state- of-the-art proportional detectors employing gases enriched in 3He (Persons and Aloise, 2011; Krane and Halliday, 1988). When a thermal neutron collides with an atomic nucleus of the detector gas, a neutron absorption reaction can occur, re- sulting in emission of charged particles, which in turn pro- duce ionization. Electrons are attracted to the anode, a cen- tral wire at a potential of ≈1000 V. Due to the steep gradient of the electrical potential towards the wire, the electrons are accelerated and collide with additional gas molecules, pro- ducing further ionization. A sensitive NPM, consisting of hy- brid analogue–digital electronics, amplifies, shapes, and fil- ters each charge pulse from the tube. The NPM further mea- sures the pulse height and records it as a neutron count if it is a valid event. The pulse is further accumulated in a pulse height spectrum (PHS). Improvements in the NPM electronics since 2013 have increased the stability of the electronic gain and the high voltage supply, as well as lowered the electronic noise floor. Therefore it is reasonable to use the whole pulse height spec- trum for the neutron counter by setting the lower discrimi- nator below the wall effect shelf (around bin 24). One of the benefits is in maximally counting all neutrons (i.e. essentially counting very close to 100 % of all neutron capture events). In addition, in such a configuration, small changes in NPM electronic gain or internal high voltage will have the most minimal effect on the count rate. 2.4.1 From neutrons to soil moisture The measured intensity of albedo neutrons depends not only on the water content in the environment but also on the inten- sity of incoming cosmic-ray neutrons. This radiation compo- nent changes with changing atmospheric conditions and also with incoming galactic cosmic rays (Zreda et al., 2012). For this reason, CRNSs are typically equipped with sensors for air pressure p, air temperature T , and relative humidity hrel. Their compound average, ⟨·⟩, was utilized to correct individ- ual neutron count rates Nraw using standard procedures: Pulse height spectra are autonomously and periodically recorded (typically daily or every several days) by the CRNS detector system, providing valuable self-diagnostics and long-term monitoring of the system health. An irregular PHS can have multiple reasons, for example collapsing high- voltage supply, gas leakage, or impurity in the detector tube, while variations at the lower end are an indication for current noise or gamma radiation. Typical CRNS systems have sta- ble, long-term sensitivity to neutrons and are maximally im- mune to environmental changes (such as temperature), elec- tronic noise, and instrumental drift. More information about neutron detectors can be found for example in Mazed et al. (2012) and Persons and Aloise (2011). N = Nraw ·  1 + α ⟨h⟩−href  · exp  β ⟨p⟩−pref  ·  1 + γ Iref/I −1  , (1) (1) where h(hrel,T ) is the absolute humidity, I is the in- coming radiation (here the average signal from neutron monitors Jungfraujoch and Kiel), href = 0 g m−3, pref = 1013.25 mbar, Iref = 150 cps, α = 0.0054, β = 0.0076, and γ = 1 (for details see Zreda et al., 2012; Rosolem et al., www.geosci-instrum-method-data-syst.net/7/83/2018/ www.geosci-instrum-method-data-syst.net/7/83/2018/ Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 2.4.3 Performance measures The spread of individual sensors around their average ⟨N⟩ can be expressed as σx(N) = " 1 9 X i |Ni −⟨N⟩|x #1/x , (6) M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environmen 2013; Hawdon et al., 2014; Schrön et al., 2015). The ac- cepted approach to convert neutron count rates to (soil) water equivalent θ uses the following relation: while the count rate N is given in units of cph. Aggregation of the time series N to longer intervals leads to the series Na (counts per a hours), where a is an arbitrary factor following τa = a τ. In order to keep cph as the standard reference unit for all neutron time series, the units can be transformed back to τ (i.e. 1 h). Then the average count rate and uncertainty become θ(N) · ϱbulk = 0.0808 N/N0 −0.372 −0.115 −θoffset , (2) (2) where ϱbulk (in g cm−3) is the soil bulk density, θoffset (in g/g) is the gravimetric water equivalent of additional hydro- gen pools (e.g. lattice water, soil organic carbon), and N0 (in counts per hour, cph) is a free calibration parameter (for details see Desilets et al., 2010; Bogena et al., 2013). The latter can be calibrated using the count rate N and inde- pendent measurements of the average water content ⟨θ⟩in the footprint. According to Schrön et al. (2017a) the CRNS probe does not measure a simple, equally weighted average of the surrounding water content due to the non-linearity of its radial sensitivity function, Wr(h,θ). Therefore, different parts of the footprint area contribute differently to the av- erage signal depending on their distance r from the sensor. This knowledge can be used to quantify the contribution of individual areas that are of specific interest. Na = 1 a Xa 1N ≈⟨N⟩, σ(Na) = 1 a qXa 1σ(N)2 ≈1 √a ⟨σ(N)⟩. (5) (5) (5) As a consequence, the average statistical error of a daily aggregated time series (in units of cph) is given as σ(⟨N⟩) = √⟨N⟩/24, which corresponds to 80 % less uncertainty com- pared to hourly resolution. 2.4.2 Counting statistics (6) Nine CRNS probes were employed for the intercomparison study and each member of the CRNS ensemble acts as an individual monitoring system. The co-location of these sen- sors, however, offers a unique opportunity to combine their signals, which leads to high total count rates and thus lower statistical noise. The average count rate ⟨N⟩and its propa- gated uncertainty σ of each ith sensor are given as where the parameter x determines the distance norm. Then, σx=1 is defined as average absolute deviation, and σx=2 ≡σ is the standard deviation. The Pearson correlation coefficient can be defined for two time series NA and NB with standard deviations σA and σB: ρ(NA,NB) = (NA −⟨NA⟩) · (NB −⟨NB⟩) σA · σB . (7) (7) ⟨N⟩= 1 9 X Ni , σ(⟨N⟩) = 1 9 qX σ(Ni)2, (3) (3) For example, ρ = 0.7 depicts that NA and NB can explain 0.72 ≈50 % of their respective variance. If those two vari- ables, NA and NB, were ranked depending on the order of their magnitude, Ni 7−→rank(Ni), the Pearson correlation turns to the so-called Spearman rank correlation: where σ(N) = √ N is given as the standard deviation of av- erage counts N using Gaussian statistics, and 9 is the number of individual sensors used in this study. Under the assump- tion that Ni ≈Nj ∀i,j ∈(1,...,9), their corresponding un- certainty will be similar as well: where σ(N) = √ N is given as the standard deviation of av- erage counts N using Gaussian statistics, and 9 is the number of individual sensors used in this study. Under the assump- tion that Ni ≈Nj ∀i,j ∈(1,...,9), their corresponding un- certainty will be similar as well: ρS(NA,NB) = 1 −6 P t  rank(NA) −rank(NB) 2 n(n2 −1) , (8) (8) σ(Ni) ≈⟨σ(Ni)⟩∀i , ⇒ σ(⟨N⟩) ≈1 9 q 9 · ⟨σ(Ni)⟩2 = 1 3 ⟨σ(Ni)⟩≈1 3 p ⟨N⟩. (4) where n is the total number of intervals and t ∈(1,...,n). This quantity can be used to identify events that changed the rank of specific sensors. (4) 2.5 The neutron transport simulator URANOS Hence, the combination of nine sensors reduces the relative statistical error by ≈67 %, thereby allowing for accurate measurements of changes of the environmental water stor- age. The generation, interaction, and detection of neutrons can be simulated with Monte Carlo codes, which are based on physically modelled interaction processes, and state-of-the- art nuclear cross section databases. The Ultra Rapid Adapt- able Neutron-Only Simulation (URANOS) has been specif- ically tailored to environmental neutrons relevant for CRNS Temporal aggregation can further reduce the standard de- viation. The measurement interval τ is usually set to 1 h, M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 87 www.geosci-instrum-method-data-syst.net/7/83/2018/ Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 88 Table 1. Two soil profiles in the grass meadow sampled nearby the profiles of the wireless sensor network (WSN) on 14 January 2016. Samples were taken with core cutters of constant volume at three depths, oven-dried, and weighted according to standard procedures. The evaporated water content is given in units of volumetric percent. series data have been utilized to calibrate the neutron signal on volumetric soil moisture using Eq. (2). WSN is a promising tool in the field of environmental sci- ence to detect and record energy and matter fluxes across Earth’s compartments (Hart and Martinwz, 2006; Zerger et al., 2010; Corke et al., 2010). The WSN used in this study was developed specifically for short-term, demand-driven ap- plications (Mollenhauer et al., 2015; Bumberger et al., 2015). The soil moisture sensors of type Truebner SMT100 used in the soil profiles directly measure electrical permittivity, ε, which is a compound quantity of the individual media (water, soil, air) and their volumetric fractions in the soil (Brovelli and Cassiani, 2008). The volumetric water content θ was de- duced from ε with the CRIM formula (Roth et al., 1990), using independent measurements of porosity and soil wa- ter temperature and assuming randomly aligned microscopic soil structures. The measurement uncertainties in units of absolute volumetric percent could be related to the device (< 2 %) and could vary from wet to dry conditions. They are highly dependent on prior calibration (Truebner, 2012) and may be related to inappropriate assumptions on the per- mittivity of quartz (< 3 %) and to the heterogeneity of soil properties and composition in the meadow (< 8 %). The lat- ter uncertainty was tested by sampling soil moisture profiles at many places within the field and is taken into account when WSN is compared to CRNS observations. Profile Depth ϱbulk in g cm−3 Porosity 2 Water θ South 7–12 cm 1.62 38 % 18 % South 15–20 cm 1.52 42 % 15 % South 25–30 cm 1.58 40 % 18 % North 5–10 cm 1.60 40 % 32 % North 15–20 cm 1.93 27 % 19 % North 25–30 cm 1.91 28 % 28 % Profile Depth ϱbulk in g cm−3 Porosity 2 Water θ research. The model was described by Köhli et al. 2.7 Measurement Strategy The urban scenario of 500m × 500m around the CRNS probes was re-enacted using 2-D images of different lay- ers that represent the different material compositions. More than 145 million neutrons were released equally distributed in heights of 80 to 50 m. The simulation domain in total cov- ered a volume of 900m × 900m and 1000 m height, where the additional padding around the area accounts for border effects. Non-sealed area was defined as grassland with an exemplary soil moisture of 30 %. Buildings were modelled as blocks of air by a 0.5 m concrete wall. Trees were mod- elled as blocks of organic material with 0.3 kg m−3 biomass stretching to heights of up to 20 m. Details of all materials used can be found in the Supplement of this paper. Simu- lated neutrons were counted in a detector layer 1.75 m above the surface, representing the typical position of cosmic-ray neutron sensors. The study site is an urban area at the Helmholtz Centre for Environmental Research – UFZ in Leipzig, Germany (51◦21′11′′ N, 12◦26′02′′ E; 116 m above sea level). The site exhibits humid climatic conditions and consists mainly of grassland patches surrounded by sealed areas such as roads and buildings (Fig. 2). In February 2014, nine cosmic-ray neutron sensors were installed in a small grass meadow to monitor the neutron den- sity in air. The sensors were co-located within a maximum distance of 15 m, which was assumed to be small relative to the sensor footprint. The individual count rates were logged every 15 min and were processed using the standard correc- tion approaches described above. Dedicated experiments were prepared (Table 2) to test the hypotheses whether a potential sensor-to-sensor variability is influenced by the location of the sensors (hypothesis A), by device-specific differences (hypothesis B), or by statistical noise (hypothesis C). In Phase I, the sensors were operated in the initial arrangement (see Fig. 2) for 3 weeks. Before entering Phase II, four sensors were swapped, while five sen- sors kept their position to serve as a reference. A comparison between Phase I and Phase II allows us to observe the effect of potential locational effects on the sensor response. After 4 weeks, detection parameters were adjusted to reduce the ob- served device-specific differences and to test their influence on the count rates when entering Phase III. In all phases, the M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment (2015) to calculate the footprint volume and spatial sensitivity of CRNS probes. It has since been successfully applied to ad- vance the method of cosmic-ray neutron sensing (Schrön et al., 2015; Schrön et al., 2017a) and also to advance re- search in nuclear physics to simulate neutron detectors and characterize their response on the millimetre scale (Köhli et al., 2016; Köhli et al., 2018). One of the unique fea- tures is the simulation of spatial neutron densities in an ar- bitrary, user-defined terrain. URANOS is very flexible and allows to input spatial bitmap information about the materi- als and geometries in the studied area. The software comes with a graphical user interface and is freely available (see www.ufz.de/uranos). 2.6 Point-scale soil moisture measurements In order to validate and calibrate the sensors against inde- pendent soil water content, two measurement methods were consulted to quantify soil moisture profiles: volume soil sam- ples (single measurement) and a mobile wireless ad hoc soil moisture network (WSN) (continuous). The measurements were taken in different depths at two locations near the CRNS probes. The corresponding soil parameters (Table 1) and time www.geosci-instrum-method-data-syst.net/7/83/2018/ Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 3.1 The influence of sensor location The nine co-located sensors were operated in their initial ar- rangement for 3 weeks (Phase I). While all sensors showed similar trends, prominent offsets were observed between in- dividual signals, particularly for sensors 3 and 4 (Fig. 3). The average deviation of all count rates from their ensemble mean exceeded the daily statistical error, σ(N24 h) ≈√600/24 = 5, by a factor of 2. Although the maximum distance between the sensors was only 15 m, it has been hypothesized that the individual loca- tions could have introduced a systematic effect on the count rate due to the steep radial sensitivity curve (Köhli et al., 2015; Schrön et al., 2017a). This hypothesis A has been tested by observing the change of neutron count rates be- fore and after the change of their position within the sensor arrangement. Figure 2. Location and arrangement of the nine cosmic-ray neu- tron sensors deployed at the small, urban meadow at UFZ Leipzig, Germany. Before the second phase positions of a subset of sensors were swapped, while others remained fixed (Fig. 4a). In order to assess the effect on their individual measurement offsets, Spearman rank correlations were applied to the time series before and after sensor permutation (see Fig. 4b). This quan- tity explains the probability with which a sensor’s count rate N is assigned to an ordered rank among the ensemble. The data showed that the favoured rank (or offset) of both fixed and swapped sensors was almost unaffected. In particular, the ranks of sensors 3 and 4 remained at their high or low levels, respectively. correlation between the sensors and its dependence on tem- poral aggregation was investigated to test the influence of re- duced statistical uncertainty. In order to fully understand implications of hypothesis A, i.e. the effects of location on the sensor response, we per- formed URANOS simulations of the site-specific neutron distribution and conducted spatial surveys in parts of the CRNS footprint area. The spatial distribution of neutrons was measured with the mobile CRNS rover detector in a car (May 2014) and on a hand wagon (July 2015). To achieve a spatial resolution in the range of a few metres, the rover was operated at walking speed. Description initial arrangement of the nine sensors permuted positions of sensors 1, 2, 3, 4, 8 adjusted detector parameters of all sensors spatial mapping of neutron heterogeneity with a CRNS rover comparison with soil moisture from a wireless ad hoc sensor networ Finally, the performance of the CRNS soil moisture prod- uct in such a complex terrain was questioned. The WSN was installed in two soil profiles near the CRNS sensor arrange- ment in order to evaluate the capabilities of the neutron sen- sor to estimate water content in the urban environment. Pond Sand Concrete Grass Tree Building CRNS 10 m 5000 m 1 9 8 7 6 5 4 2 3 Figure 2. Location and arrangement of the nine cosmic-ray neu- tron sensors deployed at the small, urban meadow at UFZ Leipzig, Germany. www.geosci-instrum-method-data-syst.net/7/83/2018/ www.geosci-instrum-method-data-syst.net/7/83/2018/ Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 3.1 The influence of sensor location Figure 3 further suggests that small-scale positioning has not been the main cause of the individual variability, as only subtle changes of the deviation of the sensor signals from their mean were found between phases I and II. Neverthe- less, the subtle changes can be quantified in more detail by looking at the counting efficiencies of the sensors (i.e. rela- Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 89 Table 2. Measurement strategy to investigate the sensor-to-sensor variability (Phase I), the influence of location (Phase II), the effects of detector parameters (Phase III), the heterogeneity of neutrons within the sensor’s footprint (Survey), and the sensor’s capabilities to monitor soil moisture in the complex, urban terrain. Experiment Period Description Phase I 2014-02-22 to 2014-03-18 initial arrangement of the nine sensors Phase II 2014-04-07 to 2014-05-08 permuted positions of sensors 1, 2, 3, 4, 8 Phase III from 2014-05-09 adjusted detector parameters of all sensors Survey 2014-05-02, 2015-07-22 spatial mapping of neutron heterogeneity with a CRNS rover Validation 2015-09-29 to 2015-10-31 comparison with soil moisture from a wireless ad hoc sensor network www.geosci-instrum-method-data-syst.net/7/83/2018/ www.geosci-instrum-method-data-syst.net/7/83/2018/ M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 90 21 Feb 10 9 8 7 6 5 4 0 10 20 30 40 50 28 Feb 7 Mar 14 Mar 11 Apr 18 Apr 25 Apr 2 May 16 May 23 May 30 May 6 Jun 13 Jun 20 Jun 27 Jun 4 Jul 11 Jul 18 Jul 25 Jul 700 650 600 Neutrons N in cph Rain in mm #1 #2 #3 #4 #5 #6 #7 #8 #9 Sensors SD from mean Stat. counting error Mean Phase III. Phase II. Phase I. Figure 3. Time series of nine sensors covering phases I (installation), II (permutation), and III (calibration) in year 2014. By removing detector-specific effects in Phase III, the standard deviation (SD) of the sensor ensemble from their mean could be reduced down to the statistical error of σ ≈5 cph. SD from mean Stat. counting error 25 Jul Figure 3. Time series of nine sensors covering phases I (installation), II (permutation), and III (calibration) in year 2014. By removing detector-specific effects in Phase III, the standard deviation (SD) of the sensor ensemble from their mean could be reduced down to the statistical error of σ ≈5 cph. 3.2 Detector-specific variability tive deviation from their mean) in Fig. 5. The efficiency can be estimated either theoretically, by the relative positions of the lower discriminator in the PHS, or empirically, by the variability of the observed neutron counts. Figure 5 shows the theoretical relative efficiency of the nine sensors before Phase III and their empirical values in phases I, II, and III. Phase III was dedicated to the diagnosis of the count rate, which is directly related to the integral of the PHS. As ex- plained in Sect. 2.3.2, the shape of the PHS and the param- eters used to determine its integral (such as the lower dis- criminator) are important for the individual sensor efficiency. Thus, consistent detection parameters are a prerequisite to assure that the same fraction of neutron capture events are counted by all detectors. The inconsistent spectra in Fig. 6 (dotted black line) indicate that this requirement was not met before Phase III. Are these device-specific differences having an influence on the intercomparability of the neutron signals (hypothesis B)? The results indicate that different components are con- tributing to the total sensor-to-sensor variability. The theo- retical, detection-specific efficiency from the PHS processor accounts for only 0.77 % mean deviation, which cannot ex- plain the high empirical values of 1.87 and 1.64 % in phases I and II, respectively. Furthermore, the fact that swapped sen- sors changed their mean efficiency by −0.37 %, while fixed sensors only changed by −0.04 % from Phase I to Phase II indicates that one of the additional variability components might be related to location. To achieve comparability of the pulse height spectra among the sensors, we set the lower discriminator consis- tently below the wall effect shelf (around bin 24) and ad- justed the high voltage and amplifier gain parameters such that the main peaks aligned approximately to bin number 100 for the sake of visual accessibility. This procedure ensured maximum count rate for the individual sensors. All in all, a small positional effect cannot be excluded (confirming hypothesis A), but the major part of the observed sensor-to-sensor variability must have originated from other sources. In Fig. 6 (left) the resulting change of the PHS is shown for all sensors, and the impact on the neutron count rate is Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 3.3 Temporal resolution for consistent observations The previous sections have shown that the CRNS probes ex- hibited small but measurable sensor-to-sensor variability that was related to positional effects and to the factory configura- tion of the neutron detector operating parameters. This sec- tion tests hypothesis C, the potential influence of statistical noise to the sensor intercomparability. The statistical vari- ability component is related to the random nature of neutron detection. According to Sect. 2.4.2, the corresponding uncer- tainty can be reduced by temporal aggregation. This is ex- pected to influence the correlation between the nine CRNS probes. While Bogena et al. (2013) calculated the uncertain- ties for several temporal resolutions theoretically, the present arrangement provides a unique opportunity for an experi- mental approach with multiple sensors. (b) Rank correlation (b) Phase I. Phase II. No. 2 No. 3 No. 4 No. 5 (öxed) No. 6 (öxed) No. 9 (öxed) No. 8 No. 7 (öxed) No. 1 Phase I. Phase II. 1 0.5 0 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 Rank 1 0.5 0 1 0.5 0 Figure 7a shows that the ensemble-averaged correlation of the nine sensors significantly increased with increasing in- tegration time across the three phases. The correlation coef- ficient was 0.12 and 0.26 for 1 h integration time and went up to 0.61 and 0.74 for 10 h in phases I and II, respectively. Since the sensor swap itself should have no effect on the cor- relation, the difference between Phase I and Phase II could be attributed solely to the meteorological dynamics in these periods. While rain events were almost absent during Phase I (compare Fig. 3), the corresponding neutron dynamics were mainly influenced by statistical and detector-specific vari- ability. In Phase II, a number of rain events led to large am- plitudes of neutron count dynamics and thus naturally to in- creased correlations. Figure 4. (a) Birds-eye view on the sensor arrangement before and after permutation of sensors 2, 3, 4, and 8. (b) Rank correlations of the nine CRNS signals before (dotted) and after (solid) permutation. Both swapped and fixed sensors showed no significant change. demonstrated exemplarily for sensor 3. The parameter ad- justments shifted the main PHS peak towards bin 100, and the reduction of the lower discriminator effectively increased the neutron count rate of the sensor. M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 91 lower amplitude neutron pulse events that were previously being filtered by the lower discriminator. No. 2 No. 3 No. 4 No. 5 (öxed) No. 6 (öxed) No. 9 (öxed) No. 8 No. 7 (öxed) Rank correlation No. 1 Phase I. Phase II. 1 0.5 0 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 Rank 1 0.5 0 1 0.5 0 1 m Phase I. Phase II. (b) (a) Sensor permutation Phase I. II. 1 9 8 7 6 5 4 2 3 1 9 8 7 6 5 4 2 3 Figure 4. (a) Birds-eye view on the sensor arrangement before and after permutation of sensors 2, 3, 4, and 8. (b) Rank correlations of the nine CRNS signals before (dotted) and after (solid) permutation. Both swapped and fixed sensors showed no significant change. Sensor permutation Phase I. II. Sensor permutation Phase I. II. Phase I. Phase II. 1 m Phase II. 1 9 8 7 6 5 4 2 3 (a) Phase I. In terms of relative variation (Fig. 5), the adjustment of the detector parameters at the beginning of Phase III caused the sensor efficiencies to change from 1.64 to 0.52 %. Thereby the detector-specific variability was almost removed and the sensors have since shown the best agreement to each other. Rank correlation Phase I. (b) (a) 1 9 8 7 6 5 4 2 3 The remaining variability could be contributed to small differences in design and geometry from the manufacturer or the sensor location. The overall variability of 0.52 % is now comparable with the standard relative error of the daily mean, σ(N)/N, which went down to 0.55 % in certain peri- ods of this study. 3.3 Temporal resolution for consistent observations After manual adjust- ment of the parameters for all sensors, most of the individual offsets vanished and the standard deviation from the mean, σ(N), was reduced by 50 % down to the order of the sta- tistical error (compare Fig. 3). Moreover, the average ab- solute deviation was reduced even below the statistical er- ror σ(N) = √N/24 of the daily aggregated time series (not shown). All in all, the instruments showed greater consis- tency in neutron counting sensitivity since the recovery of The highest correlation was achieved in Phase III, when most of the detector-specific variability was removed (Sect. 3.2). Moreover, correlation coefficients exceeded a value of 0.90 for more than 6 h of integration time and went up to 0.97 for daily aggregation. These results demonstrate the reliability of CRNS observations for integration times of at least 6 h under humid conditions, in complex terrain, and at sea level. Even higher correlations can be expected for dry regions and homogeneous terrain at high altitude, where higher neutron count rates and less structural disturbances would lead to lower noise. Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 www.geosci-instrum-method-data-syst.net/7/83/2018/ M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment Phase II. III. Detector parameter calibration 680 700 720 740 760 40 60 Bin 100 Phase II. III. Detector parameter calibration Pulse height spectra Neutrons in cph 1 May 2 May 3 May Parameter change for sensor no. 3 No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 Figure 6. Adjustment of the detector parameters harmonized the pulse height spectra of the nine sensors (before: dotted black) and increased their range towards the lower left end. The impact on the count rate is shown exemplarily for sensor 3 (orange). 40 60 Bin 100 Pulse height spectra No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 680 700 720 740 760 Neutrons in cph 1 May 2 May 3 May Parameter change for sensor no. 3 www.geosci-instrum-method-data-syst.net/7/83/2018/ www.geosci-instrum-method-data-syst.net/7/83/2018/ Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 92 -2 0 +2 1 2 3 4 5 (öxed) 6 (öxed) 7 (öxed) 8 9 (öxed) Relative efficiency in % (deviation from the mean) Sensor no. PHS 0.77 Phase I 1.87 Phase II 1.64 Phase III 0.52 Change I II -1 +1 +3 -3 Mean squared deviation in % Swapped -0.37 Fixed -0.04 Figure 5. Relative deviation of the neutron count rates around their ensemble mean, calculated before (Phase I) and after (Phase II) the swap of sensor positions and after adjusting the detector parameters (Phase III). In addition, theoretical values before Phase III have been determined from the location of the discriminator in the pulse height spectrum (PHS, black). Error bars are based on the standard deviation of the count rate for each sensor. Mean squared deviation in % Figure 5. Relative deviation of the neutron count rates around their ensemble mean, calculated before (Phase I) and after (Phase II) the swap of sensor positions and after adjusting the detector parameters (Phase III). In addition, theoretical values before Phase III have been determined from the location of the discriminator in the pulse height spectrum (PHS, black). Error bars are based on the standard deviation of the count rate for each sensor. experiments using spherical neutron detectors (Figs. 8–9 in Rühm et al., 2009). 3.4 Spatial heterogeneity in the footprint area The previous sections have confirmed that there is a mea- surable positional effect (hypothesis A), that device-specific variability exists (hypothesis B), and that statistical noise contributes to the measurement uncertainty (hypothesis C). Solutions have been found to overcome the latter two is- sues by adjusting the detector parameters or by aggregating the temporal resolution, respectively. But what can be done to better understand the influence of local structures in the CRNS footprint? Positional effects within a few metres can occur and should be taken into account, although their effect was shown to be less important than the detector-specific variability. Several of the conducted observations supported the hypo- thetical influence of local effects within the complex terrain. For example, Fig. 3 shows high variability of neutron count rates in drying periods and low variability in wetting peri- ods. This could be an effect of the dynamic size of the foot- print and of the varying rates of evaporation and dewfall. According to Köhli et al. (2015), the distance which neu- trons travelled before detection is smaller for wetter condi- tions. Thereby, distant structures could lose influence during and after rain events and thus would contribute to a harmo- nization of the nine sensor count rates. A second observation refers to Fig. 5, where noticeable changes of variability were observed for swapped sensors (phase transition I→II), while the behaviour of fixed sensors was almost unchanged. Figure 6. Adjustment of the detector parameters harmonized the pulse height spectra of the nine sensors (before: dotted black) and increased their range towards the lower left end. The impact on the count rate is shown exemplarily for sensor 3 (orange). The accuracy of the CRNS soil moisture product also im- proves for higher integration times. In Fig. 7b the effect of the temporal aggregation of neutron counts is propagated to the individual soil moisture products θ(Ni), where their root- mean-square errors (RMSEs) against the ensemble mean θ(⟨N⟩i) are plotted. For all sensors, RMSEs were reduced by 50–70 % using daily aggregation, while an accuracy of 1 % gravimetric water content was achieved beyond integration times of 6 h. These findings agree quantitatively with theo- retical calculations by Bogena et al. (2013) and with similar The two examples indicate that local effects might have the potential to influence the sensor performance. Local sen- sitivity of the neutron detectors has been augured already by M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 93 0.0 5 0.2 1.0 Pearson correlation 10 15 20 0 Integration time in h (a) 0.8 Phase I Phase II Phase III Ensemble spread 0.6 0.4 5 10 15 20 0 0.0 5 0.2 1.0 Pearson correlation RMSE (in grav. %) of sensor soil moisture against their mean 10 15 20 0 Integration time in h (a) 0.8 Phase I Phase II Phase III Ensemble spread 0 5 1 5 10 15 20 0 Integration time in h (b) 4 0.6 0.4 2 3 5 10 15 20 0 No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 Phase I. Phase II. Phase III. Figure 7. Influence of integration time, in hours (h), on the correlation and performance among the ensemble of nine sensors. (a) Ensemble- average Pearson correlation of the nine signals by twos for phases I, II, and III and temporal aggregation from 1 to 24 h. (b) Root mean square error of the individual soil moisture products against the soil moisture product of the ensemble mean ⟨N⟩. Accuracy below gravimetric water contents of 1 % can be achieved for all sensors when sensor-specific offsets were removed (Phase III) and the integration time exceeds 6 h. 5 10 15 20 0 RMSE (in grav. %) of sensor soil moisture against their mean 0 5 1 5 10 15 20 0 Integration time in h (b) 4 2 3 5 10 15 20 0 No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 Phase I. Phase II. Phase III. Pearson correlation (a) Figure 7. Influence of integration time, in hours (h), on the correlation and performance among the ensemble of nine sensors. (a) Ensemble- average Pearson correlation of the nine signals by twos for phases I, II, and III and temporal aggregation from 1 to 24 h. (b) Root mean square error of the individual soil moisture products against the soil moisture product of the ensemble mean ⟨N⟩. Accuracy below gravimetric water contents of 1 % can be achieved for all sensors when sensor-specific offsets were removed (Phase III) and the integration time exceeds 6 h. ficient to support the theory of highly heterogeneous patterns in the urban terrain. Köhli et al. M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment (2015) and could be a reasonable explanation given the heterogeneous distribution of the soil, of vegeta- tion, and of nearby structures. This section tries to further quantify the local effects in a moisture-averaging footprint of several tens of hectares, where all sensors are exposed to similar meteorological forcings. Both experimental and theoretical results clearly demon- strate that a significant neutron heterogeneity can occur within the CRNS footprint under conditions of complex ter- rain. These patterns have the potential to influence the CRNS measurements. Moreover, slight variability is evident in the small meadow (centre cross in Fig. 8), where trees and struc- tures might influence the neutron density on a scale of a few metres. This could serve as an explanation for the mi- nor position-related variability observed in the course of this study. To assess the influence of complex terrain in the urban area, neutron transport simulations were conducted with the Monte Carlo code URANOS (Sect. 2.5). The model calcu- lated the neutron response to the structures in the footprint and simulated the neutron density that could be potentially observed with CRNS detectors in the whole area. Figure 8c shows features of low and high neutron counts on the metre scale that are related to the effects of buildings, sealed areas, the pond, iron-containing structures, and vegetation. Under these conditions it is evident that local heterogeneity in the footprint can have an effect on CRNS probes located within a distance of a few metres. 3.5 Soil moisture estimation and areal correction for sealed areas Considering the revealed small-scale heterogeneity in the sensor footprint, as well as large sealed areas around the sen- sors, the important question arises whether CRNS in urban areas will be able to reliably estimate environmental water content. Therefore, we have evaluated the CRNS soil mois- ture product (Eq. 2) with time series data from two nearby profiles using the WSN. The locations (crosses) of each pro- file are shown in Fig. 9; the data were averaged and compared against the CRNS signal of sensor 7 (point). The URANOS model can help to assess those effects to support optimal sensor positioning or to explain unusual fea- tures in the spatial signal. The simulation results demonstrate the non-uniformity of the neutron density in the footprint. However, the simulated quantities are not expected to exactly match reality due to many modelling assumptions that have been put into the scenario (clean material composition, uni- form biomass density, homogeneous soil moisture). Never- theless, the modelled patterns can be assessed visually us- ing measurements from a CRNS rover (Fig. 8d, e). The rela- tive uncertainties of both the modelled and measured results are in the range of 6–9 % for ≈200 modelled neutrons per m2 and ≈200 measured neutrons per minute. A direct com- parison with the simulation results was not intended, as the low number of measurement points does not allow for metre- scale predictions of neutron density from the ordinary krig- ing interpolation. However, the collected data have been suf- Figure 9 shows that the CRNS soil moisture product (or- ange) differs significantly from the point measurements (grey dashed). Most importantly, the response to rain events ap- pears to be much more damped in the CRNS signal. A damp- ing effect can occur when a constant fraction of measured neutrons is independent of precipitation events. The CRNS probe’s footprint is much larger than the small meadow of 0.1 ha where the CRNS and the WSN probes are located. It is thus evident that the paved and sealed areas be- yond the meadow could bias the integral soil moisture signal. www.geosci-instrum-method-data-syst.net/7/83/2018/ www.geosci-instrum-method-data-syst.net/7/83/2018/ Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 94 Birds-eye view (Google Maps) 0 0 -100 -100 -200 -200 100 100 200 m 200 m iron vegetation building concrete road tar road water pool sand wood grass roof grass gas pipe (a) e d b a Abstract map of materials for input to URANOS (b) tartan ground Relative neutron density, simulated with URANOS (c) 1.0 0.9 0.8 0.7 0.6 Relative neutron density, measured with Rover, Kriging interpol. of points (d) May 2, 2014. (e) Jul 22, 2015 (d) (e) c P S P P P S S S 1.0 0.9 0.8 0.7 0.6 Neutron environment of the urban CRNS test site (centred black cross). (b) Abstract model of the area using ge ed material definitions. (c) URANOS simulation of epithermal neutrons in a detector layer above the surface. ( he mobile CRNS rover confirm heterogeneity of neutron patterns in the centred grass meadow as well as in t m Method Data S st 7 83 99 2018 geosci instr m method data s st n Figure 8. (a) Neutron environment of the urban CRNS test site (centred black cross). (b) Abstract model of the area using geometric shapes and colour-coded material definitions. (c) URANOS simulation of epithermal neutrons in a detector layer above the surface. (d, e) Measured neutrons with the mobile CRNS rover confirm heterogeneity of neutron patterns in the centred grass meadow as well as in the surrounding urban domain. Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 www.geosci-instrum-method-data-syst.net/7/83/2018/ Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 www.geosci-instrum-meth Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 www.geosci-instrum-method-data-syst.net/7/83/2018/ www.geosci-instrum-method-data-syst.net/7/83/2018/ Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 95 Pond Sand Concrete Grass Tree Building 10 m CRNS Grass area equivalent circle WSN 25 20 15 10 Sep 29 Water equiv. in % Soil probe CRNS corrected CRNS Oct 06 Oct 13 Oct 20 Oct 27 Rain Intercepted water Figure 9. Top: illustration of the circular area (dashed) around the CRNS probe 7 that confines an area equivalent to the non-sealed grassland meadow. Bottom: demonstrating the area correction ap- proach (Eq. 11). The constant contribution of neutrons from the sealed area leads to a damped signal of soil moisture dynamics mea- sured by the CRNS (orange line). The corrected signal (blue line) shows more pronounced dynamics based only on the areal contri- bution of the meadow. The remaining deviation (shaded blue) from the WSN soil moisture measurements (grey dashed) probably rep- resents intercepted water over the sealed ground during and after rain events. Pond Sand Concrete Grass Tree Building 10 m CRNS Grass area equivalent circle WSN A circular section of angle ϕ (in radiant), which is confined between radii r1 and r2, contributes the following fraction of neutrons n: n(r1,r2,ϕ) = 1 2π Z ϕ 0 Z r2 r1 Wr(h,θ) · dr · dϕ′ = ϕ 2π Z r2 r1 Wr(h,θ) · dr . (10) (10) The contribution area of the grassland meadow and sur- rounding patches is roughly equivalent to a circle of radius r2 ≈20 m. Hence, the portion of measured neutrons from this area is n(0,r2) ≈41 ± 2 %, depending on h and θ. The dry and sealed areas beyond the grass meadow are effectively damping the otherwise highly dynamic signal from the soil (orange line in Fig. 9). 25 20 15 10 Sep 29 Water equiv. in % Soil probe CRNS corrected CRNS Oct 06 Oct 13 Oct 20 Oct 27 Rain Intercepted water Intercepted water To remove this damping effect, we suggest a new method to rescale the dynamic component of the neutron signal that is influenced by both a variable and a constant patch in the footprint. At the urban test site, only 0.1 ha of the footprint contains soil, beyond which everything else is either paved area or solid building. Thus, only a small fraction n(r1,r2,ϕ) of the total neutrons is connected to soil moisture variability. M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment In order to compare these measurements with independent soil moisture sensors, we introduce an areal correction, Figure 9. Top: illustration of the circular area (dashed) around the CRNS probe 7 that confines an area equivalent to the non-sealed grassland meadow. Bottom: demonstrating the area correction ap- proach (Eq. 11). The constant contribution of neutrons from the sealed area leads to a damped signal of soil moisture dynamics mea- sured by the CRNS (orange line). The corrected signal (blue line) shows more pronounced dynamics based only on the areal contri- bution of the meadow. The remaining deviation (shaded blue) from the WSN soil moisture measurements (grey dashed) probably rep- resents intercepted water over the sealed ground during and after rain events. Ncorr = N −⟨N⟩ n(r1,r2,ϕ) + ⟨N⟩, (11) (11) that essentially scales the anomaly of neutrons by the inverse fraction of the contributing area, where ⟨·⟩denotes the tem- poral mean. Using the corrected data from the CRNS probe (blue line) and the average soil moisture from the two profiles (grey), Fig. 9 demonstrates that this scaling approach brings both signals into good agreement and is therefore helpful to interpret CRNS data with confined areal coverage. Besides the improved match of soil moisture dynamics, the area-corrected signal apparently overestimates soil moisture peaks during and after rain events. This can be interpreted as a representation of an important hydrological feature in urban areas. When the whole footprint is considered for data inter- pretation, it becomes evident that the CRNS should be sen- sitive to precipitation water ponded on buildings and paved ground before it eventually evaporates. Therefore, the addi- tional water seen by the CRNS probe following rain events can be suspected of representing the intercepted water over sealed areas. We suspect the dry soil under the sealed surface in the foot- print of having a constant and thus damping influence on the neutron signal. In the following, we aim to test the applica- tion of recent insights about the sensor’s spatial sensitivity and demonstrate how this knowledge can help to understand and even correct the biasing effect of sealed areas. Following theoretical considerations from Köhli et al. (2015) and robust evidence from Schrön et al. (2017a), the radial sensitivity function Wr(h,θ) depicts the number of de- tected neutrons that originated from the distance r under cer- tain homogeneous conditions of air humidity, h, and (soil) water equivalent, θ. M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment Its integral across all distances represents the total number of neutrons detected, N: www.geosci-instrum-method-data-syst.net/7/83/2018/ Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 9 Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/gi-7-83-2018-supplement. Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/gi-7-83-2018-supplement. C. Statistical noise was suspected to be the reason for much of the remaining variability that hinders comparability of neutron signals. We applied temporal aggregation to the neutron signals and looked at the correlation and en- semble spread of the CRNS products. Sensors showed correlations below 0.6 for hourly data and above 0.9 for aggregation of 6 h and beyond. In the same manner the RMSE between the soil moisture products improved from 2 % down to 0.9 % gravimetric percent. If multiple standard CRNS detectors are required to deliver similar results under similar conditions, a minimum temporal resolution of 6 h was found to provide acceptable com- parability for humid climate at sea level. This value can be considered as an upper limit, as it gets further re- duced by drier climates, higher altitudes, and more sen- sitive detectors. Competing interests. Darin Desilets and Gary Womack are affili- ated with Hydroinnova LLC, the manufacturer of the probes used in this study. Acknowledgements. We thank Uwe Kappelmeyer (UFZ) for providing meteorological data from a nearby UFZ-owned weather station. We acknowledge the NMDB database www.nmdb.eu, founded under the European Union’s FP7 programme (contract no. 213007) for providing data for incoming radiation, especially from monitors Jungfraujoch (Physikalisches Institut, University of Bern) and Kiel (Institute for Experimental and Applied Physics, University of Kiel). Martin Schrön acknowledges kind support by the Helmholtz Impulse and Networking Fund through Helmholtz Interdisciplinary School for Environmental Research (HIGRADE). Mandy Kasner was funded by the Helmholtz Alliance EDA – Remote Sensing and Earth System Dynamics, through the Initiative and Networking Fund of the Helmholtz Association, Germany. The research was funded and supported by Terrestrial Environmental Observatories (TERENO), which is a joint collaboration program involving several Helmholtz Research Centres in Germany. This work highlights the importance of studies on sensor- to-sensor intercomparison for geoscientific instruments. Those efforts can reveal unexpected features or systematic errors, can highly improve the understanding of the sensor response, and will thus improve their application in environ- mental sciences. One of the impacts of this study has already led to improved efforts to adjust the detection parameters during the manufacturing process. The article processing charges for this open-access publication were covered by a Research Centre of the Helmholtz Association. M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment 96 approach the influence of sealed (and thus constantly dry) ar- eas in the footprint can be quantified and the corresponding damping effects removed. The quantification of the sensitiv- ity to local patches in the footprint is particularly meaning- ful for supporting hyper-resolution land surface modelling (e.g. Chaney et al., 2016) and precision agriculture. The latter includes targeted irrigation based on information about soil properties, plant variety, and density (Hedley et al., 2013; Pan et al., 2013). In addition to soil moisture, the CRNS probe ap- peared to be sensitive to intercepted water over sealed areas. Such information could be used to actually quantify intercep- tion and evaporation processes (see e.g. Baroni and Oswald, 2015) and could eventually contribute to closing the water balance. nine CRNS measurement stations within a 5 m ×15 m grass- land area, surrounded by complex urban terrain. Three main hypotheses were investigated and the following conclusions were drawn: A. We claimed that the sensor location has an influence on the neutron measurement. The hypothesis was tested by swapping the position of four out of nine CRNS probes. We found the influence on the neutron counts is mea- surable, but insignificant within a few metres. However, mobile surveys as well as neutron simulations indicated that neutron abundance can be highly heterogeneous within the sensor’s footprint, making the signal prone to local effects on scales above a few metres. A. We claimed that the sensor location has an influence on the neutron measurement. The hypothesis was tested by swapping the position of four out of nine CRNS probes. We found the influence on the neutron counts is mea- surable, but insignificant within a few metres. However, mobile surveys as well as neutron simulations indicated that neutron abundance can be highly heterogeneous within the sensor’s footprint, making the signal prone to local effects on scales above a few metres. In future studies we would recommend to further assess the potential of cosmic-ray neutron sensors for urban hydrol- ogy. Since water in complex terrain is almost impossible to quantify with point sensors, the large-scale averaging capa- bilities of cosmic-ray neutron probes could be a promising advantage for urban sciences. B. M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment Device-specific differences were suspected of being re- sponsible for systematic variations in the CRNS signal. This was tested with the help of the manufacturers by consistently adjusting neutron detection parameters and aligning the pulse height spectra of the nine sensors. The detection parameters were found to have significant in- fluence on the count rate by up to 3 %. The adjustment led to a reduction of the total contribution of systematic errors down to the order of the statistical counting er- rors. We recommend applying this adjustment in order to achieve consistent measurements among sensors. B. Device-specific differences were suspected of being re- sponsible for systematic variations in the CRNS signal. This was tested with the help of the manufacturers by consistently adjusting neutron detection parameters and aligning the pulse height spectra of the nine sensors. The detection parameters were found to have significant in- fluence on the count rate by up to 3 %. The adjustment led to a reduction of the total contribution of systematic errors down to the order of the statistical counting er- rors. We recommend applying this adjustment in order to achieve consistent measurements among sensors. Data availability. The multi-sensor dataset is available in the Sup- plement of this paper. Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 4 Summary and conclusion This intercomparison study was motivated by the observa- tion of unknown variability in CRNS data and by the aim to understand how reliable and reproducible soil moisture data could be generated using the method of cosmic-ray neu- tron sensing. To address the open questions, we co-located N = ∞ Z 0 Wr(h,θ) · dr . (9) www.geosci-instrum-method-data-syst.net/7/83/2018/ www.geosci-instrum-method-data-syst.net/7/83/2018/ Geosci. Instrum. Method. 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Hawdon, A., McJannet, D., and Wallace, J.: Calibration and cor- rection procedures for cosmic-ray neutron soil moisture probes Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/gi-7-83-2018-supplement. The CRNS water equivalent measured in the urban en- vironment has shown remarkable agreement with indepen- dently measured soil moisture profiles when accounted for the sealed-area effect. With the proposed “areal correction” www.geosci-instrum-method-data-syst.net/7/83/2018/ Geosci. Instrum. Method. Data Syst., 7, 83–99, 2018 97 M. Schrön et al.: Intercomparison of cosmic-ray neutron sensors in an urban environment Edited by: Andrea Benedetto Edited by: Andrea Benedetto Reviewed by: two anonymous referees Corke, P., Wark, T., Jurdak, R., Hu, W., Valencia, P., and Moore, D.: Environmental Wireless Sensor Networks, P. IEEE, 89, 1903– 1917, 2010. 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Large scale patterns of genetic variation and differentiation in sugar maple from tropical Central America to temperate North America
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Large scale patterns of genetic variation and differentiation in Large scale patterns of genetic variation and differentiation in sugar maple from tropical Central America to temperate North sugar maple from tropical Central America to temperate North America America Lowell E. Urbatsch Louisiana State University David W. Foltz Louisiana State University Follow this and additional works at: https://repository.lsu.edu/biosci_pubs Faculty Publications Faculty Publications Department of Biological Sciences Faculty Publications Louisiana State University Louisiana State University LSU Scholarly Repository LSU Scholarly Repository Faculty Publications Department of Biological Science 11-19-2015 Large scale patterns of genetic variation and differentiation in Large scale patterns of genetic variation and differentiation in sugar maple from tropical Central America to temperate North sugar maple from tropical Central America to temperate North America America Yalma L. Vargas-Rodriguez Louisiana State University William J. Platt Louisiana State University Lowell E. Urbatsch Louisiana State University David W. Foltz Louisiana State University Louisiana State University Louisiana State University LSU Scholarly Repository LSU Scholarly Repository © 2015 Vargas-Rodriguez et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH ARTICLE Open Access Recommended Citation Recommended Citation Vargas-Rodriguez, Y., Platt, W., Urbatsch, L., & Foltz, D. (2015). Large scale patterns of genetic variation and differentiation in sugar maple from tropical Central America to temperate North America. BMC Evolutionary Biology, 15 (1) https://doi.org/10.1186/s12862-015-0518-7 This Article is brought to you for free and open access by the Department of Biological Sciences at LSU Scholarly Repository. It has been accepted for inclusion in Faculty Publications by an authorized administrator of LSU Scholarly Repository. For more information, please contact ir@lsu.edu. Vargas-Rodriguez et al. BMC Evolutionary Biology (2015) 15:257 DOI 10.1186/s12862-015-0518-7 Large scale patterns of genetic variation and differentiation in sugar maple from tropical Central America to temperate North America Yalma L. Vargas-Rodriguez*, William J. Platt, Lowell E. Urbatsch and David W. Foltz * Correspondence: ylvargasro@conacyt.mx Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA Background g Geological events have affected the distributions and levels of gene flow among populations of North American tree species. Repeated glacial and intergla- cial periods promoted range shifts of a number of tree species during late Pliocene and the Quaternary [1–4]. Studies indicate different genetic responses to range contraction and expansion [5, 6]. For instance, patterns of genetic diversity in temperate North American trees (e.g., Carya illinoinensis, Liquidambar styraciflua, Fagus grandifolia) include higher diversity in southern, never-glaciated areas, and lower diversity in northern areas where glaciated landmasses existed [7–11]. Furthermore, there is evidence of the impact of the Quaternary glaciations on the population genetic structure of species (e.g., Acer rubrum, A. sac- charinum, Quercus rubra) that survived in multiple refugia closer to the ice margin [10–12]. Populations of temperate tree species in the subtropics (e.g., Pinus chiapensis, Picea chihuahuana, Pseudotsuga menziesii, Fagus grandifolia var. mexicana) also show genetic differentiation related to the history of migration and isolation during glacial and interglacial periods [13–16]. Additionally, the distributions of many North American temperate species include disjunct populations in cloud forests in Mexico and Central America. These isolated populations may have originated by ancient vicariant events or through range fluctuations. These fluctuations may have involved expansions into low elevation areas at low latitudes during glacial episodes, and contractions to high elevation refuges during warmer interglacial periods [17, 18]. We investigated patterns of genetic variation and structure in extant populations of sugar maples in the tropics and explored their genetic relationships with populations in the temperate United States. We used these data to infer their evolutionary history and large-scale connectivity. We quantified within- and among-population genetic variation across the species range using chloroplast sequences and microsatellite loci. We hypothesized that ancestral haplotypes have persisted in tropical populations in unglaciated areas that have changed little over long periods, and that these populations have harbored higher genetic diver- sity. Next, we estimated divergence times of haplo- types among populations and considered whether observed genetic subdivisions were temporally consist- ent with the late Pliocene and Quaternary geologic events. Finally, we tested the hypotheses of expand- ing/contracting populations and the presence of a sin- gle refugium versus multiple refugia during episodes of glaciation. Abstract Background: Geological events in the latter Cenozoic have influenced the distribution, abundance and genetic structure of tree populations in temperate and tropical North America. The biogeographical history of temperate vegetation that spans large ranges of latitude is complex, involving multiple latitudinal shifts that might have occurred via different migration routes. We determined the regional structuring of genetic variation of sugar maple (Acer saccharum subsp. saccharum) and its only subspecies in tropical America (Acer saccharum subsp. skutchii) using nuclear and chloroplast data. The studied populations span a geographic range from Maine, USA (46°N), to El Progreso, Guatemala (15°N). We examined genetic subdivisions, explored the locations of ancestral haplotypes, analyzed genetic data to explore the presence of a single or multiple glacial refugia, and tested whether genetic lineages are temporally consistent with a Pleistocene or older divergence. Results: Nuclear and chloroplast data indicated that populations in midwestern USA and western Mexico were highly differentiated from populations in the rest of the sites. The time of the most recent common ancestor of the western Mexico haplotype lineage was dated to the Pliocene (5.9 Ma, 95 % HPD: 4.3–7.3 Ma). Splits during the Pleistocene separated the rest of the phylogroups. The most frequent and widespread haplotype occurred in half of the sites (Guatemala, eastern Mexico, southeastern USA, and Ohio). Our data also suggested that multiple Pleistocene refugia (tropics-southeastern USA, midwestern, and northeastern USA), but not western Mexico (Jalisco), contributed to post-glacial northward expansion of ranges. Current southern Mexican and Guatemalan populations have reduced population sizes, genetic bottlenecks and tend toward homozygosity, as indicated using nuclear and chloroplast markers. Conclusions: The divergence of western Mexican populations from the rest of the sugar maples likely resulted from orographic and volcanic barriers to gene flow. Past connectivity among populations in the southeastern USA and eastern Mexico and Guatemala possible occurred through gene flow during the Pleistocene. The time to the most common ancestor values revealed that populations from the Midwest and Northeast USA represented different haplotype lineages, indicating major divergence of haplotypes lineages before the Last Glacial Maximum and suggesting the existence of multiple glacial refugia. Keywords: Acer saccharum, Cloud forest, Divergence time, Gene flow, Genetic diversity, Last glacial maximum, Microsatellite, Phylogeography, Pleistocene, Pliocene, Range edge Vargas-Rodriguez et al. BMC Evolutionary Biology (2015) 15:257 Page 2 of 14 data suggest that A. Abstract saccharum underwent northward geographic expansions from a single continuous ice-free refugium in the Southern USA about 21,000 years B.P., around the beginning of the retreat of the Laurentide ice sheet [17]. The evidence of the existence and location of glacial refuges of sugar maple farther north than the Lower Mississippi Valley has been inconclusive [20]. Nonetheless, the pollen record of some temperate eastern taxa suggests the presence of small popula- tions in the upper Midwest and in the Appalachian region during glaciations [21, 22]. In addition, accord- ing to genetic data, Acer rubrum likely existed north of their Pleistocene pollen-based range limits, but, it is not yet known if the same applies to A. saccharum [21]. Acer saccharum exhibits some genetic differenti- ation in southeastern Canada and northeastern USA, but less genetic variation than other temperate trees or shrubs [23]. In addition, some sugar maple populations in Canada possess limited genetic differ- entiation, possibly due to common ancestry or recent colonization after a glacial period [24, 25]. These gen- etic studies of sugar maple in North America were conducted near its northern range limit, preventing analysis of range expansions as a result of late Plio- cene and Quaternary events. Furthermore, southern sugar maple populations with disjunct distributions from northern Mexico to Guatemala have not been considered in reconstructions of historical migration corridors. Genotyping microsatellites The sampled populations of sugar maple spanned a geographic range from Maine, USA (46°N), to El Pro- greso, Guatemala (15°N). Fresh foliage was sampled from 233 adult individuals from 16 native populations (Fig. 1, Additional file 1). Twenty-five to 41 adult Six microsatellite loci were amplified using primer pairs and polymerase chain reaction (PCR), the protocol described by Pandey et al. [27]. Six out of eight primers had been developed for Acer pseudoplatanus and showed high degree of cross-species amplification (MAP9, MAP10, a b Fig. 1 Map of collection sites and haplotype network in sugar maples. a Distribution of chloroplast haplotypes observed. Pie charts indicate the frequency of haplotypes within each population. Each circle corresponds to a locality. Haplotypes found are indicated by different colors. Barrier boundaries using nuclear data are red lines; boundaries based on chloroplast data are green lines. Boundaries, calculated in Barrier v.2.2 using the Dest differentiation estimator for nuclear microsatellites (bootstrap support is 58 % for Midwest, 45 % for West Mexico, 41 % for South Mexico) and the Tamura-Nei population distance measure DA for chloroplast haplotypes (bootstrap support is 55 % for Midwest, 50 % for South Mexico, 50 % for West Mexico, 46 % for Northeast). Black, continuous line indicates natural distribution of A. saccharum in North America. All know populations in Mexico and Guatemala of A. saccharum subsp. skutchii are represented in the map. b Network of 34 chloroplast DNA haplotypes observed. The identification letter of each haplotype is presented. The size of the circle and the number indicate the observed frequency. The colors correspond to the alleles depicted in the map a) a b a Fig. 1 Map of collection sites and haplotype network in sugar maples. a Distribution of chloroplast haplotypes observed. Pie charts indicate the frequency of haplotypes within each population. Each circle corresponds to a locality. Haplotypes found are indicated by different colors. Barrier boundaries using nuclear data are red lines; boundaries based on chloroplast data are green lines. Boundaries, calculated in Barrier v.2.2 using the Dest differentiation estimator for nuclear microsatellites (bootstrap support is 58 % for Midwest, 45 % for West Mexico, 41 % for South Mexico) and the Tamura-Nei population distance measure DA for chloroplast haplotypes (bootstrap support is 55 % for Midwest, 50 % for South Mexico, 50 % for West Mexico, 46 % for Northeast). Black, continuous line indicates natural distribution of A. Methods individuals (>40 cm diameter at breast height) per population, spaced a minimum of 50 m apart, were sampled in the USA and, where the density permit- ted, in Mexico. Because of low tree densities in Guatemala, all mature trees observed were sampled regardless of distances among them. The leaves were collected with permits granted by forest reserves. The first author performed the species identification. Leaves were immediately dried in silica gel. Dry leaf material (20–50 mg) was ground using a Mini Bead- beater 8 (BioSpec Products, Bartlesville, OK). The genomic DNA was extracted and purified using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Background Fossil pollen Page 3 of 14 Vargas-Rodriguez et al. BMC Evolutionary Biology (2015) 15:257 Field and laboratory procedures Sampling and DNA extraction Sampling of Acer saccharum subsp. saccharum was con- ducted in old-growth forests of USA. Sites where the species identity was doubted by botanists were not sam- pled. Sampling in Mexico and Guatemala included all known populations where A. saccharum subsp. skutchii has been recorded [19]. These sugar maples belong to the series Saccharodendron (Rafinesque) Murray, section Acer, characterized by apetalous flowers and connate calyces. Acer saccharum subsp. skutchii represents the only member of the series distributed in Mexico and Guatemala [26]. Both subspecies are morphologically the most similar of the series [26]. Background Based on our study, we propose the location of more than one continuous ice-age refu- gium in North America, as well as potential migration routes in relation to the complex phylogeographical patterns of North and Central American hardwood forests. Periods of connectivity and disjunction among North American and Central American refugial areas and their consequences on the genetic structure of the temperate tree populations are not known for most species. Limited geographical sampling reduces the capacity to reconstruct historical refugia and iden- tify the spatial location of genetic breaks in temperate tree species at a continental level. Assessing large- scale relationships of populations should elucidate the importance of range expansions and contractions in producing the current distribution patterns of species, as well as generate hypotheses regarding the genetic structure and diversity of temperate tree lineages af- fected by glaciation in the latter part of the Cenozoic. Acer saccharum (sugar maple) is a widespread temper- ate tree species. Study of the genetics of this species has the potential to elucidate a late Pliocene and Quaternary history that may be shared by other elements of the North America flora. The species has a continuous dis- tribution from southern Quebec to the southeastern USA, and then is disjunct in distribution between the eastern USA, Mexico, and Guatemala [19]. Fossil pollen Periods of connectivity and disjunction among North American and Central American refugial areas and their consequences on the genetic structure of the temperate tree populations are not known for most species. Limited geographical sampling reduces the capacity to reconstruct historical refugia and iden- tify the spatial location of genetic breaks in temperate tree species at a continental level. Assessing large- scale relationships of populations should elucidate the importance of range expansions and contractions in producing the current distribution patterns of species, as well as generate hypotheses regarding the genetic structure and diversity of temperate tree lineages af- fected by glaciation in the latter part of the Cenozoic. Acer saccharum (sugar maple) is a widespread temper- ate tree species. Study of the genetics of this species has the potential to elucidate a late Pliocene and Quaternary history that may be shared by other elements of the North America flora. The species has a continuous dis- tribution from southern Quebec to the southeastern USA, and then is disjunct in distribution between the eastern USA, Mexico, and Guatemala [19]. Genotyping microsatellites saccharum in North America. All know populations in Mexico and Guatemala of A. saccharum subsp. skutchii are represented in the map. b Network of 34 chloroplast DNA haplotypes observed. The identification letter of each haplotype is presented. The size of the circle and the number indicate the observed frequency. The colors correspond to the alleles depicted in the map a) Page 4 of 14 Vargas-Rodriguez et al. BMC Evolutionary Biology (2015) 15:257 MAP33, MAP34, MAP40, and MAP46). The forward primer was fluorescently labeled using 6-FAM or HEX for MAP10 (Applied Biosystems, Foster City, CA, USA) on the 5' - end for detection on an ABI 3130XL (Applied Biosystems, USA). platform (http://www.ebi.ac.uk), and manually edited in McClade v4.08 [32] as needed. The sequences have been submitted to GenBank with the accession num- bers KT933356–KT933397. PCR and fragment analysis were carried out as follows. PCR amplifications of 20 μL aliquots were performed using 20 ng of genomic DNA (quantified with a Nano- Drop 2000, Thermo Scientific, Waltham, Massachusetts, USA), 0.2 mM dNTP, 10 μM of each primer, 1.5 mM MgCl2, and 1 U HotStarTaq polymerase (HotStarTaq Master Mix, Qiagen, USA). PCR programs consisted of an initial denaturation at 95 °C for 15 min, followed by 30 cycles of 45 s at 94 °C, 45 s at appropriate annealing temperature, and 45 s at 72 °C, and a final extension of 15 min at 72 °C. Annealing temperatures were determined using a gradient PCR and were as follows: 52 °C (MAP9 and MAP33), 55 °C (MAP10), 55.8 °C (MAP34), 58 °C (MAP40), 48 °C (MAP46). Two microliters of PCR product were added to 10.8 μL of Hi-Di Formamide (Applied Bio- systems, USA) and 0.2 μL ROX 400 HD size standard and run on an ABI 3130XL automated sequencer. The allele size calling (size of fragments using decimal numbers) was done using GeneMapper v4.1 (Applied Biosystems, USA), and the binning (conversion of alleles into discrete units) was done with Autobin [28]. MICRO-CHECKER v2.2.3 was used to identify genotyping errors such as large allele dropout, stutter peaks, or null alleles [29]. Chloroplast DNA sequencing Chloroplast DNA sequencing The ndhF-rpl32R intergenic spacer in the small single-copy region and psbJ-petA intergenic spacer in the large single-copy region from the chloroplast were amplified [30]. Both regions are noted as highly vari- able and several haplotypes have been observed [30]. PCR was used to amplify the two regions following the protocol described in Shaw et al. [30] with some modifications. Each reaction (25 μL) consisted of 20 ng of genomic DNA, 10 μM of each primer, 0.2 mM dNTP, 1.5 mM MgCl2, and 1 U HotStarTaq polymerase (HotStarTaq Master Mix, Qiagen, USA). PCR programs consisted of an initial denaturation at 95 °C for 15 min, followed by 35 cycles of 1 min at 95 °C, annealing at 52 °C for 1 min, followed by a ramp of 0.3 °C/s to 65 °C, and 4 min at 65 °C, and a final extension of 10 min at 66 °C. PCR products were purified using the solid phase reversible immobilization technique and sequenced in both directions using BigDye Terminator v3.1 (Applied Biosystems, USA) on an ABI-PRISM 3730XL. Se- quence chromatograms were visually inspected and edited in Sequencher v4.1 (Gene Codes Corp., Ann Arbor, MI, USA), alignment was done in MUSCLE [31] using the European Bioinformatics Institute web y Nuclear microsatellite diversity Deviations from Hardy-Weinberg Equilibrium (HWE) and linkage disequilibrium were assessed in GENEPOP v4.1 [33]. The test was run using Markov chain parame- ters of 1000 batches and 10,000 iterations-per-batch. A sequential Bonferroni correction was applied to reduce error rates. The mean number of alleles per locus and per site and the expected (HE) and observed heterozygos- ity (HO) per locus, as well as the private allelic richness of each population were calculated in the R package “ade- genet” [34]. The inbreeding coefficient (FIS) was estimated for each population, and differences among populations were compared using Mann-Whitney U-tests. Each population was evaluated for evidence of popula- tion bottlenecks. Tests were done using the approaches described by Cornuet and Luikart [35] and Garza and Williamson [36]. Under the Cornuet and Luikart method, the estimates of expected heterozygosity based on allele frequencies (HE), and on the number of alleles and sample size (HEQ) were compared, based on the assumption that the allele diversity is reduced faster than the heterozygosity. The estimates were calculated under the infinite allele model (IAM) and stepwise-mutation model (SMM) with 1000 iterations, and the allele fre- quency graphical mode. Significance was tested using the Wilcoxon test with the Bonferroni correction, all implemented in BOTTLENECK v1.2.02 [35]. Under the Garza and Williamson method [36], the mean ratio of the total number of alleles (k) to the range in allele size (r) is calculated (M-ratio), where k is reduced faster than r. Thus, a reduced M-ratio indicates a population having been through a bottleneck. The significance of the ratio was evaluated using the critical value (Mc) under the two-phase mutation model (TPM), with the average size of non one-step mutations = 2.8, as determined by [36], and θ values from 0.01, 10, and 50. Mean M-ratio and Mc were estimated using M_P_VAL and Critical_M software, respectively [36]. Spatial genetic patterns h The genetic structure was further analyzed with the Monmonier maximum difference algorithm to confirm the significance of structure recovered with previous metrics. The algorithm was used to locate areas of maximum genetic distance within a geometric network of geo-referenced points constructed with a Delauney triangulation. Patterns of structure were also assessed by permuting allele sizes among alleles and the calculation of the FST and the RST. Permuted values of RST larger than FST indicate a contribution of stepwise mutations to differ- entiation among populations. Tests were performed in SPAGeDi v1.3 [41] using 10,000 permutations under a regression analysis restricted to population level. RST was used to account for the mutational distances among alleles under the stepwise mutation model [42]. RST is analogous to FST, but it is based on allele size rather than allele identity. Two different bootstrapped genetic distance matri- ces were used, one using nuclear microsatellite data, Dest [53], and other using chloroplast sequence data, ΦST [49]. Matrices were bootstrapped to generate 100 datasets. A multilocus weighted average ΦST estimator was computed using SPADS v1.0 [50] and visualized in BARRIER v2.2 [54] to generate a graphical output (Voronoï polygonation). The number of barrier seg- ments analyzed ranged from two to eight. The num- ber of overlapping segments between marker types was determined as well as those segments unique for a marker. A genetic differentiation estimate was calculated. The estimator Dest [39] was implemented in the application SMOGD using 1000 bootstrap replicates [43]. Dest is determined by mutation and migration rates and num- ber of populations, and it is independent of population size [44]. It is more suitable for describing allelic differ- entiation among populations than other estimates such as GST. Chloroplast diversity G i i i Genetic variation was evaluated for each population and region. The variation was given by number of polymorphic sites (S), number of haplotypes (h), haplotype diversity (Hd), average per site pairwise nu- cleotide diversity (π), and the relationship between polymorphic sites and alleles sampled (θw). The pa- rameters were estimated using DNASP v5.10.01 [37]. Page 5 of 14 Vargas-Rodriguez et al. BMC Evolutionary Biology (2015) 15:257 of genetic variation among groups of populations) that did not include any groups of a single population [49]. The analyses were carried out using SPADS v1.0 [50]. Significant genetic structure was determined using two different population-based measures. Pairwise genetic differentiation analysis was performed using FST [38] and Dest [39] with a priori groups of individuals accord- ing to their geographic position. The F estimator of genetic structure θ per locus and sample was calculated using an analysis of variance (FSTAT v2.9.3.2) [40], where the populations are weighted according to their sample size [38]. The relationships among chloroplast haplotypes were also assessed using statistical-parsimony networks imple- mented in R Statistical Software with the package PEGAS [51, 52]. Hypothesis testing: single and multiple glacial refuge models Coalescent simulations were used to test whether pat- terns of population differentiation are consistent with the single or multiple refuge models. The two a priori hypotheses of Pleistocene population structure tested were the single refugium null model and the multiple glacial refuge model. The gene-tree population-tree approach was applied, following Knowles [60] and implemented in MESQUITE v3.04 [61]. Haplotype gene trees (1000 replicates) were simulated by neutral coalescence with invariable effective population size (Ne) until total coalescence. Five arbitrary Ne were tested (10,000, 50,000, 100,000, 150,000, 225,000). The amount of discordance between the gene trees and the hypothesized populations trees was measured by the s statistic [62]. The s value for the haplotype tree was first obtained and then, the distribution of simu- lated s values for each population tree was analyzed and graphed. Genetic signatures of population bottlenecks were detected, although there was disagreement between the different tests used. The M ratio test indicated that only populations located in the northern and western Mexico and Guatemala experienced a bottleneck, likely lasting several generations. The Wilcoxon test did not indicate a significant recent bottleneck (heterozygosity excess) in any of the populations and under any models. These results were not consistent, however, with the distribu- tion of allele frequency. Eight populations (one in Mexico and two in Guatemala) displayed a mode shift, indicating that a bottleneck had occurred. In contrast, the normal L-shape distribution of allele frequencies indicated that eight populations had not experienced a recent bottleneck. Under the mutation-drift equilibrium scenario, the rarest allele class was more frequent, form- ing an L-shape graph [63]. After a bottleneck, the rarest alleles were rapidly lost, resulting in a mode-shift distor- tion (Table 1). The single refugium hypothesis assumes that extant populations of sugar maple are derived from a refu- gial population, with expansion beginning as the gla- ciers retreat. The divergence time of populations was estimated to early, middle and most recent Pleisto- cene glacial events. The null model of fragmentation of a single ancestral population is rejected if the s value for the haplotype tree falls outside of the 95 % confidence interval for that model [61]. Under the multiple glacial refuge model, the extant population structure of haplotypes results from isolation and divergence within four hypothesized glacial refugia. Nuclear microsatellite diversity Nuclear microsatellite diversity y A total of 17 alleles were detected at the six micro- satellite loci. The total number of observed alleles varied from two (MAP40) to seven (MAP34), and allelic richness ranged from 1.5 to 2.1. Private alleles were present in one locus (MAP34) in the Tamau- lipas, Chiapas, Jalisco, and Maine populations. Depar- tures from the HWE were found in two populations from western Mexico and four from the USA (mid- western: 2, northeastern: 1, and southeastern: 1) (Table 1). Observed heterozygosity ranged from 0.133 ± 0.1 to 0.340 ± 0.1 and expected heterozygosity from 0.132 ± 0.08 to 0.284 ± 0.1 (Table 1). The FIS per population ranged from -0.47 to 0.395. Two popula- tions in Mexico showed a tendency toward increased homozygosity (Guerrero 0.005, Chiapas 0.09), whereas among the USA sites, only populations from Vermont, Ohio, and Tennessee did not exhibit this homozygosity pattern (Table 1). FIS significantly differed between the tropical peripheral sites (Chiapas–Guatemala) and the populations in USA (southeastern U = 12, P = 0.039; midwestern U = 14.5, P = 0.02; northeastern U = 22.5, P = 0.04). Phylogenetic relationships and divergence of haplotypes lineages To detect the number of genetically differentiated pop- ulations without a priori assumptions of population sub- division, a Bayesian clustering analysis was used. The analysis was run under an admixture model with corre- lated allele frequencies for 10 million iterations after a burn-in period of 100,000 iterations for each value of K, and 10 replicates per run. Population structure was in- ferred using a range of K from one to six. The analysis was performed in STRUCTURE v2.3.3 [45]. The Evanno method [46] was implemented in STRUCTURE HAR- VESTER [47] to estimate the most likely K based on the likelihood scores. The analysis estimates coancestry coeffi- cients for individuals assigned to each of K populations. The relationships among chloroplast haplotypes were assessed with a phylogenetic analysis based on Bayesian inference in BEAST v1.7.5 [55]. The chloroplast dataset was concatenated to derive alleles and consisted of 34 haplotypes identified previously using the software DNASP. Outgroup sequences were generated for Dipter- onia sinensis, and Acer glabrum var. neomexicanum and downloaded from GenBank for A. buergerianum subsp. ningpoense (KF753631.1). Dipteronia and Acer are sister clades (subfamily Hippocastanoideae) [56]. The best-fit nucleotide substitution model was estimated using jMo- deltest v2.3 [57, 58]. The GTR nucleotide substitution model was chosen under an Akaike Information Criter- ion (AIC). Uncorrelated lognormal relaxed molecular clocks with a coalescent approach prior assuming con- stant population size were used. For divergence analyses, a lognormal prior was placed on the tree root, using substitution rates for the chloroplast regions that were calculated using a pairwise sequence divergence analysis (Acer-Dipteronia) with Jukes-Cantor correction. The resulting estimate (Dxy = 0.06788, SD 0.04) was divided by twice the divergence time estimated for Acer (14.6 Ma, [51]) to obtain per-lineage rate subs/site/Myr (0.06788/ Genetic differentiation in chloroplast sequence data A spatial analysis of molecular variance (SAMOVA) was conducted to identify groups of related populations based on geographical and genetic distances [48]. This approach, which does not consider a priori structure parameters, identifies groups that are geographically homogeneous and genetically differentiated. The most likely number of groups (K) was determined using 10,000 iterations and 10 repetitions for K values from two to eight groups. The most likely structure was defined by the maximum value of ΦCT (the proportion Vargas-Rodriguez et al. BMC Evolutionary Biology (2015) 15:257 Page 6 of 14 Hypothesis testing: single and multiple glacial refuge models Glacial refugia were represented by assemblages of populations from: a) West Mexico, b) Midwest USA c) Southeast USA – East Mexico – Guatemala, d) Northeast USA. We tested whether populations a and b were isolated since early Pleisto- cene (30,000 generations / 1.2 Ma) or before (98,750 generations / 3.9 Ma); and a recent split of c and d groups at 6250 generations / 250,000 ybp, or 3500 generations / 140,000 ybp or 500 generations / 20,000 ybp. Results 29.2 = 0.0023/1000000). The analysis comprised two in- dependent runs with 107generations, sampling every 1,000 generations and discarded the first 25 % as burn-in. Effective sample sizes (ESS) of parameter estimates and convergence and mixing of runs were assessed using Tracer v1.5 [59]. The results of sepa- rated runs were combined using LogCombiner v1.7.5 [55]. A maximum clade credibility tree was generated in TreeAnnotator v1.7.5 [55] and examined with FigTree v1.7.5 [55]. Chloroplast diversity A number of haplotypes were recovered from two chloroplast regions. Chloroplast sequences of the psbJ- petA and the ndhF-rpl32R intergenic spacer were 729 and 770 nucleotides long, respectively. The total number of haplotypes in the concatenated sequences was 34, and diversity Hd = 0.8609. The spacer ndhF-rpl32R had a haplotype diversity Hd = 0.667, and nucleotide diver- sity π = 0.00295 [53]; and the spacer psbJ-petA had Hd = 0.620 and π = 0.00168. Vargas-Rodriguez et al. BMC Evolutionary Biology (2015) 15:257 Page 7 of 14 Page 7 of 14 Table 1 Diversity values obtained from nuclear data (microsatellites)1 Locality HWE, P-Values Mean number of alleles Mean HO (± SE) Mean HE (±SE) FIS Bottleneck graphical shape Bottleneck under IAM (P) Bottleneck under SMM (P) Mean M value Mc Maine, U.S.A. 0.06 1.833 0.222 ± 0.14 0.263 ± 0.12 0.154 Shifted mode 0.0625 0.0625 0.4962 0.4445 Vermont, U.S.A. 0.199 1.667 0.150 ± 0.11 0.122 ± 0.08 −0.227 Normal L-shaped distribution 0.9375 0.9375 0.4648 0.4630 Pennsylvania, U.S.A. 0.013 1.833 0.270 ± 0.16 0.284 ± 0.10 0.047 Shifted mode 0.09375 0.09375 0.5204 0.4630 Ohio, U.S.A. 0.723 1.833 0.219 ± 0.14 0.170 ± 0.10 −0.283 Normal L-shaped distribution 0.875 1 0.4684 0.4630 Michigan, U.S.A. 0.033 1.667 0.133 ± 0.11 0.220 ± 0.11 0.395 Shifted mode 0.125 0.8125 0.4926 0.4630 Illinois, U.S.A. 0.011 1.833 0.183 ± 0.14 0.232 ± 0.10 0.213 Normal L-shaped distribution 0.5625 0.90625 0.5204 0.4445 Tennessee, U.S.A. 0.632 2 0.250 ± 0.13 0.247 ± 0.1 −0.011 Shifted mode 0.4375 0.84375 0.5239 0.4630 Alabama, U.S.A. Chloroplast diversity 0.019 1.5 0.133 ± 0.13 0.152 ± 0.08 0.122 Shifted mode 0.8125 0.8125 0.4891 0.4630 Tamaulipas, Mexico 0.109 2.164 0.3 ± 0.14 0.225 ± 0.10 −0.331 Normal L-shaped distribution 0.125 0.8125 0.4997 0.6359 Ojo de Agua del Cuervo, Jalisco, Mexico 0.005 1.667 0.165 ± 0.12 0.132 ± 0.08 −0.247 Normal L-shaped distribution 0.8125 0.9375 0.4648 0.6388 Sierra de Manantlan Biosphere Reserve, Jalisco, Mexico 0.015 1.833 0.196 ± 0.13 0.147 ± 0.08 −0.33 Normal L-shaped distribution 0.8125 0.9375 0.4926 0.5984 Guerrero, Mexico 0.151 2 0.214 ± 0.12 0.215 ± 0.08 0.005 Normal L-shaped distribution 0.90625 0.9375 0.5482 0.5085 Chiapas, Mexico 0.452 2.165 0.222 ± 0.11 0.244 ± 0.09 0.09 Shifted mode 0.84375 0.96875 0.5275 0.4445 Quiche, Guatemala 0.204 1.667 0.294 ± 0.14 0.210 ± 0.09 −0.401 Shifted mode 0.125 0.1875 0.4891 0.5356 Zacapa, Guatemala 0.141 2 0.340 ± 0.15 0.256 ± 0.11 −0.331 Shifted mode 0.0625 0.125 0.4962 0.6061 El Progreso, Guatemala 0.208 1.833 0.281 ± 0.16 0.191 ± 0.10 −0.47 Normal L-shaped distribution 0.8125 0.9375 0.4697 0.4630 1Hardy-Weinberg Equilibrium (HWE); Observed Heterozygosity (HO), Expected Heterozygosity (HE), Inbreeding coefficient (FIS). Bottleneck estimates under the Stepwise Mutation Model (SMM), the Infinite Allele Model (IAM), as well as the M ratio test using θ = 50. M ratio below Mc indicates a bottleneck (in italics) Table 1 Diversity values obtained from nuclear data (microsatellites)1 1Hardy-Weinberg Equilibrium (HWE); Observed Heterozygosity (HO), Expected Heterozygosity (HE), Inbreeding coefficient (FIS). Bottleneck estimates under the Stepwise Mutation Model (SMM), the Infinite Allele Model (IAM), as well as the M ratio test using θ = 50. M ratio below Mc indicates a bottleneck (in italics) Haplotype diversity was variable among populations (Hd from 0-1). A site in Guatemala had only one haplotype, whereas larger numbers of haplotypes oc- curred in the southeastern and midwestern USA (Table 2). Nucleotide diversity was low in all popula- tions (π from 0–0.00288), but the southeastern USA region had the highest (π = 0.00253), followed by the midwestern region (π = 0.00109). The site with the lowest nucleotide diversity was in western Mexico (π = 0.0009) (Table 2). (Additional file 2). A permutation test of pairwise RST and FST indicated that alleles were more related be- tween nearby populations than between distant popu- lations (slope coefficient b = 0.0075). Chloroplast diversity The results given by the estimator Dest, which accounts for small sample sizes, indicated virtually identical allele frequencies among populations in Illinois, Michigan, and Pennsylvania, as well as between populations in northern Mexico and one in Guatemala. All values of genetic differ- entiation were low, with the highest differentiation occur- ring between the northeastern USA populations and populations in Guerrero (west) and Chiapas (south) in Mexico (Additional file 2). Populations in the midwestern USA differed from those in Guerrero and Jalisco (western Mexico), as well as from Guatemalan populations (Additional file 2). Consistent with the Dest estimator, the highest differentiation, given by the FST metric, occurred Genetic differentiation in nuclear microsatellite data Genetic differentiation in nuclear microsatellite data There was variation in genetic structure across the 16 populations. The pairwise θ (FST) values ranged from -0.0703 to 0.5082 indicating differentiation among populations (Additional file 2). The values for Dest were low and varied from 0.00008 to 0.0911 Vargas-Rodriguez et al. BMC Evolutionary Biology (2015) 15:257 Page 8 of 14 Page 8 of 14 Table 2 Nucleotide polymorphism and diversity in psbJ-petA and ndhF-rpl32R chloroplast regions2. Values are given by population and by regions Locality (S) (h) (Hd) (±1 SD) (π) (±1 SD) (qw) Maine, U.S.A. 3 4 0.81 ± 0.13 0.00072 ± 0.00019 0.00084 Vermont, U.S.A. 1 2 0.4 ± 0.24 0.00028 ± 0.00016 0.00033 Pennsylvania, U.S.A. 4 5 0.786 ± 0.15 0.00082 ± 0.00023 0.00107 Ohio, U.S.A. 5 4 0.643 ± 0.18 0.00098 ± 0.00045 0.00133 Michigan, U.S.A. 1 2 0.476 ± 0.17 0.00033 ± 0.00012 0.00028 Illinois, U.S.A. 8 5 0.857 ± 0.14 0.0017 ± 0.0006 0.00225 Tennessee, U.S.A. 12 7 1 ± 0.07 0.00288 ± 0.00092 0.0034 Alabama, U.S.A. 2 2 1 ± 0.5 0.00138 ± 0.00069 0.00138 Tamaulipas, Mexico 7 3 0.378 ± 0.18 0.00096 ± 0.00063 0.00171 Ojo de Agua del Cuervo, Jalisco, Mexico 4 4 0.643 ± 0.18 0.00069 ± 0.00027 0.00106 Sierra de Manantlan Biosphere Reserve, Jalisco, Mexico 2 2 0.25 ± 0.18 0.00035 ± 0.00025 0.00053 Guerrero, Mexico 1 2 0.667 ± 0.31 0.00046 ± 0.00022 0.00046 Chiapas, Mexico 5 4 0.8 ± 0.17 0.00129 ±0.00046 0.00151 Quiche, Guatemala 3 4 0.694 ± 0.15 0.00058 ± 0.00017 0.00076 Zacapa, Guatemala 0 1 0 ± 0 0 ± 0 0 El Progreso, Guatemala 3 3 0.524 ± 0.20 0.00059 ± 0.00028 0.00084 Regions Northeast USA 5 6 0.732 ± 0.1 0.00071 ± 0.00015 0.00101 Midwest USA 8 6 0.762 ± 0.08 0.00109 ± 0.00038 0.00169 Southeast USA 13 8 0.972 ± 0.06 0.00253 ± 0.00081 0.00332 Western MX (Jalisco and Guerrero states) 9 7 0.608 ± 0.13 0.0009 ± 0.00025 0.00178 South MX and Guatemala 10 8 0.484 ± 0.11 0.00055 ± 0.00017 0.00173 2Number of polymorphic sites (S), number of haplotypes (h), haplotype diversity (Hd), average per site pairwise nucleotide diversity (π), relationship between polymorphic sites and alleles samples (qw) Table 2 Nucleotide polymorphism and diversity in psbJ-petA and ndhF-rpl32R chloroplast regions2. Genetic differentiation in nuclear microsatellite data Values are given by population d b between northeastern USA and the Mexican popula- tions from Guerrero, Jalisco (west), and Chiapas (south) (Additional file 2). The number of chloroplast haplotypes from all Acer populations was 34. Haplotype “AA” was the most frequent and widespread, occurring in half of the sites (Guatemala, northern and southern Mexico, south- eastern USA, and Ohio) (Fig. 1). The second most frequent were the haplotypes “H” and “Y”, both with the same frequency. Haplotype “Y” was exclusive to northeastern USA, whereas “H” was unique to popu- lations in western Mexico. Haplotypes “M” and “K” were frequent in the midwestern USA sites (Fig. 1). “E” and “F” were exclusive to western Mexico and “D” to northeastern USA. The remaining haplotypes were unique and confined to a single region. The Bayesian clustering analysis was moderately in- formative. The highest log probability of the data, Ln P(D) = -1158.03, inferred that the most likely number of clusters (K) was two (Additional file 3). Membership co- efficients for one cluster ranged from 0.617 to 0.816 and from 0.723 to 0.934 in the second cluster. Genetic differentiation in chloroplast sequence data p q The optimal number of groups indicated by SAMOVA was K = 4 (ΦCT = 0.545 P = 0.001; 0 singleton populations). Higher levels of K included single-population groups with only a slight increase in the index (Additional file 4). The genetic structure identified by SAMOVA (K = 4) consisted of one group composed by the populations from Guatemala, Mexico (excluding Jalisco), the Southeast USA, and Ohio. A second group included only the populations from western Mexico (Jalisco), and the Northeast and the Midwest USA populations each composed the third and fourth group. Spatial genetic patterns Geographic boundaries detected by the Monmonier algorithm supported the separation of midwestern USA and western Mexican sites. The bootstrap support of the segments ranged from 46–55 % (Fig. 1), whereas SAMOVA results, based on chloroplast data, sup- ported the regional subdivisions (ΦCT = 0.545 P = 0.001). Boundaries were also indicated using nuclear data. Strong genetic change occurred among populations in Page 9 of 14 Vargas-Rodriguez et al. BMC Evolutionary Biology (2015) 15:257 Mexico and USA and between Mexican and Guate- malan populations, suggesting current limits to gene flow (bootstrap support 41–58 %) (Fig. 1). In addition, results indicated a higher subdivision in those popula- tions in tropical regions (Fig. 1). from the midwestern USA and the phylogroups from Mexico, Guatemala, southeastern USA, and northeast- ern USA was dated to the early Pleistocene, at 2.5 Ma (95 % HPD: 1.2–5.9), whereas the Northeast USA lineage split was in the Pleistocene, at 1.4 Ma (95 % HPD: 0.2–3.8) (Fig. 2). Discussion et al. [65] found the greatest sugar maple genetic diver- sity in the southern region of Tennessee and the lowest in the northern area of Wisconsin. Latitude was sug- gested to be responsible for trends in genetic diversity [66]; no significant latitudinal pattern in genetic diversity was observed, however, when analyzing the chloroplast DNA [26]. Rates of chloroplast evolution are slow com- pared to nuclear microsatellites; thus, the chloroplast data could reflect past population sizes and past gene flow between southeastern USA and the easternmost tropical maples of Mexico and Guatemala. Data on nuclear and chloroplast DNA of sugar maple collected from North and Central America recovered signatures of past connectivity, disjunctions, and genetic structure among populations. Populations in western Mexico (Jalisco) and the midwestern USA had the high- est genetic differentiation; their haplotype lineages diverged, respectively, during the Pliocene and early Pleistocene. Further differentiation of northeastern USA haplotype lineages was dated to the pre-Illinoian glacial episode (Pleistocene). Population in the southeastern USA, eastern Mexico, and Guatemala displayed signals of past connectivity and lack genetic differentiation. In addition, most populations in the tropics had increased homozygosity and lower genetic diversity, possible as a result of bottlenecks lasting many generations. Haplo- type data suggested the presence of multiple glacial refu- gia; the tropical populations likely acted as refugia during the Quaternary glacial and interglacial events, while two other refugia were maintained in the Midwest and north of the Appalachian Mountains. The genetic structure recovered with nuclear and chloroplast DNA indicates general patterns of con- nectivity among populations. Nuclear data suggest current gene flow and larger effective population sizes in southeastern and northeast USA, whereas the op- posite is true in the tropical populations, having disjunct and geographically fragmented populations. Chloroplast data indicate that very little gene flow has occurred among the midwestern sites and the rest of the populations in the USA since some distant time in the past. Haplotypes from more southern regions may be considered ancestral, which is suggested by high fre- quency and distribution of haplotypes in southeastern USA and south Mexico and Guatemala. Haplotype lineages in western Mexico (Jalisco) were the first to diverge, during the Pliocene. Eastern Mexican and Guatemalan lineages had a time for the most com- mon ancestor similar to the northeastern USA popu- lations, dated to the pre-Illinoian glacial period. Hypothesis testing: single and multiple glacial refuge models The Bayesian topology resolved three main and well- supported clades. Relationships among the haplotypes from western Mexico (Jalisco) received high support (0.98) (Fig. 2). Haplotypes from the Midwest USA formed a well-supported monophyletic group with high posterior probability (0.98) (Fig. 2). The rest of the haplotypes formed a group with a probability of 0.84. Haplotypes from Guatemala, Mexico, and Tennes- see were related to those from the Northeast USA (Fig. 2). Divergence dating for haplotypes estimated a time for the most common ancestor between the lineage occurring in Jalisco (western Mexico) and the rest of the tropical and temperate sugar maple phylogroups as the Pliocene, at 5.4 Ma (95 % HPD: 4.1–7.3). A split between the haplotypes The single refugium null model was not supported by the data. The Slatkin and Maddison’s s statistic from the gene tree was s = 14. The observed value of s = 14 was not within the 95 % confidence interval of the simulated distribution under any of the different times of population splitting and for all Ne values. The multiple glacial refuge model could not be rejected. The observed s = 14 falls within the 95 % confi- dence interval (Additional file 5). The simulations for the estimate of Ne = 225,000 with a Southeast – Northeast USA population divergence at 250,000 ybp and population split at 1.2 Ma of the Midwest USA group were supported. Fig. 2 Maximum clade credibility tree of chloroplast haplotypes showing phylogenetic relationships among samples of sugar maples occurring in North and Central America. Posterior probabilities and mean divergence time for haplotype lineages are given. Numbers in brackets indicate the highest 95 % posterior density intervals. Colors correspond to the haplotypes depicted in Figure 1 Fig. 2 Maximum clade credibility tree of chloroplast haplotypes showing phylogenetic relationships among samples of sugar maples occurring in North and Central America. Posterior probabilities and mean divergence time for haplotype lineages are given. Numbers in brackets indicate the highest 95 % posterior density intervals. Colors correspond to the haplotypes depicted in Figure 1 Vargas-Rodriguez et al. BMC Evolutionary Biology (2015) 15:257 Page 10 of 14 Page 10 of 14 Discussion The haplotypes from the Midwest, supported as a lineage by Bayesian inference, have survived isolated since the early Pleistocene (when ice-sheets began to grow), with possible gene migrations from more central (Ohio) or southern (Alabama) ice-free areas. Samples from Iowa, Missouri, as well as sequences of other chloroplast regions, may help to clarify the routes of migration for the midwest- ern populations. Genetic diversity and geographic structure Low levels of genetic diversity from nuclear DNA data were observed in all populations. A greater ten- dency toward homozygosity occurs in maples from western and southern Mexico compared to maples Vermont, Ohio, and Tennessee. The trend could be the result of a genetic bottleneck in southern Mexico. The reduced genetic variation is also present in the Guatemalan populations. The increased homozygosity in southern tropical populations indicates that popula- tions at the most southern ends of the range of this species should be the most vulnerable to new selec- tion pressures. Populations in southern Mexico and Guatemala are also the most threatened by anthropo- genic disturbance [19]. Consistently, low values in nu- clear markers with high mutation rates reflect fragmentation patterns and reductions in population sizes in recent times. Low to moderate diversity has also been observed in Acer saccharum in populations from Canada, possibly a result of genetic drift [23–25]. Never- theless, genetic variation may be underestimated across species using the same markers, because microsatellites are subject to ascertainment bias [64]. In addition, a more accurate estimation of genetic diversity should be obtained by increasing the number of microsatellites. Pleistocene effects on populations Pleistocene effects on populations Glacial periods during the Pleistocene could have favored species expansions with continuous gene flow through eastern–southern Mexico to Guatemala, which then ended after the last glacial maximum (18,000 years B.P.). Pollen from cores of the Basin of Mexico indicates three glacial advances during 30,000–25,000 years B.P., 12,000 years B.P., and during late Holocene [74, 75]. A relative stable humidity during glacial events could have facilitated expansion and population connectivity in the tropical areas. A warm period of the middle Holocene and the “Medieval Warm Period” (1038–963 years B.P.) [76] might have helped produce the current fragmented distri- bution of Acer in cloud forests. Even though the Trans- Mexico Volcanic Belt reaches eastern Mexico, it had reduced volcanic activity along its eastern side [71] and, together with the presence of continuous mountain ridges, this might have allowed a steady connection among popu- lations in eastern–southern Mexico and Guatemala. The sites in Ohio and southern USA share the same chloro- plast haplotypes with those in northern and southern Mexico and Guatemala sites, but they are differentiated from the Jalisco and, to a lesser extent, the Guerrero populations (western Mexico). Pliocene–Quaternary glacial and interglacial effects on populations Pliocene events and western Mexico population divergence Populations of Acer saccharum could have become established in western Mexico during the Pliocene or late Miocene, when the temperatures were low. The presence of other temperate tree genera (e.g., Platanus and Populus) during the Pliocene in central Mexico is supported by the presence of macrofossils [67]. In addition, a probable arrival in Mexico during the Mio- cene/Pliocene has been suggested for other cloud forest trees, such as Liquidambar and Podocarpus matudae [68–70]. Patterns of genetic differentiation Further, the volcanic belt could have blocked northern cold fronts, creating climatic conditions that promoted growth of ample areas of dry forests in western Mexico as well as fragmenting and reducing cold and humid areas, which are most suitable for maple establishment [73]. This ancient division among maples is suggested by the high differentiation between western populations and the rest of the study sites, the presence of different haplotypes, and strong barriers, as well as by the time for the most common ancestor values. These findings suggest that the taxonomic status of the western Mexico populations needs to be reconsidered (Vargas-Rodriguez in review). reduced population sizes. Caution is needed in making inferences, however, because the accuracy of the assign- ments in STRUCTURE is affected when there are insuffi- cient microsatellite markers [45]. Populations in midwestern USA and western Mexico were highly differentiated from the rest of the sites under study. Dest and FST supported differentiation for nuclear DNA and SAMOVA for chloroplast DNA. Their isolation thus appears to be old and is supported by the time for the most common ancestor estimates for haplo- types in this study. Our data further suggest very limited present gene flow (via pollen dispersal) from Alabama to the populations in the Midwest. The nuclear DNA also indicates lack of gene flow at the present time between the northern Mexico and southern USA populations. Nonetheless, the signal of past connectivity among the Southeast USA, East Mexico, and Guatemalan populations is still present, as indicated by the low chloroplast differentiation among these populations. Similar signals of past con- nectivity have been identified between populations of Liquidambar styraciflua in the southeastern USA and Mexico [8]. Populations in Mexico also exhibit differ- entiation from each other as a result of geographic barriers and a highly fragmented landscape. Although geographically close, Guatemalan populations also dis- played a lack of pollen flow. This suggests that in addition to a mountain barrier, the population sizes are small, pollen dispersion is limited, and there are no intermediate patches of maples that might increase connectivity at the present time. Similar degrees of reproductive isolation after the last glaciation and landscape fragmentation have been recorded for other temperate trees such as Fagus grandifolia and Pinus chiapensis in Mexico [13, 15]. Patterns of genetic differentiation Patterns of genetic differentiation The results from the STRUCTURE analysis suggested that the genotyped individuals fall into two general clusters. Thus, there is one cluster from the tropics, which also includes Alabama, and one cluster com- posed of USA sites, but excludes the Southeast USA. These results contrasted with Dest and FST measures. Bayesian clustering analysis did not differentiate the west- ern Mexico populations, whereas Dest and FST gave strong support for these populations as a different group. Similarly, the population in Chiapas was assigned to the USA cluster with STRUCTURE, but it was differentiated using Dest and FST. The Chiapas population might represent an outlier, possible due to the high levels of homozygosity and Chloroplast gene diversity in most maple populations was variable. High levels of nucleotide and haplotype diversity were distributed in the southern and the mid- western regions of the USA. High nucleotide diversity (π) also was also observed in the southern population of Chiapas, Mexico. Nevertheless, populations in tropical regions contained the lowest haplotype diversity. Gunter Vargas-Rodriguez et al. BMC Evolutionary Biology (2015) 15:257 Page 11 of 14 Page 11 of 14 Quaternary, mainly along the western side [71, 72]. The volcanic activity and extensive accumulation of volcani- clastic particles during the Quaternary could have isolated western maple populations in Jalisco and Guer- rero by preventing immigration, thereby facilitating the divergence of already established populations. Further, the volcanic belt could have blocked northern cold fronts, creating climatic conditions that promoted growth of ample areas of dry forests in western Mexico as well as fragmenting and reducing cold and humid areas, which are most suitable for maple establishment [73]. This ancient division among maples is suggested by the high differentiation between western populations and the rest of the study sites, the presence of different haplotypes, and strong barriers, as well as by the time for the most common ancestor values. These findings suggest that the taxonomic status of the western Mexico populations needs to be reconsidered (Vargas-Rodriguez in review). Quaternary, mainly along the western side [71, 72]. The volcanic activity and extensive accumulation of volcani- clastic particles during the Quaternary could have isolated western maple populations in Jalisco and Guer- rero by preventing immigration, thereby facilitating the divergence of already established populations. Late Quaternary population patterns Habitat fragmentation may have resulted from increased dry and warm conditions that followed the last glacial maximum. In addition, anthropogenic habitat transform- ation has affected the cloud forests containing sugar maples in Mexico and Guatemala [19]. Reduced popula- tion sizes, lack of gene flow, genetic bottlenecks, and Hypotheses of Pleistocene refugia Low-density populations of Acer rubrum and Fagus grandifolia could also have persisted in periglacial areas in Ohio, expanding following deglaci- ation and making a more important contribution to species expansion than other populations in the southeastern USA [9, 11, 12]. Thus, the assumption of a single refugium of sugar maples in the Gulf Coastal Plain during the glaciations in the Pleistocene does not appear to be valid [1, 17]. Our results sup- ported that the idea that the midwestern USA has provided refugia most likely onward from the early Pleistocene. Parts of the area remained ice-free during the last glaciation and allowed the persistence of de- ciduous species [81]. Recent phylogeographic evidence for Smilax also supports the hypothesis that the Mid- west served as a refugium [82]. Moreover, temperate tree populations in the tropics, usually overlooked [83], have also been an important gene reservoir, act- ing as refugia during the Pleistocene. Hypotheses of Pleistocene refugia y Small maple refugia may have existed in the Midwest USA and north of the Appalachian Mountains. A population in the Midwest might have persisted through the glaciations from the beginning of Pleisto- cene, experienced possible gene flow from the sites in the south (Alabama), but remained isolated from the more eastern sites (Ohio, Tennessee). The Mississippi River Valley is hypothesized to have increased genetic The uplift of the Trans-Mexico Volcanic Belt, a major mountain barrier stretching from west to east across the country, isolated western areas of Mexico in the late Miocene. This mountain belt was characterized by major volcanic activity during the late Pliocene and the Vargas-Rodriguez et al. BMC Evolutionary Biology (2015) 15:257 Page 12 of 14 Page 12 of 14 differentiation to the east and the west of the river in Pinus and Juglans [1], and the river valley probably also affected the migration of Acer during the Pleisto- cene. Another small maple refugium should have oc- curred in Ohio, based on the diversity of haplotypes. The Ohio population also shared a larger proportion of the most abundant haplotype with the southeastern and tropical sites, and a smaller proportion of haplo- types from the northeastern site. Maples in the Northeast might have expanded from a refugium in the northern area of the Appalachian Mountains. During the last glaciations, ice-free areas existed in the Pennsylvania region, from where the maples could have migrated following deglaciation [77]. The low haplotype diversity found here suggests genetic drift in small refugial populations from the Northeast. It has been proposed that plants survived ice ages on small island-like areas protruding above the ice-sheets in the North Atlantic zone; however, climatically harsh conditions also could have prevented in situ glacial survival of maples [20, 78, 79]. Thus, maples in the Northeast likely migrated from periglacial areas of the Pennsylvania region [80]. On the other hand, physiographic conditions of northern Appalachians re- stricted maple gene flow from the south to the north- east during the last 14,000–10,000 years B.P. [22]. This barrier in gene flow is supported by the haplo- type data. Low-density populations of Acer rubrum and Fagus grandifolia could also have persisted in periglacial areas in Ohio, expanding following deglaci- ation and making a more important contribution to species expansion than other populations in the southeastern USA [9, 11, 12]. Hypotheses of Pleistocene refugia Thus, the assumption of a single refugium of sugar maples in the Gulf Coastal Plain during the glaciations in the Pleistocene does not appear to be valid [1, 17]. Our results sup- ported that the idea that the midwestern USA has provided refugia most likely onward from the early Pleistocene. Parts of the area remained ice-free during the last glaciation and allowed the persistence of de- ciduous species [81]. Recent phylogeographic evidence for Smilax also supports the hypothesis that the Mid- west served as a refugium [82]. Moreover, temperate tree populations in the tropics, usually overlooked [83], have also been an important gene reservoir, act- ing as refugia during the Pleistocene. increases in homozygosity now characterizing these pop- ulations will probably be rapidly exacerbated by ongoing anthropogenic activity. Thus, historical processes and disturbances are contracting the most southern end of the ranges of sugar maple trees. Recent strong warming conditions are promoting a northward migration of sugar maples shifting the range towards a more northern limit in Canada [84]. w v Pinus and Juglans [1], and the river valley probably also affected the migration of Acer during the Pleisto- cene. Another small maple refugium should have oc- curred in Ohio, based on the diversity of haplotypes. The Ohio population also shared a larger proportion of the most abundant haplotype with the southeastern and tropical sites, and a smaller proportion of haplo- types from the northeastern site. Maples in the Northeast might have expanded from a refugium in the northern area of the Appalachian Mountains. During the last glaciations, ice-free areas existed in the Pennsylvania region, from where the maples could have migrated following deglaciation [77]. The low haplotype diversity found here suggests genetic drift in small refugial populations from the Northeast. It has been proposed that plants survived ice ages on small island-like areas protruding above the ice-sheets in the North Atlantic zone; however, climatically harsh conditions also could have prevented in situ glacial survival of maples [20, 78, 79]. Thus, maples in the Northeast likely migrated from periglacial areas of the Pennsylvania region [80]. On the other hand, physiographic conditions of northern Appalachians re- stricted maple gene flow from the south to the north- east during the last 14,000–10,000 years B.P. [22]. This barrier in gene flow is supported by the haplo- type data. Availability of supporting data y pp g The chloroplast DNA data set supporting the results of this article is available in GenBank, with the accession numbers KT933356–KT933397. Conclusions R d d Reduced genetic variation currently is present in the most southern tropical maple populations, as indicated by nu- clear and chloroplast data. Haplotype diversity and distri- bution indicate past connectivity among populations from the Southeast USA, East Mexico, and Guatemala. Thus, gene flow and species expansion through eastern–south- ern Mexico to Guatemala was likely favored during glacial periods of stable humidity during the Quaternary. Earlier, western Mexican (Jalisco) populations diverged from the rest of the sugar maples during the Pliocene. Volcanic activity during late Pliocene and Quaternary and topo- graphic conditions in western Mexico probably promoted this early differentiation. The time for the most common ancestor estimates denote that midwestern USA popu- lations have been different lineages since the early Pleistocene and that northeastern USA lineages di- verged during the pre-Illinoian glaciation, long before the Last Glacial Maximum. Small maple refugia may have existed in the midwestern USA and north of the Appalachian Mountains, and expansion may have oc- curred from such refuges during interglacial periods. Thus, we suggest that geological events in the Plio- cene were major determinants of the current genetic structure of sugar maple populations in the area of West Mexico, whereas the geological events that occurred during the Quaternary explain the genetic structure of sugar maples in East Mexico and the USA. This study supports the notion of multiple gla- cial refugia for temperate hardwood forests in North and Central America and highlights the importance of connectivity among temperate forests in the USA and those in Central America. Author’s information 17. Delcourt PA, Delcourt HR. Long-term forest dynamics of the temperate zone. New York: Springer; 1987. YLVR present address: National Council of Science and Technology, Av. Insurgentes Sur 1582, Benito Juárez, 03940, Distrito Federal, Mexico; University of Guadalajara, Juárez 976, Colonia Centro 44100, Guadalajara, Jalisco, Mexico. YLVR present address: National Council of Science and Technology, Av. Insurgentes Sur 1582, Benito Juárez, 03940, Distrito Federal, Mexico; University of Guadalajara, Juárez 976, Colonia Centro 44100, Guadalajara, Jalisco, Mexico. 18. Hampe A, Jump AS. Climate relicts: past, present, future. Annu Rev Ecol Evol Syst. 2011;42:313–33. 19. Vargas-Rodriguez YL, Platt WJ. Remnant sugar maple (Acer saccharum subsp. skutchii) populations at their range edge: Characteristics, environmental constraints and conservation implications in tropical America. Biol Conserv. 2012;150:111–20. Received: 18 February 2015 Accepted: 22 October 2015 24. Perry DJ, Knowles P. Allozyme variation in sugar maple at the northern limit of its range in Ontario, Canada. Can J For Res. 1989;19:509–14. 25. Perry DJ, Knowles P. Spatial genetic structure within three sugar maple (Acer saccharum Marsh.) stands. Heredity. 1991;66:137–42. Competing interests 14. Ledig FT, Jacob-Cervantes V, Hodgskiss PD, Eguiluz-Piedra T. Recent evolution and divergence among populations of a rare Mexican endemic, Chihuahua spruce, following Holocene climatic warming. Evolution. 1997;51:1815–27. Authors’ contributions 15. Newton AC, Allnutt TR, Dvorak W, del Castillo R, Ennos R. Patterns of genetic variation in Pinus chiapensis, a threatened Mexican pine, detected by RAPD and mitochondrial DNA RFLP markers. Heredity. 2002;89:191–8. YLVR conceived the study. YLVR and WJP designed the study, YLVR obtained and analyzed the data and wrote the manuscript. WJP helped to draft the manuscript. LEU and DWF provided methods for the wet lab, participated in the sequence alignment, and took part in the data analysis. YLVR, WJP, LEU, and DWF critically reviewed the drafts of the manuscript. All authors have read and approved the final version of the manuscript. 16. Gugger PF, González-Rodríguez A, Rodríguez-Correa H, Sugita S, Cavender-Bares J. Southward Pleistocene migration of Douglas-fir into Mexico: phylogeography, ecological niche modeling, and conservation of "rear edge" populations. New Phytol. 2010;189:1185–99. Additional files Grauke LJ, Mendoza-Herrera MA, Miller AJ, Wood BW. Geographic patterns of genetic variation in native pecans. Tree Genet Genomes. 2011;7:917–32. 8. Morris AB, Ickert-Bond SM, Brunson B, Soltis DE, Soltis PS. Phylogeographical structure and temporal complexity in American sweetgum (Liquidambar styraciflua; Altingiaceae). Mol Ecol. 2008;17:3889–900. 9. Morris AB, Graham C, Soltis DE, Soltis PS. Reassessment of phylogeographical structure in an eastern North American tree using Monmonier’s algorithm and ecological niche modeling. J Biogeogr. 2010;37:1657–67. Additional file 4: Fixation indices and number of singleton populations obtained from the spatial analyses of molecular variance (SAMOVA) of sugar maple’s chloroplast DNA data. (DOC 28 kb) 10. Magni CR, Ducousso A, Caron H, Petit RJ, Kremer A. Chloroplast DNA variation of Quercus rubra L. in North America and comparison with other Fagaceae. Mol Ecol. 2005;14:513–24. Additional file 5: Distribution of Slatkin and Maddison’s s statistic for 1000 simulated gene trees within a population tree. 11. Saeki I, Dick CW, Barnes BV, Murakami N. Comparative phylogeography of red maple (Acer rubrum L.) and silver maple (Acer saccharinum L.): impacts of habitat specialization, hybridization and glacial history. J Biogeogr. 2011;38:992-1005. Ne = 225,000. Observed s = 14 (asterisk) indicates that the population structure is consistent with the multiple refuge hypothesis. (DOC 219 kb) Ne = 225,000. Observed s = 14 (asterisk) indicates that the population structure is consistent with the multiple refuge hypothesis. (DOC 219 kb) 12. McLachlan JS, Clark JS, Manos PS. Molecular indicators of tree migration capacity under rapid climate change. Ecology. 2005;86:2088–98. Abbreviations SAMOVA: spatial analysis of molecular variance; SMOGD: software for the measurement of genetic diversity. SAMOVA: spatial analysis of molecular variance; SMOGD: software for the measurement of genetic diversity. 13. Rowden A, Robertson A, Allnutt T, Heredia S, Williams-Linera G, Newton AC. Conservation genetics of Mexican beech, Fagus grandifolia var. mexicana. Conserv Genet. 2004;5:475–84. Competing interests The authors declare that they have no competing interests. Additional files Additional file 1: The sugar maple populations studied and the geographic information from the sampled localities. Population and ecological characteristics for Mexican and Guatemalan populations are given elsewhere [19]. (DOC 47 kb) Additional file 1: The sugar maple populations studied and the geographic information from the sampled localities. Population and ecological characteristics for Mexican and Guatemalan populations are given elsewhere [19]. (DOC 47 kb) Additional file 1: The sugar maple populations studied and the geographic information from the sampled localities. Population and ecological characteristics for Mexican and Guatemalan populations are given elsewhere [19]. (DOC 47 kb) Page 13 of 14 Page 13 of 14 Vargas-Rodriguez et al. BMC Evolutionary Biology (2015) 15:257 Additional file 2: Below diagonal, FST: the proportion of genetic diversity due to allele frequency differences among populations. Above diagonal, Dest: estimator of actual differentiation. High values are in bold and imply a degree of differentiation among populations. (DOC 49 Kb) Additional file 3: Graphical summary of Bayesian clustering. a) Geographical subdivision as inferred using STRUCTURE. Two clusters are distinguishable: one (in red) includes populations from Guatemala (PR, ZA, QC), Mexico (Tamps, Tal, Man), and Alabama (AL). Membership coefficients for the red cluster ranged from 0.617 to 0.816. A second cluster in green comprises populations from Northeast USA (ME, PA), Midwest USA (MI, IL), and the most southern site in Mexico (Chiapas, Chis). Coefficients ranged from 0.723 to 0.934 in green cluster. b) Likelihood score differences (DeltaK) with highest difference between K = 2 and K = 3. (DOC 629 kb) Additional file 4: Fixation indices and number of singleton populations obtained from the spatial analyses of molecular variance (SAMOVA) of sugar maple’s chloroplast DNA data. (DOC 28 kb) Additional file 5: Distribution of Slatkin and Maddison’s s statistic for 1000 simulated gene trees within a population tree. Ne = 225,000. Observed s = 14 (asterisk) indicates that the population structure is consistent with the multiple refuge hypothesis. (DOC 219 kb) 4. Stone JL, Crystal PA, Devlin EE, LeB Downer RH, Cameron DS. Highest genetic diversity at the northern range limit of the rare orchid Isotria medeoloides. Heredity. 2012;109:215–21. 4. Stone JL, Crystal PA, Devlin EE, LeB Downer RH, Cameron DS. Highest genetic diversity at the northern range limit of the rare orchid Isotria medeoloides. Heredity. 2012;109:215–21. Additional file 2: Below diagonal, FST: the proportion of genetic diversity due to allele frequency differences among populations. 1. Soltis DE, Morris AB, McLachlan JS, Manos PS, Soltis PS. Comparative phylogeography of unglaciated eastern North America. Mol Ecol. 2006;15:4261–93. Additional files Above diagonal, Dest: estimator of actual differentiation. High values are in bold and imply a degree of differentiation among populations. (DOC 49 Kb) Additional file 3: Graphical summary of Bayesian clustering. a) Geographical subdivision as inferred using STRUCTURE. Two clusters are distinguishable: one (in red) includes populations from Guatemala (PR, ZA, QC), Mexico (Tamps, Tal, Man), and Alabama (AL). Membership coefficients for the red cluster ranged from 0.617 to 0.816. A second cluster in green comprises populations from Northeast USA (ME, PA), Midwest USA (MI, IL), and the most southern site in Mexico (Chiapas, Chis). Coefficients ranged from 0.723 to 0.934 in green cluster. b) Likelihood score differences (DeltaK) with highest difference between K = 2 and K = 3. (DOC 629 kb) Additional file 4: Fixation indices and number of singleton populations obtained from the spatial analyses of molecular variance (SAMOVA) of sugar maple’s chloroplast DNA data. (DOC 28 kb) Additional file 5: Distribution of Slatkin and Maddison’s s statistic for 1000 simulated gene trees within a population tree. Ne = 225,000. Observed s = 14 (asterisk) indicates that the population structure is consistent with the multiple refuge hypothesis. (DOC 219 kb) 5. Excoffier L, Foll M, Petit RJ. Genetic consequences of range expansions. Annu Rev Ecol Evol Syst. 2009;40:481–501. 5. Excoffier L, Foll M, Petit RJ. Genetic consequences of range expansions. Annu Rev Ecol Evol Syst. 2009;40:481–501. Additional file 3: Graphical summary of Bayesian clustering. a) Geographical subdivision as inferred using STRUCTURE. Two clusters are distinguishable: one (in red) includes populations from Guatemala (PR, ZA, QC), Mexico (Tamps, Tal, Man), and Alabama (AL). Membership coefficients for the red cluster ranged from 0.617 to 0.816. A second cluster in green comprises populations from Northeast USA (ME, PA), Midwest USA (MI, IL), and the most southern site in Mexico (Chiapas, Chis). Coefficients ranged from 0.723 to 0.934 in green cluster. b) Likelihood score differences (DeltaK) with highest difference between K = 2 and K = 3. (DOC 629 kb) 6. Arenas M, Ray N, Currat M, Excoffier L. Consequences of range contractions and range shifts on molecular diversity. Mol Biol Evol. 2012;29:207–18. 6. Arenas M, Ray N, Currat M, Excoffier L. Consequences of range contractions and range shifts on molecular diversity. Mol Biol Evol. 2012;29:207–18. 7. Grauke LJ, Mendoza-Herrera MA, Miller AJ, Wood BW. Geographic patterns of genetic variation in native pecans. Tree Genet Genomes. 2011;7:917–32. 7. 3. Duran KL, Pardo A, Mitton JB. From middens to molecules: phylogeograp of the piñon pine Pinus edulis. J Biogeogr. 2012;39:1536–44. 2. Breen AL, Murray DF, Olson MS. Genetic consequences of glacial survival: the late Quaternary history of balsam poplar (Populus balsamifera L.) in North America. J Biogeogr. 2012;39:918–28. Acknowledgements The Mohamed bin Zayed Species Conservation Fund provided significant financial support for most of the laboratory procedures. Fieldwork was conducted through two research grants (#52583 and #80437) and a scholarship (#161695) from CONACyT to YLVR. The Louisiana Department of Wildlife & Fisheries and LSU-BioGrads also provided financial support for field and laboratory work. We thank field assistants and collecting permits granted by forest reserves. LSU Genomics Facility helped with the fragment analysis. This material is based upon work supported by HPC@LSU computing resources. Dr. Jeremy Brown gave assistance for the use of HPC resources. Dr. Kyle Dexter, Dr. Vesna Karaman-Castro and anonymous reviewers provided valuable comments on this manuscript. 20. Jackson ST, Webb RS, Anderson KH, Overpeck JT, Webb III T, Williams JW, et al. Vegetation and envirnment in Eastern North America during the Last Glacial Maximum. Quat Sci Rev. 2000;19:489–508. 21. McLachlan JS, Clark JS, Manos PS. Molecular indicators of tree migration capacity under rapid climate change. Mol Ecol. 2005;86:2088–98. 22. Miller MD, Parker K. 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Stimulating Implementation of Sustainable Development Goals and Conservation Action: Predicting Future Land Use/Cover Change in Virunga National Park, Congo
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Stimulating implementation of sustainable development goals and conservation action Predicting future land use/cover change in Virunga national park, Congo Christensen, Mads; Arsanjani, Jamal Jokar Published in: Sustainability (Switzerland) DOI (link to publication from Publisher): 10.3390/su12041570 Creative Commons License CC BY 4.0 Publication date: 2020 Document Version Publisher's PDF, also known as Version of record Link to publication from Aalborg University Citation for published version (APA): Christensen, M., & Arsanjani, J. J. (2020). Stimulating implementation of sustainable development goals and conservation action: Predicting future land use/cover change in Virunga national park, Congo. Sustainability (Switzerland), 12(4), Article 1570. https://doi.org/10.3390/su12041570 Stimulating implementation of sustainable development goals and conservation action Predicting future land use/cover change in Virunga national park, Congo Christensen, Mads; Arsanjani, Jamal Jokar Stimulating implementation of sustainable development goals and conservation action Predicting future land use/cover change in Virunga national park, Congo Christensen, Mads; Arsanjani, Jamal Jokar Published in: Sustainability (Switzerland) Citation for published version (APA): Christensen, M., & Arsanjani, J. J. (2020). Stimulating implementation of sustainable development goals and conservation action: Predicting future land use/cover change in Virunga national park, Congo. Sustainability (Switzerland), 12(4), Article 1570. https://doi.org/10.3390/su12041570 Aalborg Universitet Stimulating implementation of sustainable development goals and conservation action Predicting future land use/cover change in Virunga national park, Congo Christensen, Mads; Arsanjani, Jamal Jokar Published in: Aalborg Universitet General rights C i h d General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. - Users may download and print one copy of any publication from the public portal for the purpose of private study or research. - You may not further distribute the material or use it for any profit-making activity or commercial gain - Users may download and print one copy of any publication from the public portal for the purpose of private study Y t f th di t ib t th t i l it f fit ki ti it i l i - Users may download and print one copy of any publication from the public portal for the purpose of private study or research. - You may not further distribute the material or use it for any profit-making activity or commercial gain Users may download and print one copy of any publication from the public portal for the purpose of priv - You may not further distribute the material or use it for any profit-making activity or commercial gain - You may freely distribute the URL identifying the publication in the public portal - y p py y p p p - You may not further distribute the material or use it for any profit-making activity or co Y f l di t ib t th URL id tif i th bli ti i th bli t l Take down policy If you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access to the work immediately and investigate your claim. Take down policy If you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access to the work immediately and investigate your claim. Citation for published version (APA): Christensen, M., & Arsanjani, J. J. (2020). Stimulating implementation of sustainable development goals and conservation action: Predicting future land use/cover change in Virunga national park, Congo. Sustainability (Switzerland), 12(4), Article 1570. https://doi.org/10.3390/su12041570 Received: 7 December 2019; Accepted: 12 February 2020; Published: 19 February 2020 Abstract: The United Nations 2030 Agenda for Sustainable Development and the Sustainable Development Goals (SDG’s) presents a roadmap and a concerted platform of action towards achieving sustainable and inclusive development, leaving no one behind, while preventing environmental degradation and loss of natural resources. However, population growth, increased urbanisation, deforestation, and rapid economic development has decidedly modified the surface of the earth, resulting in dramatic land cover changes, which continue to cause significant degradation of environmental attributes. In order to reshape policies and management frameworks conforming to the objectives of the SDG’s, it is paramount to understand the driving mechanisms of land use changes and determine future patterns of change. This study aims to assess and quantify future land cover changes in Virunga National Park in the Democratic Republic of the Congo by simulating a future landscape for the SDG target year of 2030 in order to provide evidence to support data-driven decision-making processes conforming to the requirements of the SDG’s. The study follows six sequential steps: (a) creation of three land cover maps from 2010, 2015 and 2019 derived from satellite images; (b) land change analysis by cross-tabulation of land cover maps; (c) submodel creation and identification of explanatory variables and dataset creation for each variable; (d) calculation of transition potentials of major transitions within the case study area using machine learning algorithms; (e) change quantification and prediction using Markov chain analysis; and (f) prediction of a 2030 land cover. The model was successfully able to simulate future land cover and land use changes and the dynamics conclude that agricultural expansion and urban development is expected to significantly reduce Virunga’s forest and open land areas in the next 11 years. Accessibility in terms of landscape topography and proximity to existing human activities are concluded to be primary drivers of these changes. Drawing on these conclusions, the discussion provides recommendations and reflections on how the predicted future land cover changes can be used to support and underpin policy frameworks towards achieving the SDG’s and the 2030 Agenda for Sustainable Development. Keywords: land cover modelling; remote sensing; machine learning; sustainable development goals; Virunga National Park Article Stimulating Implementation of Sustainable Development Goals and Conservation Action: Predicting Future Land Use/Cover Change in Virunga National Park, Congo Mads Christensen and Jamal Jokar Arsanjani * Geoinformatics Research Group, Department of Planning and Development, Aalborg University Copenhagen, A.C. Meyers Vænge 15, DK-2450 Copenhagen, Denmark; madc@dhigroup.com * Correspondence: jja@plan.aau.dk Mads Christensen and Jamal Jokar Arsanjani * Geoinformatics Research Group, Department of Planning and Development, Aalborg University Copenhagen, A.C. Meyers Vænge 15, DK-2450 Copenhagen, Denmark; madc@dhigroup.com * Correspondence: jja@plan.aau.dk www.mdpi.com/journal/sustainability Downloaded from vbn.aau.dk on: October 24, 2024 Downloaded from vbn.aau.dk on: October 24, 2024 sustainability sustainability sustainability Sustainability 2020, 12, 1570; doi:10.3390/su12041570 1. Introduction Established in 1925 as the first National Park (NP) in Africa, the Virunga NP is located in the Albertine Rift Valley in the eastern part of the Democratic Republic of the Congo [1]. Along with the Mgahinga Gorilla NP in Uganda and the Parc Nationale Des Volcans in Rwanda, Virunga is part of a triangle of NP’s in central Africa, principally designated in order to enhance conservation efforts to Sustainability 2020, 12, 1570; doi:10.3390/su12041570 2 of 28 Sustainability 2020, 12, 1570 protect the critically endangered mountain gorilla (Gorilla Beringei Beringei) [2]. The park covers an area of 790,000 ha [3], and hosts the majority of the fragments of the last remaining habitat suitable for the mountain gorilla. Furthermore, the multitude of variety in nature and climate variables, including large lakes, open land savannah, vast forest areas, snow-covered mountain tops and erupting volcanoes, provides critical habitats for a great variety of the other large species of mammals we associate with Africa [1]. For this reason, the park was inscribed as a United Nations (UN) Educational, Scientific and Cultural Organization (UNESCO) World Heritage site in 1979. However, the NP is located in one of the most densely populated regions in sub-Saharan Africa, which has been the scene of prolonged political turmoil and social conflict [4], causing severe pressure on the ecological integrity of the landscape and its biodiversity. Moreover, the rich volcanic soil and high rainfall within the Virunga NP catchment makes it highly suitable for agriculture, and thus an attractive opportunity to underpin subsistence and commercial farming operations [2]. The rapidly increasing population has significantly increased the demand for natural resources (land, water energy, food, etc.), causing rapid land clearing for agriculture and grazing, removal of plants for different purposes, including artisanal mining operations, and house building [4]. Despite the efforts of authorities to protect the integrity of the NP, multiple pressures continue to threaten its habitats and biodiversity, including: civil unrest; illegal activities; land conversion and encroachment; livestock farming / grazing of domesticated animals; widespread depletion of forests in the lowlands and; a massive influx of 1 million refugees occupying adjacent areas of the park [5]. 1. Introduction Militia leaders and prospectors are threatening the borders of the park in search for the vast deposits of diamonds, gold, uranium and other coveted minerals, while the vast influx of destitute refugees resorts to poaching and charcoal production, resulting in further fragmentation and degradation of the forest landscape [1]. In fact, the majority of the total population of nearly 6 million people in the surrounding province of North Kivu rely entirely on charcoal for their cooking needs, and an estimated three-quarter of this charcoal is sourced from the Virunga catchment, most of it illicitly from within the NP [6]. Thus, the region is highly important, both ecologically and economically, and the conflicting demands for socioeconomic development while maintaining the ecological integrity of the NP has underpinned the need to ensure continued conservation efforts and sustainable natural resource management. This is critical in order to safeguard the biodiversity and habitats of the NP, while preserving the foundation of the livelihoods of millions of people. This agenda is fortified through the UN 2030 Agenda for Sustainable Development and the 17 Sustainable Development Goals (SDG)’s that were adopted at the UN general assembly in 2015. The SDG’s call on concerted action to pursue economic development while ensuring social inclusion and environmental sustainability on the basis of good governance. The SDG framework provides a comprehensive agenda through which to mainstream policies and derive targeted actions for addressing core sustainability challenges. However, the ability to target policies and actions to address conservation issues, while pursuing economic development and prosperity, leaving no one behind, is hampered by lacking scientific evidence and data to direct and support informed decision making. In order to derive targeted policies and actions to support effective land use planning, management and ecological restoration conforming to the requirements of the SDG’s, it is imperative to understand the underlying processes of change [7]. Up-to-date information on current land cover and land use provides critical information that can be used to underpin decision-making processes, while modelled predictions about plausible future land use/land cover (LULC) scenarios provide indications of potential trajectories and thus a platform for identifying interventions. Changes in land use and land cover can be described and projected through the use of land change models, which can be used to explain and assess the dynamics of land cover- and broader system change [8]. 1. Introduction Spatial land change models thus provide platforms for exploring potential future scenarios, which can be used to guide land use decision making and planning [8]. The purpose of the study is to assess and quantify past and plausible future land use and land cover changes and dynamics within the Virunga case study area. The primary analysis will be guided 3 of 28 Sustainability 2020, 12, 1570 by a change analysis of classified satellite imagery to quantify past changes, and the development of a land change model, applying a coupled machine learning—the Markov chain approach—to derive a future land cover prediction for the year 2030. The aim is to assess the plausible future evolution of the landscape within the Virunga case study area and address an existing data gap in order to provide evidence to support data-based decision-making processes conforming to the requirements of the SDG’s. Problem Statement and Research Questions While several studies have already successfully applied predictive land change modelling to support land use management and decision-making processes [9–12], a thorough literature review indicates that such an approach has been applied in just a few case studies and hence it is necessary to explore further cases in order to assure its applicability across different landscapes. Therefore, a remote area is targeted in Africa covering the Virunga NP and its immediate vicinity. This study aims to apply remotely sensed data and geospatial and modelling tools to detect, quantify, analyse, and predict future land change in the Virunga NP and its immediate vicinity. Hence, the following research questions are targeted: • Have there been major land cover changes within the study area in the last 10 years? And if so, what kind of land cover changes? • Have there been major land cover changes within the study area in the last 10 years? And if so, what kind of land cover changes? • What has the spatial extent of the land cover change been and which areas have experienced the highest rate of changes? • What has the spatial extent of the land cover change been and which areas have experienced the highest rate of changes? • What are the major driving forces behind these changes? • What will the extent of land change be by 2030? • How can the future land cover prediction for the Virunga study area be used to support and underpin policy frameworks towards achieving the SDG’s? • How can the future land cover prediction for the Virunga study area be used to support and underpin policy frameworks towards achieving the SDG’s? 2. Study Area The Virunga NP is located in Central Africa, in the Eastern part of the Democratic Republic of the Congo, on the border with Uganda and Rwanda. It is located in the equatorial zone, within the Albertine Rift, of the Great African Rift Valley [3]. In this study, The Virunga NP and its immediate vicinity was used as case study area in order to assess the dynamics within the NP and the landscape dynamics of the entire Virunga catchment. The study area is created from a minimum-bounding rectangle encompassing the entire NP, clipped at the Rwandan border. It was considered critical to include the border areas around the NP in order to explore socioeconomic changes, primarily in the form of urban development and cropland expansion, outside of the NP, and assess how these land cover dynamics could potentially impede conservation efforts and sustainable land management planning. The study area, as shown in Figure 1, covers a total of 14,810 km2 of which 7779 km2 is within the Virunga NP. Figure 1. Study area around the Virunga NP in the Democratic Republic of the Congo. 3. Methodology  Figure 1. Study area around the Virunga NP in the Democratic Republic of the Congo. Figure 1. Study area around the Virunga NP in the Democratic Republic of the Congo. 3 Methodology Figure 1. Study area around the Virunga NP in the Democratic Republic of the Congo. 4 of 28 Sustainability 2020, 12, 1570 Figure 1 Stu 3. Methodology 3. Methodology The methodological framework utilized in this study to predict the future landscape around the Virunga NP was developed using a variety of different tools and the theoretical framework outlined below. The workflow is illustrated in Figure 2. The methodological framework utilized in this study to predict the future landscape around the  Virunga NP was developed using a variety of different tools and the theoretical framework outlined  below. The workflow is illustrated in Figure 2. Figure 2. Land Change Modelling workflow to predict land cover change in Virunga in 2030. Figure 2. Land Change Modelling workflow to predict land cover change in Virunga in 2030. Figure 2. Land Change Modelling workflow to predict land cover change in Virunga in 2030. Figure 2. Land Change Modelling workflow to predict land cover change in Virunga in 2030. In this section, the methodology applied in this study to derive land cover predictions for the year 2030, conforming to this sequential stepwise approach is described. All datasets were either created in, or re-projected to, a Reseau Geodesique de la République Democratique du Congo (RDC) 2005 Transverse Mercator (TM) Zone 18 (EPSG:4051) projected coordinate system, recommended for use in the Democratic Republic of the Congo. ArcGIS was used as a primary tool for data preprocessing and visualisation of results. 3.1. Land Cover Classification Google Earth Engine provides a cloud-based platform for accessing and processing large amounts of both current and historical satellite imagery, including those acquired by the Landsat-7 and Landsat-8 satellites. The advantages of seamless integration of archived, and pre-processed satellite imagery, along with a powerful cloud-processing platform made Google Earth Engine an ideal platform for conducting the land cover classification. The land classification in Google Earth Engine is composed of several different steps; • Choosing an appropriate satellite imagery dataset, fitting the objective of the study, • Choosing an appropriate satellite imagery dataset, fitting the objective of the study, • Define land cover classes and collect training data to train the supervised classification algorithm, • Define land cover classes and collect training data to train the supervised classification algorithm, • Developing a JavaScript code to acquire, process and classify the satellite imagery based on the choice of classification algorithm. • Define land cover classes and collect training data to train the supervised classification algorithm, • Developing a JavaScript code to acquire, process and classify the satellite imagery based on the choice of classification algorithm. • Developing a JavaScript code to acquire, process and classify the satellite imagery based on the choice of classification algorithm. 5 of 28 Sustainability 2020, 12, 1570 3.1.2. Collecting Training/Validation Data As a first step in preparing a training dataset for the land classification, the definition of a nomenclature of land cover classes fitting the objective of the study needed to be defined. Accordingly, the 5 following land cover classes were enough to ensure a sufficient representation of the spatiotemporal variety of land cover changes and identify the primary drivers contributing to forest change dynamics. 1. Forest: afforested and primary forest areas. (Note: While the authors acknowledge the significant difference between afforested areas and primary forest, further separation of the two classes would have caused significant classification errors, due to the spectral similarity of the two classes. Accordingly, the two classes were merged into a combined forest cover class to increase classification accuracy of forest cover.) 2. Water: lakes and rivers. 3. Urban areas: developed residential or industrial areas, roads and urban fringes. 4. Cropland: planted or bare crop fields. 5. Open land/grassland: areas with sparse vegetation, characterized by open grasslands, bare soil or volcanic ash. To train and validate the land-cover classifications, a reference training dataset was collected within the study area. The minimum samples for machine learning based algorithms to perform optimally should be at least 10 times the number of land cover classes. Thus, the training data samples should be at least 50. These reference training datasets were collected by drawing polygons and clicking points within the Google Earth Engine map interface. The points and polygons were randomly collected throughout the case study area, collecting multiple instances of training data samples for each land cover class. From the collection of training data polygons and points, a subsample of 500 points was used to train the model. An additional point dataset consisting of 50 individually sampled points were collected and used for validation. The validation dataset (i.e., ground truth) was sampled using high-resolution images available in the Google Earth archive from representative years between 2010–2019. 3.1.1. Satellite Imagery In this study, three land cover maps were created, one for 2010, 2015 and 2019. As the National Aeronautics and Space Administration (NASA)’s Landsat satellites provides an archived and freely available dataset covering the entire study period with high resolution (30 m) multispectral imagery, these were selected for this study. Google Earth Engine provides integrated access to analysis-ready (already geometrically corrected and orthorectified), surface reflectance Landsat data from the Tier-1 collection. For the 2010 land cover map, tier-1 data from the Landsat 7 Enhanced Thematic Mappers (ETM+) sensor were selected, while tier-1 data from the Landsat 8 Observation Land Images (OLI) were chosen for the 2015 and 2019 land cover maps. For all three land cover maps, cloud-free composite images were generated from a stack of multiple images collected from the preceding three years (i.e., the 2010 land cover map is based on images from 2008–2010). Only images with less than 30% cloud cover were used in the compositing process, resulting in approximately 60–70 useable tiles for each three-year period. 3.2.1. Modelling Transition Potentials: Submodels The second step in the LULC change prediction process is to model the transition potentials, which are in essence maps of suitability/likelihood of one land cover changing into another [13]. Following [14] the land cover transitions can be grouped together into empirically evaluated transition submodels when the common underlying drivers are assumed to be the same. The submodels can consist from a single land cover transition (e.g., from open land to cropland) or from multiple transitions, grouped together based on the assumption that transitions are caused by the same underlying drivers of change. The explanatory variables are used to model the historical change process based on the empirical relationship between the measured change and the explanatory variable. Based on the major land class transitions identified in the previous step, the 12 predominant transitions were grouped together based on transition type to form six individual submodels. The composition of transition groups and a description of the types of changes under each submodel can be seen in Table 1. Although persistence, i.e., areas that did not change, can be considered a trajectory, it cannot be considered as a transition class, and thus areas of persistence are ignored in LCM [10]. Table 1. Transition submodels and descriptions. Transition Submodel Description Land Cover Transitions Abandonment/reclamation Urban and agricultural areas converted to grassland and open land • Urban to open land • Cropland to open land Afforestation Land cover classes converted to tree plantation • Cropland to forest • Open land to forest Agricultural intensification Agricultural areas substituting grasslands and open land areas • Open land to cropland Deforestation Forested areas converted into other land class types • Forest to cropland • Forest to open land Natural dynamics Areas where natural changes cause land conversion • Forest to water • Water to forest • Open land to water Urban intensification Urban areas substitute other land classes • Cropland to urban • Open land to urban Table 1. Transition submodels and descriptions. 3.1.3. Land Cover Classification within the Google Earth Engine IDE In order to create the three land cover maps, three individual scripts were prepared within the Google Earth Engine Integrated Development Environment (IDE), one for each of the three years. The JavaScript source code for the land cover classification is included in the Appendix A. The first component of the script was to import the area of interest (AOI) as table data. Secondly, five empty containers for geometry collections for the training datasets were imported as variables. Subsequently, the cloud-free composite of satellite images was imported using the JavaScript code. The function ‘maskClouds’ generates a cloud and a cloud shadow mask for the imported Landsat 6 of 28 Sustainability 2020, 12, 1570 collection. Furthermore, within this function, the Normalized Difference Vegetation Index (NDVI) was calculated and added to the band collection of the satellite image composite. The NDVI was added to the band collection to enhance the contribution of vegetation in the spectral response for the classification. In addition to NDVI, the first seven bands of the Landsat 8 composite were used in the classification algorithm for the 2015 and 2019 land cover maps. Bands 1-5, band 7 and the added NDVI band were used for the 2010 land cover map, based on the Landsat 7 composite. Thereafter, 500 sample points for each training layer were generated by looping over each training dataset and creating random points within the geometries of the training data layers. Lastly, a confusion matrix was created in order to assess the performance of the classification algorithm. 3.2. LULC Modelling and Prediction The Land Change Module (LCM) within TerrSet (Geospatial Monitoring and Modeling Software) was used to conduct the sequential steps conforming to the requirements of LULC modelling using a multi-layer perceptron (MLP)-Markov chain approach. In this section, each step of the LULC modelling process is described. 3.2.2. Explanatory Variables LULC change processes are dynamic and result from the interaction between a range of different, primarily biophysical and socioeconomic criteria. In LULC change modelling, these criteria are also 7 of 28 Sustainability 2020, 12, 1570 referred to as ‘explanatory’ variables, as these explain the components of the causal relationships determining the land cover dynamics and they form a critical prerequisite for developing a realistic land change model. The explanatory variables sum up the “knowledge” that the model will use to simulate future land cover scenarios [15]. Each explanatory variable was tested for its potential explanatory value using Cramer’s V scores. Cramer’s V is a coarse statistic measure of the strength of association or dependency between two variables and it ranges from 0.0 to 1.0 in values. Generally, variables with a total Cramer’s V score higher than 0.15 are considered useful and those with a score over 0.4 are considered good [13]. In choosing explanatory variables, the processes contributing to land cover change needs to be visualised in the form of a spatial dataset representing the underlying changes, at a spatial resolution consistent with the land cover maps (30 m). GIS data sets (further described in Table 2) were identified to describe the transitions in the case study area and geoprocessing was performed to derive spatial datasets to, either directly or as a proxy, explain the underlying changes for each transition. According to [16] variables cannot be categorical and thus needs to be continuous and quantitative. The drivers that were used in this study include; elevation (Digital Elevation Model (DEM)), aspect (Asp), slope, Evidence Likelihood (EL), distance from artisanal mines (D_am), distance from disturbance (D_disturb), distance from cities (D_cities), distance from forests (D_forest), distance from mining concessions (D_mining), distance from roads (D_roads), distance from waterways (D_water). For each land cover class, evidence likelihood answers the question, “How likely is it that you would have this if you were an area that would experience change?” [13], meaning that it established the suitability of each pixel to transform into urban areas or cropland. To do this, evidence likelihood transforms a categorical variable into a continuous surface, based on the relative frequency of pixels belonging to the different categories within the areas of change [17]. In this study, evidence likelihood is a quantitative measure of the frequency of change between urban areas and cropland (also called disturbance) and all other land classes from 2010–2015. 3.2.2. Explanatory Variables Thus, it represents the relative frequency of the different land cover classes that occurred in the areas that transitioned to urban or cropland. This variable aims to explain the geospatial processes that determine urban expansion and agricultural intensification. The distance drivers represent the proximity of pixels to forces that either constraints or incentivise land cover changes. Mining activities is one of the primary driver of deforestation within the Virunga area “Artisanal mining operations are unregulated and often occur in riparian zones, removing forest and vegetation cover to process the mineral soil.” [18]. Accordingly, there is a documented relationship between deforestation and mining operations, thus, distance from artisanal mines and distance from mining concessions are included as proxy drivers of forest conversion, the rationale being that the closer in proximity a forested area is to known mining operations, the more likely it is to be deforested. Likewise, these drivers will likely positively correlate with an increase of open land, urban areas and cropland nearer to the mining concessions. Distance to disturbance is a spatial driver made from extracting Euclidian distances from areas that were urban or cropland in 2010. The hypothesis is that future anthropogenic disturbance is believed to be closer to areas of existing disturbance, and thus distances to existing disturbances are believed to be closely correlated with urbanisation processes and agricultural expansion. Table 2 below describes the input datasets as well as the geoprocessing steps of each of the explanatory variables. 8 of 28 8 of 28 Sustainability 2020, 12, 1570 Table 2. Description of potential explanatory variables. 3.2.3. Modelling Transition Potential—MLP Calibration 3.2.3. Modelling Transition Potential—MLP Calibration Artificial neural networks are types of computational frameworks for a collection of units or nodes (also called neurons or perceptrons), which aim to mimic the human brain [19]. In this study, an MLP neural network was trained in order to model the nonlinear relationships between land cover change and the explanatory variables, thus deriving the transition potential for each type of land cover change. Operationally, within the LCM, MLP creates a random sample of cells that transitioned and a sample of cells that persisted and use half of the samples to train the model and develop multivariate functions (adjusting the weights) to predict the potential for change based on the value of the conditions at each location [15]. The other half of the subset sample of cells that transitioned and persisted is used to test the performance of the model (validation). The 12 major transitions which occurred in the period between 2010 and 2015 were grouped together in 6 different submodels, namely; Abandonment/reclamation, Afforestation, Agricultural intensification, Deforestation, Natural dynamics and Urban intensification. The next step in modelling the transition potential was to assign the explanatory variables to each submodel. Variables can be added to the model either as static, meaning that they do not change over time, such as slope, or dynamic, meaning that they do change over time, such as proximity to roads (assuming dynamic road development). Static variables are unchanging over time and express aspects of basic suitability for transitions under consideration, while dynamic variables are time-dependent, such as proximity to existing forest areas or road networks, and are recalculated during the course of a future land cover simulation [13]. In this study DEM, slope, aspect, EL, D_am, D_mining and D_water was used as static variables, while D_disturb, D_cities. D_forests and D_roads were designated as dynamic variables. In contrast to the other distance-related variables, D_am and D_mining are defined as static, under the assumption that the geographic location of the mining operations remain fixed and are not expanding over time. g p p g An iterative approach was used to establish the most appropriate, and accurate, combination of driver variables for each submodel, while avoiding overfitting. 3.2.2. Explanatory Variables Variable DEM Asp Slope EL D_am D_disturb D_cities D_forests D_mining D_roads D_water Data origin SRTM 90m Digital Elevation Database v4 Land cover 2010 + 2015 International Peace Information Service (IPIS) Land cover 2010 World Resources Institute Land cover 2010 World Resources Institute Data format Raster (GeoTiff) Raster (GeoTiff) Shapefile (points) Raster (GeoTiff) Shapefile (points) Raster (GeoTiff Shapefile (polygons) Shapefile (lines) Shapefile (lines) Native coordinate system WGS 84 EPSG:4051 WGS 84 EPSG:4051 WGS 84 EPSG:4051 WGS 84 Spatial resolution (m) 90 m cell resolution resampled to a 30 m resolution 30 m 1: 50 000 vector scale converted to 30 m cell resolution 30 m 1: 50 000 vector scale converted to 30 m cell resolution 30 m 1: 50 000 vector scale converted to 30 m cell resolution Temporal resolution 2008 2010–2015 2009–2016 2010 2009 2010 2013 2009 2009 Geoprocessing Reproject Reproject; computed from DEM Computed from land cover maps Reproject; clip to AOI; Euclidian distance from all artisanal mines (rasterize) Reclassify boolean (urban/cropland); Euclidian distance from disturbed areas Reproject; clip to AOI; Euclidian distance from all cities (rasterize) Reclassify boolean (forest); Euclidian distance from forest areas Reproject; clip to AOI; Euclidian distance from all mining concessions (rasterize) Reproject; clip to AOI; Euclidian distance from all roads (rasterize) Reproject; clip to AOI; Euclidian distance from all waterways (rasterize) Table 2. Description of potential explanatory variables. 9 of 28 Sustainability 2020, 12, 1570 3.2.4. Change Prediction and Model Validation Following the transition submodel development, the 12 transition potential maps were used as input in a Markov chain model to simulate future LULC changes (Table 3). The Markov chain determines the amount of change using the earlier and later land cover map along with a pre-specified future year [13]. The Markov module produces a transition probability matrix, which is a matrix that records the probability of each land cover class to change into every other land class category. It also creates a transition areas matrix which is a record of the number of pixels that are expected to change from each land cover class over the specified time frame [13]. Finally, the Markov chain creates a set of conditional probability images which reports the probability of a land cover type to be found at each pixel after the specified prediction date [13]. However, as the matrices only determine the quantity of change, the transition potential (suitability) maps are utilized within the Markov analysis to spatially allocate changes in order to make a land cover prediction for a future year [13]. Table 3. Markov chain transition probability matrix. Given: Probability of Changing to: Forest Water Urban Cropland Open Land Forest 0.3169 0.0017 0.0014 0.6271 0.0528 Water 0.019 0.9953 0.0000 0.0017 0.0011 Urban 0.0208 0.0016 0.4025 0.2786 0.2965 Cropland 0.1029 0.0005 0.0035 0.8022 0.0910 Open land 0.0701 0.0035 0.0268 0.6451 0.2545 Table 3. Markov chain transition probability matrix. Consequently, Markov chain analysis was used to make a LULC prediction for 2019 and subsequently validated by using the actual 2019 land cover map for comparison. Yearly recalculation stages were assigned in the model to specify the frequency of which the dynamic variables are recalculated in the model. This means that the D_disturb, D_cities. D_forests and D_roads explanatory variables are updated in the model every year until the prediction year. Besides visually inspecting how the predicted land cover compare with the actual 2019 land cover map, different Kappa Index of Agreement (KIA) scores was used to assess the overall accuracy of the prediction. The KIA scores can be used to test the agreement between a ‘comparison’ map and a ‘reference map’, both in terms of the quantity of cells in each land cover category and the agreement in terms of location of these cells [13]. 3.2.3. Modelling Transition Potential—MLP Calibration Each submodel was fitted with all 11 explanatory drivers to being with, and an iterative approach was used to remove the driver with the least explanatory potential, while assessing the accuracy score and skill level of the model after each iteration. The accuracy score provides a value in percentage that indicates how well the model is able to predict the changes that happened between 2010 and 2015, accounting for both change and persistence. The skill measure compares the number of correct predictions, minus those attributable to random guessing, to that of a hypothetical perfect prediction [10]. Thus, the skill measure provides an indication of how the explanatory drivers will explain past changes. The skill is measured on a scale from −1 to 1, where values less than 0 indicates that the model performs worse than what would be expected by random guessing, 0 indicates that the model performs as well as random guessing while values between 0 and 1 indicates that the performance of the model exceeds what is expected by pure chance. After each iteration of calibrating individual submodels using MLP, a report about the nature of the model performance is created. This provides critical information on the overall accuracy and skill of the model, the skill measure broken down by component (transition and persistence type) and the explanatory power of each variable. In step 1, the variable with the lowest negative effect on the skill is held constant, and this provides information on the explanatory potential of this variable. If the accuracy and skill of the model don’t decrease by much, when holding the variable constant, this suggests that the variable has little value and can be removed [13]. On each iteration of the calibration of each submodel, the variable with the least explanatory potential was removed until a combination of 5-6 of the variables with the strongest explanatory potential was left under each submodel. Consequently, the final selected variables were loaded into the submodel structure to execute the final iteration of the MLP training. Sustainability 2020, 12, 1570 10 of 28 10 of 28 3.2.4. Change Prediction and Model Validation The Kappa Standard (Kstandard) is equivalent to kappa and indicates the proportion of correctly assigned pixels versus the proportion that is correct by chance. The Kappa for no information (Kno) indicates the overall agreement between the simulated and reference map [13]. The Kappa for location (Klocation) is a measure of the spatial accuracy in the overall landscape, due to the correct assignment of values in each category between the simulated and reference map [13]. The Kappa for stratum-level location (KlocationStrata) is a measure of the spatial accuracy within preidentified strata, and it indicates how well the grid cells are located within the strata. The combination of Kstandard, Kno, Klocation and KlocationStrata scores allows for a comprehensive assessment of the overall accuracy both in terms of location and quantity. All KIA scores range from 0 to 1 (or 0% to 100%), where 0 indicates that agreement is equal to agreement due to chance and 1 (or 100%) indicates perfect agreement. 4.1. Land Cover Classification Using the workflow described in Section 3.1, the three land cover maps for 2010, 2015 and 2019 was generated, using Landsat data. The resulting land cover classifications can be seen in Figure 3 below. 11 of 28 11 of 28 Sustainability 2020, 12, 1570 Figure 3. Generated land cover maps. Figure 3. Generated land cover maps. Figure 3. Generated land cover maps. overall accuracy, producers- and users’ accuracy for each of t n Table 4, and the quantified land cover area under each land Fig ll d ps. ch of the th ee la d co e Figure 3. Generated land cover maps. Figure 3. Generated land cover maps. y ed land cover area under each Figure 3. Generated land cover maps. Figure 3. Generated land cover maps. y ed land cover area under each The overall accuracy, producers‐ and users’ accuracy for each of the three land cover maps can  be seen in Table 4, and the quantified land cover area under each land class, and for each year, can  be seen from the graph presented in Figure 4. The overall accuracy, producers- and users’ accuracy for each of the three land cover maps can be seen in Table 4, and the quantified land cover area under each land class, and for each year, can be seen from the graph presented in Figure 4. from the graph presented in Figure 4. Table 4. Accuracy scores for the 2010, 2015 and 2019 land cover maps. Table 4. Accuracy scores for the 2010, 2015 and 2019 land cover maps. Table 4. Accuracy scores for the 2010, 2015 and 2019 land cover maps. Overall Accuracy Producer’s Accuracy Us Table 4. Accuracy scores for the 2010, 2015 and 2019 land cover maps. Overall Accuracy  Producer’s Accuracy  User’s Accuracy  Land Cover Class  2010  2015    2019  2010  2015    2019  2010  2015    2019  Forest        96  90.9  100  96  100  86  Water  100  100  100  100  100  100  Urban  95.5  97.8  97.9  84  90  92  Cropland  91.8  100  80  90  82  88  Open Land  82.5  84.5  85.5  94  98  94  Total (in %)  92.8  94  92  93.2  94.6  92.7  92.8  94  92  Table 4. Accuracy scores for the 2010, 2015 and 2019 land cover maps. the spatiotemporal changes between 2010 and 2015, the e s‐tabulated The cross‐tabulation table shown in Table 5 sh ure 4. Land cover area per class in 2010, 2015 and 20 Figure 4. Land cover area per class in 2010, 2015 and 2019. 4.1. Land Cover Classification Overall Accuracy Producer’s Accuracy User’s Accuracy Land Cover Class 2010 2015 2019 2010 2015 2019 2010 2015 2019 Forest 96 90.9 100 96 100 86 Water 100 100 100 100 100 100 Urban 95.5 97.8 97.9 84 90 92 Cropland 91.8 100 80 90 82 88 Open Land 82.5 84.5 85.5 94 98 94 Total (in %) 92.8 94 92 93.2 94.6 92.7 92.8 94 92 Overall Accuracy Producer s Accuracy User s Accuracy Land Cover Class 2010 2015 2019 2010 2015 2019 2010 2015 2019 Forest 96 90.9 100 96 100 86 Water 100 100 100 100 100 100 Urban 95.5 97.8 97.9 84 90 92 Cropland 91.8 100 80 90 82 88 Open Land 82.5 84.5 85.5 94 98 94 Total (in %) 92.8 94 92 93.2 94.6 92.7 92.8 94 92 Figure 4. Land cover area per class in 2010, 2015 and 2019. nd Changes  In order to assess the spatiotemporal changes between 2010 and 2015, the earlier and latter land t b l t d Th t b l ti t bl h i T bl 5 h th f i Forest Water Urban Cropland Open land 2010 5113 1773 18 4538 3035 2015 3646 1780 76 6878 2098 2019 3358 1767 69 7105 2154 0 1000 2000 3000 4000 5000 6000 7000 8000 Area (km2) Area under each land cover class in 2010, 2015 and 2019 Figure 4. Land cover area per class in 2010, 2015 and 2019. Forest Water Urban Cropland Open land 2010 5113 1773 18 4538 3035 2015 3646 1780 76 6878 2098 2019 3358 1767 69 7105 2154 0 1000 2000 3000 4000 5000 6000 7000 8000 Area (km2) Area under each land cover class in 2010, 2015 and 2019 Figure 4. Land cover area per class in 2010, 2015 and 2019. Sustainability 2020, 12, 1570 12 of 28 12 of 28 Land Changes The majority of the net loss of the open land class (1109.2 km2) is associated with agricultural expansion, while another 59,5 km2 is attributed to urbanisation processes. A net loss of 69.8 km2 is associated with afforestation processes. 5. The water bodies remained largely unchanged, which is to be expected as there has been no waterworks (e.g., dam construction) in the study period. Thus, the water bodies, largely consisting from the two major lakes in the study area, Lake Édouard and Lake Kivu, have remained relatively consistent. 5. The water bodies remained largely unchanged, which is to be expected as there has been no waterworks (e.g., dam construction) in the study period. Thus, the water bodies, largely consisting from the two major lakes in the study area, Lake Édouard and Lake Kivu, have remained relatively consistent. 5. Table 5. LULC change matrix for the period from 2010 to 2015 (km2) class. Table 5. LULC change matrix for the period from 2010 to 2015 (km2) class. LC_2010 Land Class Forest Water Urban Cropland Open Land Total (km2) LC_2015 Forest 3327.5 1.6 0.0 247.5 69.8 3646.4 Water 3.8 1770.2 0.0 0.3 5.2 1779.5 Urban 0.6 0.0 13.0 2.5 59.5 75.6 Cropland 1715.4 0.3 0.9 4051.8 1109.2 6877.6 Open land 66.1 0.9 3.8 236.2 1791.2 2098.3 Total (km2) 5113.4 1773.0 17.8 4538.4 3035.0 14,477.5 The spatial trends of the changes outlined above are illustrated in Figure 5 below. The spatial trends of the changes outlined above are illustrated in Figure 5 below. The spatial trends of the changes outlined above are illustrated in Figure 5 below. Land Changes In order to assess the spatiotemporal changes between 2010 and 2015, the earlier and latter land cover maps were cross-tabulated. The cross-tabulation table shown in Table 5 shows the frequencies with which the land classes remained the same (Diagonal) or changed into other categories (off-diagonal frequencies). The table represents quantities of conversion from the earlier to the later land cover data, and it clearly depicts significant changes, primarily between forest and cropland. The following information was obtained about the changes in each class from the table: 1. Between 2010 and 2015 the forest cover was reduced by 28.7% from 5113.4 km2 in 2010 to 3646.4 km2 in 2015. Even though there was a forest gain of 318.9 km2 largely caused by afforestation from cropland and open land, the net loss of 1467 km2 is almost exclusively attributed to forest conversion into cropland. 2. Accounting for the least prevalent land class in the case study area, urban areas have experienced a large increase between 2010 and 2015, from 17.8 km2 to 75.6 km2, resulting in a 57.9 km2 net gain. This is largely attributed with rapid urbanisation processes in the Democratic Republic of Congo in general, which has an estimated average annual urban population growth rate of 4.3% [20]. The population of the capital city in the North Kivu province, Goma, located in the south-eastern corner of the case study area, increased from 150,000 people in 1990 to more than one million in 2017 [6]. Thus, the majority of the urban class increase is caused by the expansion of Goma. 3. Cropland is the most dynamic land class in the case study area and represents the most dominant land cover type. The total area under cultivation increased by 51.5%, from 4538.4 km2 in 2010 to 6877.6 km2 in 2015. As mentioned previously, cropland is the main driver of deforestation and thus the majority of the agricultural expansion is caused by forest conversion. However, another 47% of cropland expansion is attributed with the cultivation of previously open land/grassland areas. 4. The open land cover class was reduced by 30.8%, from 3035.0 km2 in 2010 to 2098.3 km2 in 2015. Even though the open land class received a total net loss, 236.2 km2 was gained, caused by agricultural abandonment. Another 66 km2 gain of open land is attributed to deforestation. 4.2. LULC Model Using the 2010 and 2015 land cover maps and the explanatory variables as input data, the model described in the methods section was trained to make a land cover prediction for each year between 13 of 28 Sustainability 2020, 12, 1570 2020 and 2030. Table 6 provides a list of the Cramer’s V scores of each explanatory variable used in the model and Figure 6 illustrates all the processed variables. Sustainability 2020, 11, x FOR PEER REVIEW  14  of  31 Figure 5. Class transitions between 2010 and 2015. Figure 5. Class transitions between 2010 and 2015. 20, 11, x FOR PEER REVIEW Figure 5. Class transitions between 2010 and 2015. Figure 5. Class transitions between 2010 and 2015. , 11, x FOR PEER REVIEW 4.2. LULC Model  Using the 2010 and 2015 land cover maps and the explanatory variables as input data, the model described in the methods section was trained to make a land cover prediction for each year between 2020 and 2030. Table 6 provides a list of the Cramer’s V scores of each explanatory variable used in the model and Figure 6 illustrates all the processed variables. Figure 6. Processed explanatory variable datasets used as input for the MLP modelling. Table  7  below  provides  information  on  the  accuracy  and  skill  measure  of  the  model  when  Figure 6. Processed explanatory variable datasets used as input for the MLP modelling. 15 land cover maps and the explanatory variables ection was trained to make a land cover predicti Backwards Stepwise Constant Forcing Model Variables Included Accuracy (%) Skill Measure With all variables All variables 72.25 0.6300 Step 1: var.[5] constant [1,2,3,4,6,7,8,9,10] 72.25 0.6300 Step 2: var.[5,10] constant [1,2,3,4,6,7,8,9] 72.23 0.6298 Step 3: var.[5,10,7] constant [1,2,3,4,6,8,9] 72.06 0.6275 Step 4: var.[5,10,7,9] constant [1,2,3,4,6,8] 69.86 0.5981 Step 5: var.[5,10,7,9,4] constant [1,2,3,6,8] 65.48 0.5397 Step 6: var.[5,10,7,9,4,8] constant [1,2,3,6] 62.59 0.4999 Step 7: var.[5,10,7,9,4,8,3] constant [1,2,6] 54.55 0.3940 Step 8: var.[5,10,7,9,4,8,3,1] constant [2,6] 47.77 0.3036 Step 9: var.[5,10,7,9,4,8,3,1,6] constant [2] 29.40 0.0587 Backwards Stepwise Constant Forcing  Model  Variables Included  Accuracy (%)  Skill Measure  With all variables  All variables  72.25  0.6300  Step 1: var.[5] constant  [1,2,3,4,6,7,8,9,10]  72.25  0.6300  Step 2: var.[5,10] constant  [1,2,3,4,6,7,8,9]  72.23  0.6298  Step 3: var.[5,10,7] constant  [1,2,3,4,6,8,9]  72.06  0.6275  Step 4: var.[5,10,7,9] constant  [1,2,3,4,6,8]  69.86  0.5981  Step 5: var.[5,10,7,9,4] constant  [1,2,3,6,8]  65.48  0.5397  Step 6: var.[5,10,7,9,4,8] constant  [1,2,3,6]  62.59  0.4999  Step 7: var.[5,10,7,9,4,8,3] constant  [1,2,6]  54.55  0.3940  Step 8: var.[5,10,7,9,4,8,3,1] constant  [2,6]  47.77  0.3036  St 9 [5 10 7 9 4 8 3 1 6] t t [2] 29 40 0 0587 The final skill measure and accuracy rate of each model calculated through MLP is summarized in Figure 7 and the explanatory drivers used under each submodel and selected performance scores is provided in Table 8. The final skill measure and accuracy rate of each model calculated through MLP is summarized  in Figure 7 and the explanatory drivers used under each submodel and selected performance scores  is provided in Table 8. The final skill measure and accuracy rate of each model calculated through MLP is summarized in Figure 7 and the explanatory drivers used under each submodel and selected performance scores is provided in Table 8. The final skill measure and accuracy rate of each model calculated through MLP is summarized  in Figure 7 and the explanatory drivers used under each submodel and selected performance scores  is provided in Table 8. Figure 7. Submodel accuracy and skill measure from MLP. Figure 7. Submodel accuracy and skill measure from MLP. Figure 7. Submodel accuracy and skill measure from MLP. Figure 7. Submodel accuracy and skill measure from MLP. The accuracy and skill measure reveal some disparity between the level of confidence of the  transition modelling under each submodel, however overall the values are fairly consistent, ranging  from 75% to 93%. Abandonment/reclamation has the lowest accuracy score (75.12%), followed by  deforestation (77.61%), afforestation (77.79%) and agricultural intensification (78.23%). 15 land cover maps and the explanatory variables ection was trained to make a land cover predicti ng the 2010 and 2015 land cover maps and the explanatory variables as input data, t ed in the methods section was trained to make a land cover prediction for each year d 2030. Table 6 provides a list of the Cramer’s V scores of each explanatory variab del and Figure 6 illustrates all the processed variables. Figure 6. Processed explanatory variable datasets used as input for the MLP modelling. Table 7 below provides information on the accuracy and skill measure of the model when Figure 6. Processed explanatory variable datasets used as input for the MLP modelling. 14 of 28 Sustainability 2020, 12, 1570 Table 6. Cramer’s V scores for each of the explanatory variables. Variable DEM Asp Slope EL D_am D_disturb D_cities D_forests D_mining D_roads D_water Forest 0.52 0.21 0.26 0.68 0.17 0.40 0.17 0.43 0.14 0.26 0.27 Water 0.61 0.83 0.57 0.42 0.32 0.68 0.54 0.82 0.27 0.57 0.30 Urban 0.14 0.07 0.06 0.10 0.08 0.04 0.06 0.04 0.09 0.09 0.07 Cropland 0.42 0.32 0.34 0.29 0.31 0.52 0.20 0.32 0.31 0.34 0.29 Open land 0.23 0.17 0.24 0.65 0.20 0.15 0.18 0.30 0.24 0.11 0.08 Cramer’s V Overall 0.42 0.42 0.33 0.48 0.22 0.42 0.28 0.46 0.22 0.32 0.20 Sustainability 2020, 12, 1570 15 of 28 Table 7 below provides information on the accuracy and skill measure of the model when holding one or more variables constant. This relationship was used to identify the explanatory variables with the strongest explanation potential for each transition type. Sustainability 2020, 11, x FOR PEER REVIEW  17  of  31 Table 7. Extract from the calibration report indicating accuracy scores and skill measure of the model when holding variables constant. Table 7. Extract from the calibration report indicating accuracy scores and skill measure of the model  when holding variables constant. Table 7. Extract from the calibration report indicating accuracy scores and skill measure of the model when holding variables constant. Table 7. Extract from the calibration report indicating accuracy scores and skill measure of the model  when holding variables constant. 15 land cover maps and the explanatory variables ection was trained to make a land cover predicti Agricultural  intensification, however, has the lowest skill measure of all the submodels (0.56). Natural dynamics  and urban intensification performed best, with accuracies of 93.90% and 83.41%, respectively. The  skill  measure  of  these  two  submodels  was  also  the  highest  among  all  six,  with  0.93  for  natural  dynamics and 0.78 for urban intensification. The outcome of the transition potential modelling is a  series  of  transition  potential  maps,  describing  the  suitability  for  each  of  the  12  major  transitions  included in the submodels. These maps can be seen in Figure 8. The accuracy and skill measure reveal some disparity between the level of confidence of the transition modelling under each submodel, however overall the values are fairly consistent, ranging from 75% to 93%. Abandonment/reclamation has the lowest accuracy score (75.12%), followed by deforestation (77.61%), afforestation (77.79%) and agricultural intensification (78.23%). Agricultural intensification, however, has the lowest skill measure of all the submodels (0.56). Natural dynamics and urban intensification performed best, with accuracies of 93.90% and 83.41%, respectively. The skill measure of these two submodels was also the highest among all six, with 0.93 for natural dynamics and 0.78 for urban intensification. The outcome of the transition potential modelling is a series of transition potential maps, describing the suitability for each of the 12 major transitions included in the submodels. These maps can be seen in Figure 8. 16 of 28 16 of 28 Sustainability 2020, 12, 1570 Table 8. Submodels included in MLP with associated explanatory variables and selected performance indicators. 15 land cover maps and the explanatory variables ection was trained to make a land cover predicti Submodel Explanatory Variables Transition/Persistence Class Class Skill Measure (ratio) Submodel Accuracy Submodel Skill RMS Training Testing Abandonment/reclamation DEM; Slope; D_am; D_cities; D_mining; D_water Urban to Openland 0.8134 75.12% 0.6682 0.2980 0.3071 Cropland to Openland 0.5741 Persistence: Urban 0.7401 Persistence: Cropland 0.5398 Afforestation DEM; Slope; EL; D_disturb; D_forests; D_water Cropland to Forest 0.5181 77.79% 0.7038 0.2751 0.2737 Openland to Forest 0.8918 Persistence: Cropland 0.6536 Persistence: Openland 0.7515 Agricultural intensification DEM; D_am; D_disturb; D_mining; D_roads; D_water Openland to Cropland 0.5961 78.23% 0.5646 0.3899 0.3906 Persistence: Openland 0.5329 Deforestation DEM; D_am; D_disturb; D_mining; D_roads; D_water Forest to Cropland 0.6103 77.61% 0.6642 0.3358 0.3369 Forest to Openland 0.8300 Persistence: Forest 0.5516 Natural dynamics DEM; Slope; EL; D_forests; D_water Forest to Water 0.9848 93.90% 0.9269 0.1207 0.1281 Water to Forest 0.9096 Openland to Water 0.8707 Persistence: Forest 0.8677 Persistence: Water 0.9849 Persistence: Openland 0.9441 Urban intensification Slope; EL; D_am; D_cities; D_mining; D_roads Cropland to Urban 0.8664 83.41% 0.7788 0.2536 0.2489 Openland to Urban 0.6564 Persistence: Cropland 0.8294 Persistence: Openland 0.7630 Submodels included in MLP with associated explanatory variables and selected performance indicators. Table 8. Submodels included in MLP with associated explanatory variables and selected perform 17 of 28 1  of  31 Sustainability 2020, 12, 1570 Sustainability 2020, 12, x FOR Figure 8. Transition potentials. Figure 8. Transition potentials. Figure 8. Transition potentials. Figure 8. Transition potentials. Figure 8. Transition potentials. Figure 8. Transition potentials. 4.2.1. Validating Results  4.2.1. Validating Results Figure 9 below shows the actual and the predicted land cover map for 2019. The actual 2019 land  cover  map  was  created  using  Landsat  data  composites  from  the  years  2017–2019,  applying  the  process described in Section 3.1.3. A visual inspection indicates that the predicted land cover map,  overall, looks fairly similar to the actual land cover map, however there are localised discrepancies  where  the  model  failed  to  predict  changes/persistence,  for  example,  in  the  mid‐west  where  the  simulation predicted cropland to replace large open land areas, when in actuality it did not. Figure 9 below shows the actual and the predicted land cover map for 2019. The actual 2019 land cover map was created using Landsat data composites from the years 2017–2019, applying the process described in Section 3.1.3. A visual inspection indicates that the predicted land cover map, overall, looks fairly similar to the actual land cover map, however there are localised discrepancies where the model failed to predict changes/persistence, for example, in the mid-west where the simulation predicted cropland to replace large open land areas, when in actuality it did not. p p p g p y K scores was used to comprehensively assess how the simulated 2019 land cover map ‘comparison’ compare with the actual 2019 land cover map ‘reference’. The K scores are provided in Table 9. Table 9. K scores for 2019. K INDICATORS 2019 KSTANDARD 0.8828 KNO 0.9224 KLOCATION 0.9001 KLOCATIONSTRATA 0.9001 Table 9. K scores for 2019. K INDICATORS 2019 KSTANDARD 0.8828 KNO 0.9224 KLOCATION 0.9001 KLOCATIONSTRATA 0.9001 The statistics from the k scores shows that Kno is 0.9224, Klocation is 0.9001, KlocationStrata is 0.9001 and the overall Kstandard is 0.8828. According to [21], a model is valid if the overall Kappa (Kstandard) Sustainability 2020, 12, 1570 18 of 28 score exceeds 70% (or 0.7). The Kstandard score, close to 90%, is a very strong indicator of the overall accuracy and performance of the model, and the remaining k scores, all exceeding 85%, indicate that there are almost no, or very small quantification and location errors between the predicted and the actual land cover map for 2019. Thus, the simulation has a strong ability to predict both the quantity and the locations of change. Sustainability 2020, 12, x FOR PEER REVIEW  2  of  31 Figure 9. Actual land cover map for 2019 versus the predicted 2019 land cover map. Figure 9. 4.2.1. Validating Results  4.2.1. Validating Results Actual land cover map for 2019 versus the predicted 2019 land cover map. Figure 9. Actual land cover map for 2019 versus the predicted 2019 land cover map. Figure 9. Actual land cover map for 2019 versus the predicted 2019 land cover map. K scores was used to c 4.2.2. Land Cover Prediction p y p ‘comparison’ compare with the actual 2019 land cover map ‘reference’. The K scores are provided in  Table 9. The resulting compilation of land cover predictions from 2020 through to 2030 can be seen from Figure 10, while the predicted land cover for 2030 is presented in Figure 11. Sustainability 2020, 12, x FOR PEER REVIEW  3  of  31 p y p ‘comparison’ compare with the actual 2019 land cover map ‘reference’. The K scores are provided in  Table 9. The resulting compilation of land cover predictions from 2020 through to 2030 can be seen from Figure 10, while the predicted land cover for 2030 is presented in Figure 11. Sustainability 2020, 12, x FOR PEER REVIEW  3  of  31 Table 9. K scores for 2019. K INDICATORS  2019  KSTANDARD  0.8828  KNO  0.9224  KLOCATION  0.9001  KLOCATIONSTRATA  0.9001  The statistics from the k scores shows that Kno is 0.9224, Klocation is 0.9001, KlocationStrata is 0.90 verall Kstandard is 0.8828. According to [21], a model is valid if the overall Kappa (Kstandar eds 70% (or 0.7). The Kstandard score, close to 90%, is a very strong indicator of the overall ac performance of the model, and the remaining k scores, all exceeding 85%, indicate that th st no, or very small quantification and location errors between the predicted and the actu r map for 2019. Thus, the simulation has a strong ability to predict both the quantity a ions of change. Land Cover Prediction  The resulting compilation of land cover predictions from 2020 through to 2030 can be see re 10, while the predicted land cover for 2030 is presented in Figure 11. Figure 10. Predicted land cover maps from 2020 to 2030. Figure 10. Predicted land cover maps from 2020 to 2030. Table 9. K scores for 2019. K INDICATORS  2019  KSTANDARD  0.8828  KNO  0.9224  KLOCATION  0.9001  KLOCATIONSTRATA  0.9001  The statistics from the k scores shows that Kno is 0.9224, Klocation is 0.9001, KlocationStrata i overall Kstandard is 0.8828. According to [21], a model is valid if the overall Kappa (K ] o 90% dicator ry stro Figure 10. Predicted land cover maps from 2020 to 2030. Figure 10. Predicted land cover maps from 2020 to 2030. 19 of 28 Sustainability 2020, 12, 1570 Figu Figure 11. Predicted 2030 land cover in Virunga. Figure 11. Predicted 2030 land cover in Virunga. re 11. 5. Discussi 5. Discussion Understanding LULC changes, transitions, landscape risks and dynamics is paramount in order  to inform policies, planning interventions and actions aiming to ensure sustainable development in  all dimensions (economic, social and environmental) conforming to the objective of the UN SDG’s  and the 2030 agenda for sustainable development. In this study, a combined MLP‐Markov chain  approach has been used to simulate future land cover changes in the period from 2020 to 2030, in a  case study area covering the Virunga NP in the Democratic Republic of the Congo and its immediate  vicinity. Two  simulations  were  carried  out. The  first  (2019)  was  used  for  model  validation  and  accuracy assessment, and the second (2030) was used to predict landscape change within the case  study area  covering  the  Virunga  NP  catchment. The  assessment  of  the  spatial  patterns  of  LULC  change derived through a change analysis of historical trends combined with the development of a  plausible  future  land  cover  scenario  for  the  Virunga  catchment  will  help  to  improve  the  understanding of the land system and establish cause‐effect relationships between driver variables  and land cover dynamics. Thus, the LULC change model aims to contribute to informing policy  Understanding LULC changes, transitions, landscape risks and dynamics is paramount in order to inform policies, planning interventions and actions aiming to ensure sustainable development in all dimensions (economic, social and environmental) conforming to the objective of the UN SDG’s and the 2030 agenda for sustainable development. In this study, a combined MLP-Markov chain approach has been used to simulate future land cover changes in the period from 2020 to 2030, in a case study area covering the Virunga NP in the Democratic Republic of the Congo and its immediate vicinity. Two simulations were carried out. The first (2019) was used for model validation and accuracy assessment, and the second (2030) was used to predict landscape change within the case study area covering the Virunga NP catchment. The assessment of the spatial patterns of LULC change derived through a change analysis of historical trends combined with the development of a plausible future land cover scenario for the Virunga catchment will help to improve the understanding of the land system and establish cause-effect relationships between driver variables and land cover dynamics. Thus, the LULC change model aims to contribute to informing policy responses aiming to support sustainable land management and landscape planning decisions within the Virunga NP. K scores was used to c 4.2.2. Land Cover Prediction Predicted 2030 land cover in Viru Figure 11. Predicted 2030 land cover in Virunga. eries of land cover predictions covering 2020 to 2030, and the final land cover m in Figure 11 clearly illustrates that the model predicts continuous cropland  at the expense of forest areas and existing open lands. The model also predicts c elopment, particularly around existing settlements. The collective change per cl nt change per year, is illustrated in Figure 12. As depicted in the graph, the fores The series of land cover predictions covering 2020 to 2030, and the final land cover map for 2030 presented in Figure 11 clearly illustrates that the model predicts continuous cropland expansion, primarily at the expense of forest areas and existing open lands. The model also predicts continuous urban development, particularly around existing settlements. The collective change per class in total, and percent change per year, is illustrated in Figure 12. As depicted in the graph, the forest cover will continue to decrease throughout the 10-year period, with an average annual loss of 4.21% and a total area loss of 1085 km2, from 3104 km2 in 2020 to 2019 km2 in 2030. Water coverage will, as expected, remain largely the same, gaining a negligible average of 0.04% per year. Urban expansion and development of new settlements will continue, gaining an average annual of 3.44%. The total urban area is predicted to increase by 38 km2, from 95 km2 in 2020 to 133 km2 in 2030, and looking at the predicted land cover map, most of this is expected to be as a result of urban sprawl around the main city of Goma in south-eastern Virunga. As also visually apparent, cropland expansion will continue throughout the 10-year period, gaining an average annual area of 1.83%, and a total area gain of 1522 km2, from 7636 km2 to 9161 km2 class coverage. Along with forest areas, open land/grassland zones are expected to decrease the most, by 2.96% per year, losing a total of 482 km2 in the 10-year period, from 1857 km2 in 2020 to 1375 km2 in 2030. Sustainability 2020, 12, 1570 km , from 7636 km are expected to decr 20 of 28 nes  od, Figure 12. Predicted land cover (km2) for each land cover class for each year between 2020–2030 and  % yearly (gain/loss) per class. K scores was used to c 4.2.2. Land Cover Prediction The horizontal lines indicate the yearly gain/loss in percent for each  class, while the columns indicate the total land cover in km2 for each class for each year. 5 Discussion ‐6 ‐4 ‐2 0 2 4 ‐500 500 1,500 2,500 3,500 4,500 5,500 6,500 7,500 8,500 9,500 2020 2021 2022 2023 2024 2025 2026 2027 2028 2029 2030 % gain/loss per year Km2 per class Land cover change 2020 ‐ 2030 Forest Water Urban Cropland Open land Forest Water Urban Cropland Open land Figure 12. Predicted land cover (km2) for each land cover class for each year between 2020–2030 and % yearly (gain/loss) per class. The horizontal lines indicate the yearly gain/loss in percent for each class, while the columns indicate the total land cover in km2 for each class for each year. Di i Land cover change 2020 ‐ 2030 Figure 12. Predicted land cover (km2) for each land cover class for each year between 2020–2030 and  % yearly (gain/loss) per class. The horizontal lines indicate the yearly gain/loss in percent for each  class, while the columns indicate the total land cover in km2 for each class for each year. Figure 12. Predicted land cover (km2) for each land cover class for each year between 2020–2030 and % yearly (gain/loss) per class. The horizontal lines indicate the yearly gain/loss in percent for each class, while the columns indicate the total land cover in km2 for each class for each year. 5. Discussi 5. Discussion It aims to provide a blueprint for quantified policy responses aiming to address existing challenges. Follow-up research should aim at applying the LULC model developed for this study for scenario-based analysis of policy response impacts. As formerly mentioned, the LULC model developed in this study predicts a future land cover state, based on a business as usual scenario. Past land cover changes within the Virunga catchment has been largely linked with charcoal production and cropland expansion, which have impeded conservation efforts and put critical pressure on the ecological integrity of the landscape and its biodiversity. By cross-tabulating two land cover maps for 2010 and 2015, this study aimed to quantify past land cover changes and identify spatial trends of change. It concludes that forest conversion into cropland is the most common and frequent type of landcover change, contributing to the majority of the total net forest loss of 28.7% between 2010 and 2015. The most significant forest loss occurred around 21 of 28 21 of 28 Sustainability 2020, 12, 1570 the perimeter of the forest areas in the northern sector of the case study area, however, the forest areas just north of the North Kivu provincial capital of Goma, also experienced substantial losses. While a 318.9 km2 forest cover gain was also identified in the change assessment, these gains cannot be qualified in existing literature, and as these gains are largely located within, or close by, existing cropland areas, they may likely be the result of misclassified pixels, possibly classifying plantation development as forest. While the urban cover is the least predominant land class type within the case study area, the cross-tabulation indicated that the urban land cover quadrupled between 2010 and 2015. The majority of the urban gain, however, is associated with significant urban development around Goma, located in the southeastern corner of the case study area. Another major land cover change results from the conversion of open land/grassland areas into cropland. In total, open land areas were reduced by 30.8% in the period from 2010 to 2015 and the majority of these transitions were located just north of Lake Edward. If unchecked and unregulated, the LULC change model developed in this study indicates that the landscape within and outside the NP will continue to change dramatically in the next 10 years. 5. Discussi 5. Discussion While Figure 12 in the previous section quantified the collective amount of changes, per land class per year, from 2020 to 2030, Table 10 below quantifies the projected land changes from 2019 to 2030, between land classes. As can be seen from the cross tabulation, forests are attributed with the most significant land class loss in the period from 2019-2030, and almost all forest loss (1579.5 km2 of a total of 1651.1 km2) is associated with cropland expansion. Open land is also projected to experience significant land class loss (1162.8 km2) the majority of which (1081.8 km2) is also attributed to cropland expansion. While urban areas are projected to continue to be the minority land class in the Virunga NP catchment, the total urban area cover is projected to almost double, from 68,5 km2 to 132,7 km2 in the 11-year period. Most of this is attributed to the conversion of cropland and open land areas. Thus, the majority of all transitions, gains and losses, are for the most part attributed with the expansion of agricultural lands, and largely at the expense of forest areas. Table 10. Cross-tabulation between actual 2019 land cover and the simulated land cover for 2030. LC_2019 Land Class Forest Water Urban Cropland Open Land Total (km2) Simulated LC_2030 Forest 1707.2 0.2 0.5 289.7 15.8 2019.1 Water 4.8 1766.3 0.3 3.6 13.9 1789.6 Urban 0.9 0.0 45.7 34.0 51.3 132.7 Cropland 1579.6 0.5 15.7 6469.5 1081.8 9161.3 Open land 65.8 0.2 6.3 308.3 991.3 1374.9 Total (km2) 3358.3 1767.2 68.5 7105.1 2154.0 39,605.0 Table 10. Cross-tabulation between actual 2019 land cover and the simulated land cover for 2030. Addressing forest loss is a primary component of conservation efforts and land management planning within the Virunga NP catchment. Therefore, it is critical to determine not just the amount of forest cover loss, but also the spatial extent and location of forest dynamics. Figure 13 below illustrates the spatial location of the dynamics of forest land, and as seen from the figure, forest loss is largely concentrated in the northern part of the study area, and particularly the north-eastern margin of the NP. 5. Discussi 5. Discussion This change is consistent with past deforestation patterns, which has historically been more predominant in the north where larger and more remote forest areas are located, and literature (i.e., [22]) indicate that illegal slashing of old growth forest to produce carbonized wood has been particularly predominant in the northern sector. This is largely caused by rebel groups operating near the city of Beni, supplying local villages and larger cities in the outskirts of the national park with charcoal [22]. Besides charcoal, army groups have also been known to transport illegal timber along the Kamango Route, linking the Democratic Republic of the Congo with Uganda, causing further forest loss and fragmentation [22]. Conversion of cleared forest areas and slashing of trees to plant subsistence crops, such as cassava and maize, is another primary driver of forest loss, particularly in the south [22]. While forest loss is also expected to continue in the southern part of the NP, particularly just north of 22 of 28 da,  g of Sustainability 2020, 12, 1570 timber  along  the  K causing further fore Goma, the high montane forests to the northeast of Goma is predicted to remain largely intact, likely protected by its high altitude and steep terrain, making the area less accessible and thus less likely to be logged. particularly in the south [22]. While forest loss is also expected to continue in the southern part of the  NP, particularly just north of Goma, the high montane forests to the northeast of Goma is predicted  to remain largely intact, likely protected by its high altitude and steep terrain, making the area less  ibl d th l lik l t b l d Goma, the high montane forests to the northeast of Goma is predicted to remain largely intact, likely protected by its high altitude and steep terrain, making the area less accessible and thus less likely to be logged. particularly in the south [22]. While forest loss is also expected to continue in the southern part of the  NP, particularly just north of Goma, the high montane forests to the northeast of Goma is predicted  to remain largely intact, likely protected by its high altitude and steep terrain, making the area less  accessible and thus less likely to be logged Figure 13. Spatial location of forest loss/gain from 2019 to 2030. 5. Discussi 5. Discussion R O ti Pl i I t ti d SDG I l t ti Figure 13. Spatial location of forest loss/gain from 2019 to 2030. l d l Figure 13. Spatial location of forest loss/gain from 2019 to 2030. Figure 13. Spatial location of forest loss/gain from 2019 to 2030. Policy Response Options, Planning Interventions and SDG Implementation  5.1. Policy Response Options, Planning Interventions and SDG Implementation Policy Response Options, Planning Interventions and SDG Implementation  5.1. Policy Response Options, Planning Interventions and SDG Implementation As the LULC change model developed in the context of this study is an empirical‐statistical  projection of past changes into the future, the outcome represents likely LULC changes as a reflection  of a business as usual scenario. Thus, the results help to understand and illustrate how the intrinsic  drivers of change will continue to drive land cover changes, if unchecked, while providing valuable  information on possible future LULC configurations in the Virunga catchment and thus an indication  As the LULC change model developed in the context of this study is an empirical-statistical projection of past changes into the future, the outcome represents likely LULC changes as a reflection of a business as usual scenario. Thus, the results help to understand and illustrate how the intrinsic drivers of change will continue to drive land cover changes, if unchecked, while providing valuable information on possible future LULC configurations in the Virunga catchment and thus an indication of the causes and consequences of land-use change. In the absence of reformed regulatory policies, legal frameworks and planning interventions, Virunga NP will continue to be threatened by encroachment and deforestation, primarily caused by cropland expansion and persistent conflict. The high population density and a continuous population growth believed to be around 3% in the Virunga region [2], will inevitably result in fewer resources outside the Virunga NP, which will ultimately put more pressure within the park, resulting in further damaging human impact. The large-scale deforestation and conversion to agriculture caused by human activities will severely alter the integrity of the landscape and cause strong negative impacts on biodiversity and soil degradation, while undermining the natural resource foundation on which the local livelihoods depend. The formulation of adequate spatial policies in the Virunga catchment must balance the competing needs for land to feed the accelerating population and provide energy and resources, while reducing the loss of ecosystems and biodiversity. The SDGs 23 of 28 Sustainability 2020, 12, 1570 provide the blueprint for such policy planning and interventions, aiming to balance prosperity for both people and the planet. The direct exploitation of resources and expansion of cropland activities is intimately linked with the economic situation of the people [2], and thus in order to protect the biodiversity and integrity of the Virunga NP, policies should aim to improve and strengthen the economic security and livelihoods of the people living in its vicinity. 5.1.1. Incentivising Alternatives to Charcoal As mentioned previously, the vast deforestation in the northern and southern sectors of the park, visualized in the simulated 2030 LULC map, is largely believed to be a reflection of illegal charcoal production and land clearing for agricultural expansion. The major demand for charcoal is located within the major villages, refugee camps and the capital city of Goma in particular. As the majority of the population in Goma rely on charcoal for their entire energy consumption, the prediction of a total clearing of the forest just north of Goma is highly probable and inherently linked with charcoal production and cropland expansion. Electricity is recognized to have substantial benefits for poverty reduction, health and education, and thus access to electricity should be incentivised and subsidized. Realising the US$10 million potential of hydropower in Virunga NP alone, could potentially contribute to provide job opportunities and tax revenue, and more importantly, if sustainably planned and implemented, release pressure on forests to obtain charcoal. Furthermore, the affordability and availability of modern cooking fuels and practices could be subsidised through regulatory reforms, i.e., reducing costs on kerosene stoves and cylinders [24], or through the establishment of microcredit systems. As evident from the LULC change model cropland expansion cause the majority of the land transformation and will continue to grow. Thus, adopting measures to support the development of sustainable biomass production initiatives, i.e., by improving linkages to agriculture, animal husbandry, agroforestry, etc. could be another approach to reduce the dependency on charcoal. Such policy initiatives would not only contribute to promote conservation action, and thus contribute to realizing SDG 15 (Life on land), but also contribute to the realisation of multiple SDG’s, including SDG 1 (No poverty), SDG 3 (Good health and well-being), SDG 8 (Decent work and economic growth), SDG 13 (Climate action) and SDG 7 which aims to ensure access to affordable, reliable, sustainable and modern energy for all. Policy Response Options, Planning Interventions and SDG Implementation  5.1. Policy Response Options, Planning Interventions and SDG Implementation The Virunga NP exists between the extremes of economic poverty and natural wealth, which has made it a target for all of those who aim to profit from its resources. A 2013 report by the World Wildlife Fund (WWF) entitled ‘The economic value of Virunga national park’ concluded that the “direct use of Virunga’s ecosystem could generate US$348 million per year and help diversify DRC’s economy [23]”. The main direct contributors to this value are tourism (US$235 million), fisheries (US$90 million) and hydropower (US$10 million), while another US$63.8 million, primarily attributed to carbon sequestration and erosion control, can be generated through the provision of ecosystem services [23]. If sustainably managed, the outstanding natural value of the Virunga NP could contribute significantly to the local economy, while providing livelihoods for 45.000 people through the provision of job opportunities [23]. Thus, policies should aim to strengthen conservation action by creating an alternative economy that incorporates and enables the surrounding communities from a thriving and well managed national park, while embracing the framework of the SDGs. 5.1.2. Community Development Land grabbing for subsistence agriculture has been another primary driver of change, historically, and unregulated and illegal encroachment has threatened the fringes of the Virunga NP. The LULC change model predicts that vast expanses of the NP will be subject to cropland expansion, at the expense of forests, savannahs and grassland, in 2030. To counteract the infringement, enforcement of existing legislation needs to be strengthened while at the same time community development efforts should aim to build capacity to pave the way for an alternative, and more sustainable, livelihood options 24 of 28 Sustainability 2020, 12, 1570 for the increasing population [23]. Community-based planning and management is undoubtedly a cornerstone of conservation action and SDG implementation, as local communities are effectively custodians of their environment. Consequently, the local communities should be involved in the wider planning framework in order to maximise the development potential and environmental benefits. Thus, in order to contribute to the conservation of the NP and reduce land grabbing, economic development in the region, communal development projects and community involvement should be promoted, e.g., expanding the fragmented and desolate road infrastructure in order to improve market access, and thus increase revenue potential from agricultural and artisanal productions. Other communal development projects could support the promotion of alternative income generating activities, such as ecotourism development or educational programmes which could facilitate access to the tourism industry, such as free public park ranger or guide training programmes. Depending on the nature of community development programmes, successful implementation of initiatives such as those outlined above could potentially contribute to the realisation of SDG 1 (No poverty), SDG 3 (Good health and well-being), SDG 4 (Quality education), SDG 8 (Decent work and economic growth), SDG 9 (Industry, Innovation and Infrastructure), SDG 11 (Sustainable cities and communities) and SDG 15 (Life on land). 5.1.3. Utilising the LULC Change Model to Gain Intergovernmental Support and Mobilise Resour While underpinning the need for reformative action to counteract the impact of deforestation and land degradation in Virunga, it is vital to realize that the majority of the policies and actions suggested will require significant investments. Accordingly, the Democratic Republic of the Congo will, to some extent, be relying on support and engagement from donor countries in order to forge strong bilateral relationships through which investments can be sourced and policies framed. 5.1.2. Community Development Furthermore, collective international support can be forged using the framework of existing Multilateral Environmental Agreements (MEA)’s in order to better integrate conflict-concerns into the implementation and priorities and attain earmarked funding for targeted capacity building and conservation activities. For this purpose, the LULC change model and the simulated land cover for 2030 is not only an effective policy support tool to inform spatial planning and policy-making, but also a vital instrument which can be used for lobbying activities in order to gather support for conservation and poverty reduction activities and strategies at the intergovernmental level. Insight into a probable future LULC scenario within one of the most biodiverse world heritage sites in the world, which indicates that most of the forest resources within the NP will be gone by 2030, may provide further traction to support collective action and mobilisation of resources to preserve the integrity of the park and the biodiversity within it. The fortification of these bilateral and multilateral relationships will be vital in order to mainstream and finance conservation actions across sectoral policies, contributing to sustainable energy production, poverty reduction, education, health etc., thus underpinning a coordinated strategy providing political and economic governance while increasing human capacity and wellbeing. While potentially contributing to realise the majority of the SDG’s, development and revitalisation of global partnerships to strengthen the implementation of the SDG’s is the overall objective of SDG 17 (Partnerships for the Goals). 6. Conclusions The Virunga catchment in the eastern part of the Democratic Republic of the Congo is subject to dramatic deforestation rates and land grabbing, causing significant changes to the land cover dynamics in one of the most biodiverse regions of Africa. In order to inform conservation actions and management practices to protect the diversity and integrity of the Virunga NP, while developing sustainable land managing policies and socioeconomic reforms it is vital to understand the drivers and dynamics of LULC changes. This study was successfully able to use a combination of cloud processing platforms (Google Earth Engine), GIS software (ArcGIS) and LULC modelling tools (LCM in TerrSet) to simulate future deforestation and land change patterns in the Virunga catchment. It provides a good understanding of Sustainability 2020, 12, 1570 25 of 28 25 of 28 the predicted LULC changes, under a business-as-usual scenario, over the next ten years, and thus presents an effective policy support tool for decision makers and administrative bodies aiming to strengthen SDG implementation while preserving park resources. The LULC model predicted that the largest shift between classes is attributed with the conversion of forest areas into cropland and the overall general trend is a significant increase in cropland with a net gain of more than 2000 km2. The increase in cropland is primarily located in the north of the Virunga catchment where a substantial proportion of the remaining forest areas is predicted to be replaced by cropland. The primary drivers of deforestation were identified as distance to artisanal mines and mining concessions and distance to cropland and cities, distance to roads and distance to water. These drivers all reflect the inherent relationship between accessibility to forested areas and proximity to human activities, which is consistent with literature and consistent with the hypothesis that charcoal production and land clearing for mining, urban expansion and subsistence agriculture are the primary contributors to deforestation within the Virunga NP. Author Contributions: M.C. conducted the study, drafted and revised the paper. J.J.A. co-designed the paper and supervised the paper. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. Source code for the 2019 land cover classification in Google Earth Engine Source code for the 2019 land cover classification in Google Earth Engine Map.centerObject(AOI, 9); Map.addLayer(AOI, {}, ’aoi’); //For Landsat surface reflectance product cloud masking function maskclouds(image) { var cloudShadowBitMask = 1 << 3; // cloud shadow var cloudsBitMask = 1 << 5; // cloud var qa = image.select(’pixel_qa’); var date = image.get(’system:time_start’); var mask = qa.bitwiseAnd(cloudShadowBitMask).eq(0) .and(qa.bitwiseAnd(cloudsBitMask).eq(0)); var ndvi = image.normalizedDifference([’B5’,’B4’]).multiply(10000).rename(’NDVI’); return image.addBands(ndvi).updateMask(mask).divide(10000).set(’system:time_start’,date); } //Landsat 8 image collection var L8collection = ee.ImageCollection(’LANDSAT/LC08/C01/T1_SR’) .filterDate(’2017-01-01’, ’2019-03-23’) .filterBounds(AOI) .filter(ee.Filter.lt(’CLOUD_COVER’, 35)) .map(maskclouds); print (L8collection); var testimage = L8collection.median().clip(AOI); Map.addLayer(testimage.select([’B5’, ’B4’, ’B3’]), {min:0, max:0.4}, ’false color’); Map.addLayer(testimage.select([’B4’, ’B3’, ’B2’]), {min:0, max:0.4}, ’true color color’); // Subsample training polygons with random points // this ensures all classes have same sample size // also EE can’t handle too many cells at once var trainingLayers = [forest, water, city, cropland, openland2]; var n = 500; // loop over training layers Source code for the 2019 land cover classification in Google Earth Engine Map.centerObject(AOI, 9); Map.addLayer(AOI, {}, ’aoi’); //For Landsat surface reflectance product cloud masking function maskclouds(image) { var cloudShadowBitMask = 1 << 3; // cloud shadow var cloudsBitMask = 1 << 5; // cloud var qa = image.select(’pixel_qa’); var date = image.get(’system:time_start’); var mask = qa.bitwiseAnd(cloudShadowBitMask).eq(0) .and(qa.bitwiseAnd(cloudsBitMask).eq(0)); var ndvi = image.normalizedDifference([’B5’,’B4’]).multiply(10000).rename(’NDVI’); return image.addBands(ndvi).updateMask(mask).divide(10000).set(’system:time_start’,date); } //Landsat 8 image collection var L8collection = ee.ImageCollection(’LANDSAT/LC08/C01/T1_SR’) .filterDate(’2017-01-01’, ’2019-03-23’) .filterBounds(AOI) .filter(ee.Filter.lt(’CLOUD_COVER’, 35)) .map(maskclouds); print (L8collection); var testimage = L8collection.median().clip(AOI); Map.addLayer(testimage.select([’B5’, ’B4’, ’B3’]), {min:0, max:0.4}, ’false color’); Map.addLayer(testimage.select([’B4’, ’B3’, ’B2’]), {min:0, max:0.4}, ’true color color’); // Subsample training polygons with random points // this ensures all classes have same sample size // also EE can’t handle too many cells at once var trainingLayers = [forest, water, city, cropland, openland2]; var n = 500; // loop over training layers Sustainability 2020, 12, 1570 26 of 28 for (var i = 0; i < trainingLayers.length; i++) { // sample points within training polygons var pts = ee.FeatureCollection .randomPoints(trainingLayers[i].geometry(), n); // add class var thisClass = trainingLayers[i].get(’class’); pts = pts.map(function(f) { return f.set({class: thisClass}); }); // extract raster cell values var training = testimage.sampleRegions(pts, [’class’], 30); // combine trainging regions together if (i === 0) { var trainingData = training; } else { trainingData = trainingData.merge(training); } } print (trainingData); //// classify with random forests // use bands 1-7+NDVI var bands = [’B1’, ’B2’, ’B3’, ’B4’, ’B5’,’B6’, ’B7’, ’NDVI’]; // fit a random forests model var classifier = ee.Classifier.randomForest(500) .train(trainingData, ’class’, bands); // produce the land cover map var classified = testimage.classify(classifier); var p = [’00ff00’, ’ff0000’, ’000000’, ’0000ff’, ’orange’,]; // display Map.addLayer(classified, {palette: p, min: 1, max: 5}, ’classification’); //Accuracy assessment //Test the classifiers’ accuracy. Source code for the 2019 land cover classification in Google Earth Engine Export.image.toDrive({ image: classified, description: ’VirungaLC_2018’, scale: 30, folder: ’Master thesis’, region: AOI.geometry().bounds(), maxPixels: 2091108075, crs:’EPSG:4051’ }); Source code for the 2019 land cover classification in Google Earth Engine (data, y, X) var trainingClassifier = classifier.train(training, ’class’, bands); //Separate validation var testingsep = forestvali.merge(watervali).merge(cityvali).merge(croplandvali).merge(openlandvali); // Add reducer output to the Features in the collection. testingsep = testimage.sampleRegions(testingsep, [’class’], 30); //print (testingsep) var validation_sep = testingsep.classify(trainingClassifier); //print (validation_sep) var errorMatrix_sep = validation_sep.errorMatrix(’class’, ’classification’); //print(’Error Matrix:’, errorMatrix_sep); var ft = ee.FeatureCollection([ee.Feature(null, {’Accuracy’: errorMatrix_sep.accuracy(), ’Producer Accuracy’:errorMatrix_sep.producersAccuracy(), ’User Accuracy’:errorMatrix_sep.consumersAccuracy(), ’Kappa’: errorMatrix_sep.kappa(), ’Error Matrix’:errorMatrix_sep.array()})]); for (var i = 0; i < trainingLayers.length; i++) { // sample points within training polygons var pts = ee.FeatureCollection .randomPoints(trainingLayers[i].geometry(), n); // add class var thisClass = trainingLayers[i].get(’class’); pts = pts.map(function(f) { return f.set({class: thisClass}); }); // extract raster cell values var training = testimage.sampleRegions(pts, [’class’], 30); // combine trainging regions together if (i === 0) { var trainingData = training; } else { trainingData = trainingData.merge(training); } } print (trainingData); //// classify with random forests // use bands 1-7+NDVI var bands = [’B1’, ’B2’, ’B3’, ’B4’, ’B5’,’B6’, ’B7’, ’NDVI’]; // fit a random forests model var classifier = ee.Classifier.randomForest(500) .train(trainingData, ’class’, bands); // produce the land cover map var classified = testimage.classify(classifier); var p = [’00ff00’, ’ff0000’, ’000000’, ’0000ff’, ’orange’,]; // display Map.addLayer(classified, {palette: p, min: 1, max: 5}, ’classification’); //Accuracy assessment //Test the classifiers’ accuracy. (data, y, X) var trainingClassifier = classifier.train(training, ’class’, bands); //Separate validation var testingsep = forestvali.merge(watervali).merge(cityvali).merge(cr // Add reducer output to the Features in the collection. testingsep = testimage.sampleRegions(testingsep, [’class’], 30); //print (testingsep) var validation_sep = testingsep.classify(trainingClassifier); //print (validation_sep) var errorMatrix_sep = validation_sep.errorMatrix(’class’, ’classificatio //print(’Error Matrix:’, errorMatrix_sep); var ft = ee.FeatureCollection([ee.Feature(null, {’Accuracy’: errorMatri Accuracy’:errorMatrix_sep.producersAccuracy(), ’User Accuracy’:erro ’Kappa’: errorMatrix_sep.kappa(), ’Error Matrix’:errorMatrix_sep.arra 27 of 28 27 of 28 Sustainability 2020, 12, 1570 // Define customization options. var options = { title: ’Landsat 8’, hAxis: {title: ’Wavelength (micrometers)’}, vAxis: {title: ’Reflectance’}, lineWidth: 1, pointSize: 4, series: { 0: {color: ’00FF00’}, // forest 1: {color: ’0000FF’}, // water 2: {color: ’FF0000’}, // city 3: {color: ’orange’}, // openland1 4: {color: ’grey’}, // cropland 5: {color: ’yellow’}, // openland2 }}; // Define a list of Landsat 8 wavelengths for X-axis labels. var wavelengths = [0.44, 0.48, 0.56, 0.65, 0.86, 1.61, 2.2, 2.5]; // Create the chart and set options. var spectraChart = ui.Chart.image.regions( testimage.select(bands), trainingLayers, ee.Reducer.mean(), 30, ’class’, wavelengths) .setChartType(’ScatterChart’) .setOptions(options); // Display the chart. print(spectraChart); Export.table.toDrive({collection: ft, description: ’accu_2018’, fileNamePrefix: ’accu_2018’, folder: ’Master thesis’, selectors: [’User Accuracy’, ’Producer Accuracy’, ’Accuracy’,’Kappa’, ’Error Matrix’]}); // Export the image, specifying scale and region. 4. Rainer, H.; Lanjouw, A.; Kayitare, A.; Rutagarama, E.; Sivha, M.; Asuma, S.; Kalpers, J. Beyond Boundaries: Transboundary Natural Resource Management for Mountain Gorillas in the Virunga-Bwindi Region; Biodiversity Support Program: Washington, DC, USA, 2001. References 1. Andersen, F. Virunga National Park, the heart of darkness as UNESCO World Heritage. Cont. Manuscr. 2018, 11 1–15 [CrossRef] 1. Andersen, F. Virunga National Park, the heart of darkness as UNESCO World Heritage. Cont. Manuscr. 2018, 11, 1–15. [CrossRef] 2. Kayijamahe, E. Spatial Modelling of Mountain Gorilla (Gorilla Beringei Beringei) Habitat Suitability and Human Impact Virunga Volcanoes Mountains, Rwanda, Uganda and Democratic Republic of Congo; International Institute for Geo-information Science and Earth Observation: Enschede, The Netherlands, 2008. 2. Kayijamahe, E. Spatial Modelling of Mountain Gorilla (Gorilla Beringei Beringei) Habitat Suitability and Human Impact Virunga Volcanoes Mountains, Rwanda, Uganda and Democratic Republic of Congo; International Institute for Geo-information Science and Earth Observation: Enschede, The Netherlands, 2008. 3. UNESCO. Virunga National Park. 2019. Available online: https://whc.unesco.org/en/list/63/ (accessed on 9 May 2019). 4. Rainer, H.; Lanjouw, A.; Kayitare, A.; Rutagarama, E.; Sivha, M.; Asuma, S.; Kalpers, J. Beyond Boundaries: Transboundary Natural Resource Management for Mountain Gorillas in the Virunga-Bwindi Region; Biodiversity Support Program: Washington, DC, USA, 2001. 4. Rainer, H.; Lanjouw, A.; Kayitare, A.; Rutagarama, E.; Sivha, M.; Asuma, S.; Kalpers, J. Beyond Boundaries: Transboundary Natural Resource Management for Mountain Gorillas in the Virunga-Bwindi Region; Biodiversity Support Program: Washington, DC, USA, 2001. 28 of 28 Sustainability 2020, 12, 1570 28 of 28 5. UNESCO. Virunga National Park (Democratic Republic of the Congo). 2018. Available online: https: //whc.unesco.org/en/soc/3815 (accessed on 9 May 2019). 6. Yee, A. In Africa’s Oldest Park, Seeking Solutions to A Destructive Charcoal Trade; YaleEnvironment360: New Haven, CT, USA, 14 September 2017. 7. Liping, C.; Yujun, S.; Saeed, S. Monitoring and predicting land use and land cover changes using remote sensing and GIS techniques-A case study of a hilly area, Jiangle, China. PLoS ONE 2018, 13, e0200493. [CrossRef] 8. National Research Council. Advancing Land Change Modeling: Opportunities and Research Requirements; National Academies Press: Washington, DC, USA, 2014. [CrossRef] 9. Shade, C.; Kremer, P. Predicting land use changes in philadelphia following green infrastructure policies. Land 2019, 8, 28. [CrossRef] 10. Gibson, L.; Münch, Z.; Palmer, A.; Mantel, S. Future land cover change scenarios in South African grasslands—Implications of altered biophysical drivers on land management. Heliyon 2018, 4, e00693. [CrossRef] [PubMed] 11. Guerrero, G.; Masera, O.; Mas, J.-F. Land use/Land cover change dynamics in the Mexican highlands: Current situation and long term scenarios. In Modelling Environmental Dynamics; Springer: Berlin/Heidelberg, Germany, 2008; pp. 57–76. [CrossRef] 12. References Jokar Arsanjani, J.; Helbich, M.; Kainz, W.; Bloorani, A.D.; Darvishi Boloorani, A. Integration of logistic regression, Markov chain and cellular automata models to simulate urban expansion. Int. J. Appl. Earth Obs. Geoinf. 2013, 21, 265–275. [CrossRef] 13. Eastman, R.J. TerrSet Geospatial Monitoring and Modeling System—Manual; Clark University: Worchester, MA, USA, 2016. 14. Pérez-Vega, A.; Mas, J.-F.; Ligmann-Zielinska, A. Comparing two approaches to land use/cover change modeling and their implications for the assessment of biodiversity loss in a deciduous tropical forest. Environ. Model. Softw. 2012, 29, 11–23. [CrossRef] 15. Allan, R.; Förstner, U.; Salomons, W.; Paegelow, M.; Olmedo, M.T.C. Model Environmental Dynamics: Advances in Geomatic Solutions; Springer: Berlin/Heidelberg, Germany, 2008. 6. Eastman, R.J. Terrset Geospatial Monitoring and Modelling System—Tutorial; Clark University: Worchester, USA, 2016. 17. Mirici, M.E. Land use/cover change modelling in a mediterranean rural landscape using multi-layer perceptron and markov chain (mlp-mc). Appl. Ecol. Environ. Res. 2018, 16, 467–486. [CrossRef] 18. Institute for Environmental Security. Mining, Forest Change and Conflict in the Kivus, Eastern Democratic Republic of Congo—Outcome of a Short Study within the IES-ESPA Programme; Institute for Environmental Security: The Hague, The Netherlands, 2008. 19. Yang, Z.R. Multi-layer perceptron. In Machine Learning Approaches to Bioinformatics; Yang, Z.R., Ed.; World Scientific: Singapore, 2010; pp. 133–153. [CrossRef] 20. Central Intelligence Agency. The World Factbook 2020; Central Intelligence Agency: Washington, DC, USA, 2020. 21. Zadbagher, E.; Becek, K. Modeling land use/land cover change using remote sensing and geographic information systems: Case study of the Seyhan. Environ. Monit. Assess. 2018, 190, 494. [PubMed] 22. Jones, B. Deforestation surges in Virunga National Park in the wake of violence. Mongabay. 2018. Available online: https://news.mongabay.com/2018/10/deforestation-surges-in-virunga-national-park-in-the-wake- of-violence/ (accessed on 17 February 2020). 23. World Wildlife Fund (WWF). The Economic Value Of Virunga National Park Report; WWF: Grand, Switzerland, 2013. 24. Crawford, A.; Bernstein, J. MEAs, Conservation, and Conflict—A Case Study of Virunga National Park, DRC; International Institute for Sustainable Development: Winnipeg, MB, Canada, 2008; Volume 1. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. 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Effects of Non-Driving Related Tasks During Self-Driving Mode
IEEE transactions on intelligent transportation systems
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Effects of Non-Driving Related Tasks During Self-Driving Mode Saad Minhas , Student Member, IEEE, Aura Hernández-Sabaté , Shoaib Ehsan , Senior Member, IEEE, and Klaus D. McDonald-Maier , Senior Member, IEEE Abstract—Perception reaction time and mental workload have proven to be crucial in manual driving. Moreover, in highly automated cars, where most of the research is focusing on Level 4 Autonomous driving, take-over performance is also a key factor when taking road safety into account. This study aims to investigate how the immersion in non-driving related tasks affects the take-over performance of drivers in given scenarios. The paper also highlights the use of virtual simulators to gather efficient data that can be crucial in easing the transition between manual and autonomous driving scenarios. The use of Computer Aided Simulations is of absolute importance in this day and age since the automotive industry is rapidly moving towards Autonomous technology. An experiment comprising of 40 subjects was performed to examine the reaction times of driver and the influence of other variables in the success of take-over performance in highly automated driving under different cir- cumstances within a highway virtual environment. The results reflect the relationship between reaction times under different scenarios that the drivers might face under the circumstances stated above as well as the importance of variables such as velocity in the success on regaining car control after automated driving. The implications of the results acquired are important for understanding the criteria needed for designing Human Machine Interfaces specifically aimed towards automated driving conditions. Understanding the need to keep drivers in the loop during automation, whilst allowing drivers to safely engage in other non-driving related tasks is an important research area which can be aided by the proposed study. The development today reflects that there is still quite a long way to go in achieving fully autonomous driving under all road environments as well as weather conditions [1]. The SAE International standard J3016 [2] defines six levels of car automation, from totally manual (level 0) until totally automated (level 5). Far from being at level 5, most current research efforts focus on level 3 and level 4 (like autonomous minibuses CabiBus [3]). These levels include situations in which the car handles all the dynamic driving aspects except the driver taking back control when required (conditionally automated) or in some driving modes. Saad Minhas, Shoaib Ehsan, and Klaus D. McDonald-Maier are with the School of Computer Science and Electronic Engineering, Essex University, Colchester CO4 3SQ, U.K. (e-mail: ssminh@essex.ac.uk; sehsan@essex.ac.uk; kdm@essex.ac.uk). 1391 1391 IEEE TRANSACTIONS ON INTELLIGENT TRANSPORTATION SYSTEMS, VOL. 23, NO. 2, FEBRUARY 2022 Manuscript received September 21, 2019; revised March 14, 2020 and August 1, 2020; accepted September 8, 2020. Date of publication October 1, 2020; date of current version February 2, 2022. This work was supported by the Engineering and Physical Sciences Research Council (EPSRC). The Associate Editor for this article was D. F. Wolf. (Corresponding author: Saad Minhas.) Aura Hernández-Sabaté is with the Computer Science Department, Univer- sitat Autònoma de Barcelona, 08193 Barcelona, Spain, and also with the Com- puter Vision Center, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain (e-mail: aura@cvc.uab.cat). This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecom Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ Effects of Non-Driving Related Tasks During Self-Driving Mode This, in turn, frees up the driver to indulge in other Non-Driving Related tasks (NDR-tasks) while the car is autonomously driving, although the driver must be able to regain the control as soon as the system requires it. At this point, it is necessary to have a comprehensive knowledge under which circumstances the system should require the driver to regain control of the car, taking into account the system boundary and the situational awareness and capabilities of the driver. Perception Reaction Time (PRT) and mental workload have proven to be crucial in manual driving [4]–[6]. Meanwhile, in highly automated cars, Take-Over Performance (TOP) is an addition variable to take into account for road safety [7]. In these cases, the mental workload is closely related to immersing the driver in NDR-tasks (s)he is performing while the car is driving autonomously [8]. Previous studies found impairments in take-over performance while engaged in NDR-tasks, but little is known about the impact of specific task characteristics [9], [10]. Index Terms—Mental work capacity, human–computer inter- action, vehicle ergonomics, perception, virtual environment. Manuscript received September 21, 2019; revised March 14, 2020 and August 1, 2020; accepted September 8, 2020. Date of publication October 1, 2020; date of current version February 2, 2022. This work was supported by the Engineering and Physical Sciences Research Council (EPSRC). The Associate Editor for this article was D. F. Wolf. (Corresponding author: Saad Minhas.) Saad Minhas, Shoaib Ehsan, and Klaus D. McDonald-Maier are with the School of Computer Science and Electronic Engineering, Essex University, Colchester CO4 3SQ, U.K. (e-mail: ssminh@essex.ac.uk; sehsan@essex.ac.uk; kdm@essex.ac.uk). Aura Hernández-Sabaté is with the Computer Science Department, Univer- sitat Autònoma de Barcelona, 08193 Barcelona, Spain, and also with the Com- puter Vision Center, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain (e-mail: aura@cvc.uab.cat). Digital Object Identifier 10.1109/TITS.2020.3025542 Digital Object Identifier 10.1109/TITS.2020.3025542 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ A. Perception Reaction Time A. Perception Reaction Time PRT of human drivers is an active research area within the manual driving performance domain because it plays a central role in different road incidents. Several studies have been carried out to deepen the com- prehension of PRT’s role in crash risks [4], [11]–[14]. The methodologies range from proposing accident situations involving surprise factor to examine the reaction times of drivers and also analyzing reaction times as a factor to take into account within crash surrogate indicators. The main results found in these studies are that the reaction time of drivers seem to be approximately a linear function of Time To Collision (TTC), and the mean reaction time and inter-individual variability progressively increases with age although some other factors such as driver gender, cognitive load, and urgency might influence in human perception-brake reaction time. However, the most influential factor is driver expectation. Besides, [29] suggests that a take-over request with lead time at 10–60 s led to lower crash rate, greater mini- mum TTC, and lower lateral acceleration. However, both stud- ies do not account for critical control transitions. Some other experiments [9], [30] exposed drivers to critical take-over situations and showed evidence that cognitive load on its own might not influence takeover time but have effects on the takeover quality. As well, reaction times might be in line with the driver’s perception of emergency. All of the above studies were conducted under manual driving conditions, so they do not take into account the PRT when the driver is carrying NDR-tasks. I. INTRODUCTION T T HE automotive industry is moving at a rapid phase towards autonomous vehicles. This involves the cars to make complex judgments depending upon the road scenarios in hand. Several big-name companies such as Waymo, Tesla, and Uber are pushing immense resources into achieving the most efficient autonomous vehicle technology on the market. This study aims to explore how the immersion in NDR-tasks affects the success of TOP of drivers in a highway critical situation and evaluate the influence of several variables, such as PRT, on this success. For that, 40 participants (mean age, 31 years old) were selected to evaluate their feet and hands reaction times under different circumstances in a sim- ulation environment. In particular, they had to avoid a crash after taking back the control of the car that previously was autonomously driving when an alarm was triggered due to the detection of an obstacle on the road in front of the car. The remaining article is structured as follows. Section II summarizes the state-of-the-art related with the paper. Section III is devoted the experiment, detailing the data set, the simulation environment, the procedure and the measures used for the study. Finally, section IV shows the results IEEE TRANSACTIONS ON INTELLIGENT TRANSPORTATION SYSTEMS, VOL. 23, NO. 2, FEBRUARY 2022 1392 obtained and section V discuss the results and betrays the further lines. obtained and section V discuss the results and betrays the further lines. assessing these variables under a controlled simulated autonomous car environment is quite crucial in exploring their effects further. II. RELATED WORK II. RELATED WORK The state of the art is described for the following three categories. A. Participants Forty participants (10 female, 30 male) between 19 and 45 years old (mean = 30.73, std = 7.086) were recruited. All of them held a valid driver’s license at the time of the experiment with a seniority of at least 1 year and at most 26 years (mean = 9.725, std = 7.66). Informed consent was obtained from each participant before the trials. C. Control Switching Last but not least, in highly automated cars, the process of getting the driver back into the loop is very important. In this fashion, some authors [25]–[27] explore different ways to get the driver back to the driving task in a safe manner, either focusing on signal modalities [25] or designing complete human-machine interfaces [26], [27]. But still, the automated system needs to know how far in advance and under which circumstances it has to warn the driver, depending on the NDR-tasks the driver is doing or can do, so that the analy- sis of drivers’ take-over performance is crucial. Concerning the lead time to safely allow the driver to regain control. Eriksson et al. [28] review several papers exploring driver control transitions, although they not take into account sec- ondary tasks, and carry on an experiment involving secondary tasks. On one hand, they claim that the reviewed results differ depending on the emergency the driver perceives (s)he has to cover on, and, on the other one, they find significant differences when drivers are engaged in secondary tasks. B. Mental Workload Other studies have been centred on the influence of mental workload as a crash risk factor [15]–[19]. Mental workload is not only related to being stressed, fatigued or drowsy but performing a divided-attention task causes an increase of mental workload and task demands can exceed the driver’s attention resources. These studies explore several indicators from many external sensors, such as pulse rate, skin electric potential activity or surface temperature, to better determine the physical and psychological state under different provoked circumstances. Although the general workload is not well defined psychometrically [20], all of them coincide that exces- sive (related to stress) or too low (related to vigilance) mental workload could derail the quality of driving [21]. In case of being behind the wheel of an autonomous car such as Tesla S [31], although the drivers were also told that they were responsible for the safe operations of the vehicle regardless of it’s driving mode, the recorded data demonstrated behaviour indicative of complacency and over-trust. Still, prospectively evaluate the expected limitations caused by NDR tasks on the driver’s ability to take control of an autonomous vehicle [32], more research is needed so that different aspects of NDR tasks can be translated into a modelling of a framework to predict takeover time or quality. This makes our present article more relevant as we explore in detail the different effects that the NDR tasks have on reaction times of drivers in an autonomous scenario. Besides, human performance can either deteriorate or improve depending on the degree of automation which is introduced in that particular environment [22], so that men- tal workload should also be taken into account in highly automated cars. Indeed, PRT and mental workload can be closely related [23], [24], since increasing workload of the driver reduces the driver’s ability to process information at different distances and thus deteriorates driving performances and increases reaction times. The first study shows that mental calculations increase the average reaction time for each age group, while the second one suggests that reaction times can increase by 40%-87% due to increased fatigue levels, giving valuable insight into how reaction times are taken into account via visual perception. III. EXPERIMENTS B. Simulated Environment and Scenarios All the experiments studying conditions of the drivers and involving crash accidents are dangerous to approach on real The above studies do appear to be invaluable in assess- ing the relationship between PRT and mental workload but MINHAS et al.: EFFECTS OF NON-DRIVING RELATED TASKS DURING SELF-DRIVING MODE MINHAS et al.: EFFECTS OF NON-DRIVING RELATED TASKS DURING SELF-DRIVING MODE 1393 Fig. 1. Driver simulator setup. Fig. 2. Simulator recorded data. Fig. 3. Road surface model. Fig. 4. Traffic cars. Fig. 1. Driver simulator setup. Fig. 3. Road surface model. Fig. 3. Road surface model. Fig. 4. Traffic cars. Fig. 5. Simulator interior model. Fig. 3. Road surface model. Fig. 1. Driver simulator setup. Fig. 1. Driver simulator setup. Fig. 4. Traffic cars. Fig. 2. Simulator recorded data. Fig. 4. Traffic cars. Fig. 4. Traffic cars. Fig. Fig. 2. Simulator recorded data. Fig. 2. Simulator recorded data. Fig. 5. Simulator interior model. roads. Simulators are an effective tool in recognizing and assessing problematic driving scenarios, and hence can provide vital data that can be used to design more efficient Advanced Driver Assistance Systems (ADAS), from predicting driver steering behaviours [33] to evaluating the mental workload of the driver [19], [34]. Our simulated environment is an efficient and inexpensive real looking driving environment that immerses the driver into driving on the highway [35]. The assets required are fairly standard and upgrade-able, which makes the setup quite extendable. The setup consists of one camera facing the driver straight on for capturing the important aspects of driver’s facial features, a second camera looking towards the steering and a third one recording precisely the driver’s feet placement. Consequently, the three cameras can generate a detailed picture of exactly what the driver is doing at any given instance in time during a trial. Figure 1 shows the general simulator setup. Fig. 5. Simulator interior model. the curved road section. These two sections were then used in such a pattern that would result in a continuous looping road environment. By doing a looping road environment we achieved a never-ending motorway section that could be used for as long as an experiment is required. The road section environment is roughly 4 miles long and proves to be a base for experimenting in the virtual world. Besides the road, basic tree models were then placed to simulate nature. B. Simulated Environment and Scenarios The system records several variables (reaction times of hands and feet, distance to the front car at the alarm triggering and after a successful stop, and cruise distance of the car in automated mode) that are visualized on a primary screen as shown in Figure 2. The traffic cars as shown in Figure 4 are populated via a third-party plug-in called ITS (Intelligent Traffic System). This enables the cars to spawn at a random location every time a new trial is executed. The cars can overtake other cars and their speeds can be controlled depending on their placement on the road i.e. the cars on the fast lane can have a top speed ranging from 70-80 mph, cars in the middle lane can range from 60-70 mph and cars in the third lane can range from 50-60 mph. Moreover, the traffic cars can keep a safe distance from the cars ahead, and when the front car stops due to an incident the other traffic cars respond accordingly. The software side consists of Unity3d, which is used for all the programming of the driver simulator, and other third-party softwares, like Autodesk 3dsmax and Adobe Photoshop, are used to generate the required 3d and 2d assets. This includes virtual cars and road surface assets. The road surface was modelled to reflect as close as possible the London Orbital Motorway. To target a modular approach towards the road environments only two road sections were produced, the first one being a straight section and the second one being a 25-degree curved road section. Figure 3 shows Figure 5 shows the virtual interior of the driver car. All the useful elements of the main virtual car were rigged, ranging IEEE TRANSACTIONS ON INTELLIGENT TRANSPORTATION SYSTEMS, VOL. 23, NO. 2, FEBRUARY 2022 1394 Fig. 6. Simulator proximity model. autonomous car. Following a previous experiment performed by Eriksson et al. [36], in which he used a newspaper reading scenario while the car was in autonomous mode, the challenge was to enable the driver to lose focus on the road ahead while engaging in secondary tasks inside the car. Secondary tasks were selected in such a way that they resembled real-life situations as closely as possible. Hence keeping that in mind, three different scenarios for the driver were posed while the car was in autonomous driving mode. B. Simulated Environment and Scenarios The first scenario, henceforth Default, is termed as the base scenario in which the driver was aware to the road with the hands and feet ready to react (hands on the wheel but without touching them and feet on a marked place very close to the pedals). The second scenario, henceforth Social, deals with the fact that the driver was not paying attention down the road, but was freely immersed in social media activities on her/his smartphone. Since immersion to social media could be total and the system is automatically driving, to suppress the variable off-road glance time, the driver was not allowed to look at the road. In the third scenario, namely Immersive Question and Answers (IMQA), the driver was answering a list of questions via a smartphone. These were relatively basic questions consisting of the driver’s name, age, hobbies, etc and more cognitive loaded ones such as some basic mathematical addition and subtraction questions. Strict guidelines were provided to the drivers so that they won’t even peek at the road ahead while the car was driving automatically and the driver is indulging in secondary tasks (second and third scenarios), the penalty for which was to start the trial again if the driver looked at the road during specific scenarios. Fig. 6. Simulator proximity model. from the RPM needle in the dashboard to the brake and accelerator pedals underneath. The setup consists of both a hardware steering wheel as well as a virtual steering wheel. The visibility of the virtual steering wheel behaves like a bridge between the virtual and the real-world counterpart. Apart from providing visual feedback, the virtual steering also acts as a somewhat intrusive figure further simulating the real-world steering position. The input hardware was rigged accordingly to respond to the slightest movement from the driver. The rear-view mirrors were also rigged up to properly simulate the mirror functionality. Besides, a fully functional adaptive cruise control was rigged, which allowed the driver car to keep its lane on the motorway and also allows the car to maintain a specific distance from the car ahead. The Adaptive cruise control is split up into two proximity zones, namely Primary Proximity zone and Secondary Proximity Zone as shown in the figure 6. C. Simulated Situation The simulated situation consisted of an infinite three-lane motorway of 4 miles loop, as explained in the subsection above. The car was driving in autonomous mode and, sud- denly, the vehicle detected an invisible obstacle, an alarm was triggered and all the cars in front of it stopped. The detection time for obstacles randomly varies from trial to trial but usually, it happens between 2-5 minutes during a trial. At that moment, all the cars in front stopped and the driver had to take over the control of the car in order to avoid a collision. The average distance and the standard deviation of the main car to the car in front at the time the alarm was triggered was 40.64 ± 13.34 meters and the velocity of the car was 45.2 ± 10.97 mph. The traffic can be turned ON on both sides of the road, but this was not a requirement for the current experimental setup. Moreover, the drivers had the instruction of only braking or dodging if needed, depending on the situation. B. Simulated Environment and Scenarios The Primary Proximity Zone forces the autonomous car to maintain an average speed cruise control resulting in approx 70MPH speed whereas the Secondary Proximity Zone forces the autonomous car to brake and increase the distance to the car in front. Each driver performed the experiment four times in each scenario under different circumstances, that is a combina- tion of enabling to dodge or not and a threshold on the initial distance to the car in front during the autonomous driving (the approximate distance that the car would keep from the car in front in autonomous mode). Thus, there are a total of 12 trials that were carried out on each subject, although we group the four trials per driver and scenario as 4 different samples. There are a number of variables that are being recorded: Hands/feet PRT, cruise distance threshold to the front car, distance to the front car at which the alarm is triggered, final distance to the front car after the car comes to a stop, the speed at which the alarm is triggered, any traffic cars to the left and right side of the driver’s car, crashed or not, and if the driver tried to dodge and save himself. The experiment also includes other basic variables like the age and gender of the driver and the seniority of his/her license. The autonomous driving model uses a basic path follow algorithm with an obstacle avoidance system simulating the Lidar component. As the study was based mostly on the driver’s perception there was little to no need for a complex autonomous model for this experiment. E. Objective Measures During the experiment, we recorded several objective vari- ables to be explored under different scenarios. where μi, i = 1, 2, 3 corresponds to the mean of the objective variable for each scenario. 1) PRT: We understand the PRT as the time that elapses from the instant that the driver recognizes the existence of a hazard on the road to the instant that the driver takes appropriate action. The hazard can result from traffic cars in front of the driver’s car suddenly stopping and queuing up, which would alert the driver of a possible hazard. In this case, the hazard recognition is perceived thanks to an acoustic alarm. Since we do not have a tool to measure the time from when the alarm is triggered until the driver perceives it and the time elapsed from when the driver perceives it until (s)he acts, we can only measure the elapsed time from the moment the alarm is triggered until the moment when the driver reacts. 1) PRT: We understand the PRT as the time that elapses from the instant that the driver recognizes the existence of a hazard on the road to the instant that the driver takes appropriate action. The hazard can result from traffic cars in front of the driver’s car suddenly stopping and queuing up, which would alert the driver of a possible hazard. In this case, the hazard recognition is perceived thanks to an acoustic alarm. Since we do not have a tool to measure the time from when the alarm is triggered until the driver perceives it and the time elapsed from when the driver perceives it until (s)he acts, we can only measure the elapsed time from the moment the alarm is triggered until the moment when the driver reacts. A requirement for applying an ANOVA is that data is normally distributed, which can be contrasted by means of a Kolmogorov–Smirnov test. In particular, the Lilliefors test is a normality test based on the Kolmogorov–Smirnov one that compares the empirical distribution of the data with a normal distribution without any expected value and variance of the distribution [37]. In case the data does not follow a normal distribution we can use a non-parametric statistical test, instead of a parametric one, which analyzes differences among group medians instead of means. D. Experimental Design After a brief introduction to the driver simulator, the par- ticipants were given a trial run with the autonomous mode disabled just so that they could get the feel and sensitivity of the steering wheel as well as the pedals. Afterwards, the autonomous mode was turned on and the participants were told to take back control as soon as the audio/visual alarm was triggered by the simulator. Moreover, the participants were also instructed to place their hands and feet in a neutral To carry out the experiment, a series of plausible scenarios were needed that a driver would find behind the wheel of an MINHAS et al.: EFFECTS OF NON-DRIVING RELATED TASKS DURING SELF-DRIVING MODE 1395 can be analyzed across the scenarios to explore if they are affected by the current scenario. position during this time. This gave them the initial confidence in tackling the simulated event and the baseline scenario (Default). For the next scenario, Social Media, the participants were briefed to indulge themselves in social media activities on their smartphones while behind the wheel of the virtual autonomous car. For the Immersive Question and Answers scenario, the participants were only given a short briefing regarding the type of questions that would be asked for this part of the experiment. All the data recorded during the trials was used in an anonymous form. To decide if PRTH and PRT are affected by the scenario, a one-way ANOVA should be computed for each variable. This test is usually used to detect significant differences between the distributions of more than two factors (in this case the different scenarios). That is, its hypothesis test associated considers as null hypothesis H0 meaning the factor has no effect, and as an alternative that it does. In terms of parameters, the ANOVA test can be written as follows:  H0 : μ1 = μ2 = μ3 H1 : ∃μi s.t. μi ̸= μ j for some j = 1, . . ., 3 IV. RESULTS For each variable recorded there is a total of 480 sam- ples (40 participants x 4 trials x 3 scenarios). In all tests, the significance level is 0.05. As well, none of the variables follow a normal distribution because the null hypothesis of the Lilliefors test is rejected with a p-value less than 0.05. E. Objective Measures In this case, the results are categorized as small effect = [0.10, 0.30), medium effect = [0.30, 0.50) and large effect = [0.50, 1]. b) PRT of feet (PRTF): The appropriate action for feet is braking, so that, the system records the reaction time as soon as it detects pressure on the brake pedal. 2) Velocity of the car at the time the alarm is triggered. 3) Distance of the car to the one in front at the time the alarm is triggered. 4) Success of TOP: We consider a success when the car does not crash. To answer how the immersion in NDR-tasks affects the TOP of drivers we analyze the PRTH and PRTF as soon as the alarm is triggered across different scenarios. As well, we explore the relationship between the velocity and distance at the moment the alarm is triggered. E. Objective Measures In particular, since each subject repeats the test for all scenarios in our experimental design, we can consider a repeated measure one way ANOVA, so that we use a Friedman test [38]. [ ] To measure the strength of agreement between subjects (effect size) we also compute Kendall’s W, defined as W = χ2/N(k −1), where χ2 is the test statistic, N the number of samples (160) and k the number of scenarios (3). The results can be categorized as small, medium and large, which in our case will be [0, 0.10), [0.10, 0.30) and [0.30, 1], respectively. To analyze the influence of an objective variable on the TOP success in each scenario, we also need to compute a non-parametric test in case the data does not follow a normal distribution. In this case the Wilcoxon rank-sum test [39] is an alternative to the Student’s t-test for independent (unpaired) samples and the effect size is computed as r = ∥Z/N0.5∥, where Z is the Z-statistic, and N is the number of par- ticipants. In this case, the results are categorized as small effect = [0.10, 0.30), medium effect = [0.30, 0.50) and large effect = [0.50, 1]. a) PRT of hands (PRTH): The appropriate action for hands is steering the wheel. The system only records the reaction times of the steering once it receives at least 1 degree of input in either direction from the driver. Notice that the alarm is only triggered during straight road sections, so that normal steering inputs on a curve are absent. To measure the strength of agreement between subjects (effect size) we also compute Kendall’s W, defined as W = χ2/N(k −1), where χ2 is the test statistic, N the number of samples (160) and k the number of scenarios (3). The results can be categorized as small, medium and large, which in our case will be [0, 0.10), [0.10, 0.30) and [0.30, 1], respectively. To analyze the influence of an objective variable on the TOP success in each scenario, we also need to compute a non-parametric test in case the data does not follow a normal distribution. In this case the Wilcoxon rank-sum test [39] is an alternative to the Student’s t-test for independent (unpaired) samples and the effect size is computed as r = ∥Z/N0.5∥, where Z is the Z-statistic, and N is the number of par- ticipants. F. Statistical Analysis The results of the test verify the above visual observation with a global p-value of 6.2954e-33, ensuring the significant difference between PRT for hands and feet. This is due to the fact that drivers, when suddenly encountering an obstacle, tend to prioritize to use the brake pedal before putting any input into the steering wheel hence resulting in the above higher reaction times for hands. Still, another interesting point is the success of the TOP action by itself, which is reflected in table II. We can appreci- ate a slight peak of failures in the Social scenario, although it is not significant (Pearson’s chi-squared test [42]: χ2 = 3.2881, p −value = 0.193 at significant level of 0.05). There are no significant differences in PRT among Social and IMQA scenarios either, but probably social activities provokes a deepen immersion than questions and answers, so that there are more crashes. If we separate by gender, we can observe that the peak in social scenario remains, as table III suggest. As well, we can notice that females have less crashes than male, although these data could be biased since the number of females is 1 3 than males. Still, the global average reaction time was 3.51 seconds for hands and 2.47 seconds for feet which coincides with the minimum amount of time described in [40] in which drivers can take over the control of vehicle safely and comfortably in this situation. The results of Friedman test for PRTH show that there are no significant effects among the 3 scenarios, χ2(2, N = 160) = 3.6904, p = 0.1580, W = 0.0115, although the strength of agreement among drivers is very small. That means that drivers take more or less the same time to steer the wheel in both scenarios. However, in the case of feet there are significant differences between at least one of the scenarios, with a medium strength of agreement: χ2(2, N = 160) = 44.3974, p = 2.2868e −10, W = 0.1387. To know what scenario is different from the rest, a multiple comparison test has been computed using the output of the Friedman test and shown in Figure 8. Two means are significantly different if their intervals are disjoint, and are not significantly different if their intervals overlap, so that the significant difference in PRTF is on the default scenario against the other two. F. Statistical Analysis To have a visual idea of the distributions of PRT on each scenario, figure 7 shows the boxplots of the 3 global scenarios (Default, Social and IMQA) for PRT of hands and feet. The first observation is that the hands reaction times are higher as compared to their feet counterpart in all scenarios. Outliers, in this case are reflecting the non-normality of the data. One plausible explanation about this non-normality is that there According to the objective variables explained in the above subsection notice that success of TOP can be considered as a dependent variable, while the remaining ones are indepen- dent, so that we analyze how such independent variables can influence on the success of TOP. Also, since the scenario can influence on the performance of the driver, PRTH and PRTF IEEE TRANSACTIONS ON INTELLIGENT TRANSPORTATION SYSTEMS, VOL. 23, NO. 2, FEBRUARY 2022 1396 Fig. 8. Multiple comparison test of PRTF among the three scenarios. Fig. 7. BoxPlots of PRTH and PRTF among the three scenarios. TABLE I MEAN ± STD, MEDIAN (IN SECONDS) AND p-VALUES FOR PRTH AND PRTF Fig. 8. Multiple comparison test of PRTF among the three scenarios. TABLE II PERCENTAGE OF SUCCESSES OF TOP AMONG THE 3 GLOBAL SCENARIOS TABLE III PERCENTAGE OF SUCCESSES OF TOP BY GENDER AMONG THE 3 GLOBAL SCENARIOS TABLE II PERCENTAGE OF SUCCESSES OF TOP AMONG THE 3 GLOBAL SCENARIOS were some instances where the drivers failed to input any motion within the steering or the feet resulting in a crash, the reaction times were recorded only after the user performed any input, this caused the system to record higher than normal reaction times, hence the outliers. The corresponding measures of central tendency are reported in Table I. For each scenario we report the ranges (mean ± std), medians and p-values of the Wilcoxon Signed-rank test, which is the same as Wilcoxon rank-sum test, but for paired data. that the alarm is triggered at the same time the cars in the front stop. In this way, as we pointed out before, the reaction time of hands is not relevant in any scenario, but drivers’ reaction time of feet might be slower in NDR-tasks scenarios due to their cognitive processing [41], and the driver’s lack of attention to the road. TABLE VII WILCOXON RANK-SUM TEST FOR THE RELATIONSHIP BETWEEN PRTF AND CRASHING TABLE VII WILCOXON RANK-SUM TEST FOR THE RELATIONSHIP BETWEEN PRTF AND CRASHING If we focus on the relationship between PRT and success of TOP we can observe that PRTH maintain the little rele- vance they already had across the scenarios. Table VI shows that p-values are much greater than the significance level. It makes sense in the light of the foregoing. On the contrary, PRTF seems to impact on the success of TOP, since there are significant differences between crashing or not in most of the scenarios, with a medium effect size. In the case of Social scenario, the null hypothesis can not be rejected. but the p-value is very close to the significance level. F. Statistical Analysis 3 As well, we can assess the relationship between variables such as velocity, distance or PRTH and PRTF and the success of TOP in each scenario. Global descriptive statistics from figure 9 show that the clearest variable that has significant differences between crashing or not is the velocity. Other variables like Occlusion and aggressive traffic cars had little part to play because the experiment was targeted towards a controlled study of driver’s perceptions in a given scenario. That is, the bihistogram of velocity is the most asymmetric one, having most of the no-crash samples lower velocity than most of the crash ones. These results make sense from the point of view that drivers have to avoid a sudden hazard on the road, so probably the first instinctive action would be braking, taking into account This fact is proved by means of the Wilcoxon rank-sum test, summarized in table IV. In all the scenarios we can reject the MINHAS et al.: EFFECTS OF NON-DRIVING RELATED TASKS DURING SELF-DRIVING MODE 1397 MINHAS et al.: EFFECTS OF NON-DRIVING RELATED TASKS DURING S Fig. 9. Bihistograms of the distributions of the different variables abou success of TOP. TABLE IV WILCOXON RANK-SUM TEST FOR THE RELATIONSHIP BETWEEN VELOCITY AND CRASHING REFERENCES [1] L. Kelion. (2016) Tesla Autopilot Involved in a Crash. [Online]. Avail- able: http://www.bbc.com/news/technology-36783345 Since another important variable, which is out of the scope of this article, is the lead time [29], further lines will be focused on the relationship between lead time, velocity and NDR-tasks. Indeed, the implications of these results are impor- tant for understanding the criteria needed for the appropriate design of Human Machine Interfaces in automated driving conditions, to ensure that messages regarding the transfer of control are given in a timely and appropriate manner, which is another important area in need of further research [10]. Understanding how to keep drivers in the loop during such automation will allow drivers to safely engage in other NDR-tasks. [2] SAE, J3016. (2019). Sae Levels of Driving Automation. [Online]. Available: https://www.sae.org/news/2019/01/sae-updates-j3016- automated-driving-graphic [3] (2019). Cabibus, the Vehicle. [Online]. Available: https://www.cabibus. com/cabibus_vehicle.html [4] Y. Kuang, X. Qu, J. Weng, and A. Etemad-Shahidi, “How does the driver’s perception reaction time affect the performances of crash surrogate measures?” PLoS ONE, vol. 10, no. 9, Sep. 2015, Art. no. e0138617. [5] N. Lyu, L. Xie, C. Wu, Q. Fu, and C. Deng, “Driver’s cognitive workload and driving performance under traffic sign information exposure in complex environments: A case study of the highways in China,” Int. J. Environ. Res. Public Health, vol. 14, no. 2, p. 203, Feb. 2017. J. Environ. Res. Public Health, vol. 14, no. 2, p. 203, Feb. 2 [6] C. Dijksterhuis, K. A. Brookhuis, and D. De Waard, “Effects of steering demand on lane keeping behaviour, self-reports, and physiology. A sim- ulator study,” Accident Anal. Prevention, vol. 43, no. 3, pp. 1074–1081, May 2011. Finally, the experiment also forwards the notion that using simulators is a vital part of pushing forward the development of new systems that can provide the drivers of future with necessary assistance in keeping themselves and other road users safe [19]. The resulting data is quite beneficial and it will serve as a dataset for future research like convolutional neural networks development and analysis to test new ADAS algorithms. As well, we have recorded data from 3 webcams monitoring the head, hands and the feet, that can be used to further analyze driver performance with regards to driver psychology and mental workload under autonomous driving conditions. VI. KEY POINTS • Experiment shows that not all secondary tasks result in higher Perception Reaction Times. • Experiment shows that the Perception Reaction Times have a global average of 3.51 seconds for hands and 2.47 seconds for feet. Experiments also shed a light on the fact that the increase in reaction times due to secondary tasks can affect the quality of driving of subjects in any given scenario [43], as well as other variables that can play an important role. In this way, our results sustain the hypothesis that the velocity is one of the most influential variables in the success of TOP as we previously claimed [35]. • The study shows that any secondary tasks while driving result in deterioration of quality of driving. • The implications of the results are important in under- standing the criteria needed for designing Human Machine Interfaces for autonomous driving vehicles. This can include entities such as Driver’s awareness to his/her surroundings which can be monitored by ADAS resulting in a more enhanced driving experience. From the point of view of safety for passengers, we can observe that the most influential variable for safe driving is speed, whether the car drives automatically or the driver does. Besides, drivers have a shorter reaction time with their feet than with their hands when trying to regain control after performing NDR-tasks, so PRTF could be the second most influential variable for safe driving under that conditions. At this point, gender should be another factor to consider, having balanced data to infer. • The study also highlights the fact that the true self-driving cars can be hazardous if they lack the proper systems that can observe the driver’s behavior in a timely manner. IEEE TRANSACTIONS ON INTELLIGENT TRANSPORTATION SYSTEMS, VOL. 23, NO. 2, FEBRUARY 2022 1398 data can then serve in designing complex Advanced Driver Assistance Systems for future Autonomous Vehicles. This study provides significant insight into how driver’s would react when they are behind the wheel of an increas- ingly autonomous car in a real life scenario. It also reflects the implications of poor driving behaviors which can result in fatalities for the future drivers. Better acoustic alarms, cognitive screening of driver’s interactions and better lead time calculations can all result in a more effective and safe autonomous driving systems. One interesting aspect of the recorded results indicate that PRTF of subjects that were involved in NDR-tasks has sig- nificantly longer reaction times as compared to subjects who were paying attention to the road. The experiment also seems to show that not all secondary tasks result in higher PRTH. In accordance to that, the results reflect the fact that the reac- tion times of Social Media usage is higher than the reaction times for IMQA, although these results are not significant. The results also show that the reaction times for subjects are within the 5-second mark, which was noted in other previous research papers [34]. V. CONCLUSION AND FUTURE DIRECTIONS Fig. 9. Bihistograms of the distributions of the different variables about their success of TOP. In this paper, we explore the influence of several variables related to driver-vehicle interactions in an automated driving mode under different scenarios in a simulated environment. In particular, we assess the reaction times of drivers after an unforeseen hazard appears in front while the car is in autonomous driving mode during which the driver is engaged in NDR-tasks. This hazard is alerted by an acoustic alarm and the driver has to regain car control to avoid a crash with the cars stopped in front. This experiment has proven to be a step up from the last version of the driver simulator [35]. Significant modifications were made to get the most amount of usable data from the 40 subjects. As well, the recorded null hypothesis so that we have evidence that the medians of the velocity when crashing or not differ. Table V for the variable of the initial distance of the car to the one in front at the time the alarm is triggered shows that, depending on the scenario, their medians when crashing or not significantly differ, although p-value is very close to the significant level. If we do not take into account the scenario, the p-value obtained rejects the null hypothesis, but we can appreciate that the effect size is small, unlike in the case of velocity. REFERENCES Green, “‘How long does it take to stop?’ methodological analysis of driver perception-brake times,” Transp. Hum. Factors, vol. 2, no. 3, pp. 195–216, 2000. [36] A. Eriksson and N. A. Stanton, “Driving performance after self-regulated control transitions in highly automated vehicles,” Hum. Factors, J. Hum. Factors Ergonom. Soc., vol. 59, no. 8, pp. 1233–1248, Dec. 2017. pp [15] J. Paxion, E. Galy, and C. Berthelon, “Mental workload and driving,” Frontiers Psychol., vol. 5, p. 1344, Dec. 2014. [37] H. W. 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Wickens, “Situation aware- ness, mental workload, and trust in automation: Viable, empirically sup- ported cognitive engineering constructs,” J. Cognit. Eng. Decis. Making, vol. 2, no. 2, pp. 140–160, Jun. 2008, doi: 10.1518/155534308X284417. [23] H. Makishita and K. Matsunaga, “Differences of drivers’ reaction times according to age and mental workload,” Accident Anal. Prevention, vol. 40, no. 2, pp. 567–575, Mar. 2008. [24] R. Abbasi-Kesbi, H. Memarzadeh-Tehran, and M. J. Deen, “Technique to estimate human reaction time based on visual perception,” Healthcare Technol. Lett., vol. 4, no. 2, p. 73, 2017. [25] S. Petermeijer, P. Bazilinskyy, K. Bengler, and J. de Winter, “Take- over again: Investigating multimodal and directional TORs to get the driver back into the loop,” Appl. Ergonom., vol. 62, pp. 204–215, Jul. 2017. Aura Hernández-Sabaté received the degree in mathematics in 2002, and the Ph.D. degree (cum laude) in Computer Science in 2009. She is cur- rently a member of the Computer Vision Center and an Associate Professor with the Computer Science Department, Universitat Autònoma de Barcelona. She has published more than 50 papers in refereed journals and international conferences. Her main research interests include performance evaluation and driver behavior analysis. Aura Hernández-Sabaté received the degree in mathematics in 2002, and the Ph.D. degree (cum laude) in Computer Science in 2009. She is cur- rently a member of the Computer Vision Center and an Associate Professor with the Computer Science Department, Universitat Autònoma de Barcelona. She has published more than 50 papers in refereed journals and international conferences. Her main research interests include performance evaluation and driver behavior analysis. [26] S. S. Borojeni, L. Chuang, W. Heuten, and S. REFERENCES This is particularly important as described by Gaku Iizuka [19], who claims that changes in driver behaviour are observed in all parameters such as blood oxygen levels and gaze perception. Monitoring the cognitive state of the driver could replace several parameters from other convoluted sensors. [7] C. Gold, D. Damböck, L. Lorenz, and K. Bengler, “‘Take over!’ How long does it take to get the driver back into the loop,” in Proc. Hum. Factors Ergonom. Soc. Annual Meeting, vol. 57, no. 1. Los Angeles, CA, USA: SAGE, 2013, pp. 1938–1942. pp [8] B. Wandtner, N. Schömig, and G. Schmidt, “Effects of non-driving related task modalities on takeover performance in highly automated driving,” Hum. Factors, J. Hum. Factors Ergonom. Soc., vol. 60, no. 6, pp. 870–881, Sep. 2018. [9] B. Wandtner, G. Schmidt, N. Schoemig, and W. Kunde, “Non-driving related tasks in highly automated driving-effects of task modalities and cognitive workload on take-over performance,” in Proc. AmE Automot. 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Stocklein, “Recon- figurable driving simulator for testing and training of advanced driver assistance systems,” in Proc. IEEE Int. Symp. Assem. Manuf. (ISAM), Jul. 2013, pp. 337–339. [13] M. Svetina, “The reaction times of drivers aged 20 to 80 during a divided attention driving,” Traffic Injury Prevention, vol. 17, no. 8, pp. 810–814, Nov. 2016. [35] S. Minhas et al., “LEE: A photorealistic virtual environment for assessing driver-vehicle interactions in self-driving mode,” in Proc. Eur. Conf. Comput. Vis. (ECCV) Workshops. Amsterdam, The Netherlands: Springer, Oct. 2016, pp. 894–900. [14] M. REFERENCES Boll, “Assisting drivers with ambient take-over requests in highly automated driving,” in Proc. 8th Int. Conf. Automot. User Interface Interact. Veh. Appl. Automo- tive’UI, 2016, pp. 237–244. [27] A. Eriksson, S. M. Petermeijer, M. Zimmermann, J. C. F. de Winter, K. J. Bengler, and N. A. Stanton, “Rolling out the red (and green) carpet: Supporting driver decision making in automation-to-manual transitions,” IEEE Trans. Human-Mach. Syst., vol. 49, no. 1, pp. 20–31, Feb. 2019. Shoaib Ehsan (Senior Member, IEEE) is currently a Research Scientist with extensive experience and major research accolades, including the BMVA Sul- livan Doctoral Thesis Prize in 2013, and recognition as a "Researcher with Exceptional Promise" by the Royal Academy of Engineering in 2014. He has made significant contributions with his expertise in multiple fields, especially computer vision, embed- ded systems, and big data. He has received funding as a Researcher Co-Investigator for £12.69M in total, and a ‘Named Researcher’ for £5.5M in total. [28] A. Eriksson and N. A. Stanton, “Takeover time in highly automated vehicles: Noncritical transitions to and from manual control,” Hum. Factors, J. Hum. Factors Ergonom. Soc., vol. 59, no. 4, pp. 689–705, Jun. 2017. [29] J. Wan and C. Wu, “The effects of lead time of take-over request and nondriving tasks on taking-over control of automated vehicles,” IEEE Trans. Human-Mach. Syst., vol. 48, no. 6, pp. 582–591, Dec. 2018. [30] C. Gold, M. Körber, D. Lechner, and K. Bengler, “Taking over control from highly automated vehicles in complex traffic situations: The role of traffic density,” Hum. Factors, J. Hum. Factors Ergonom. Soc., vol. 58, no. 4, pp. 642–652, Jun. 2016. Klaus D. McDonald-Maier (Senior Member, IEEE) is currently a Professor with the School of Computer Science and Electronic Engineering (CSEE), Uni- versity of Essex, where he leads the Embedded and Intelligent Systems (EIS) Research Group. He is also a Visiting Professor with ESIGELEC, and the CTO of UltraSoC Technologies Ltd. He has authored or coauthored over 200 publications and 15 pending patents. His research has been funded by ESRC and EPRSC grants with substantial contributions from industry, as well as multiple EU grants, several other ects. Klaus D. McDonald-Maier (Senior Member, IEEE) is currently a Professor with the School of Computer Science and Electronic Engineering (CSEE), Uni- versity of Essex, where he leads the Embedded and Intelligent Systems (EIS) Research Group. REFERENCES He is also a Visiting Professor with ESIGELEC, and the CTO of UltraSoC Technologies Ltd. He has authored or coauthored over 200 publications and 15 pending patents. His research has been funded by ESRC and EPRSC grants with substantial contributions from industry, as well as multiple EU grants, several other grants, and funded projects. Klaus D. McDonald-Maier (Senior Member, IEEE) is currently a Professor with the School of Computer Science and Electronic Engineering (CSEE), Uni- versity of Essex, where he leads the Embedded and Intelligent Systems (EIS) Research Group. He is also a Visiting Professor with ESIGELEC, and the CTO of UltraSoC Technologies Ltd. He has authored or coauthored over 200 publications and 15 pending patents. His research has been funded by ESRC and EPRSC grants with substantial contributions from industry, as well as multiple EU grants, several other cts. Klaus D. McDonald-Maier (Senior Member, IEEE) is currently a Professor with the School of Computer Science and Electronic Engineering (CSEE), Uni- versity of Essex, where he leads the Embedded and Intelligent Systems (EIS) Research Group. He is also a Visiting Professor with ESIGELEC, and the CTO of UltraSoC Technologies Ltd. He has authored or coauthored over 200 publications and 15 pending patents. His research has been funded by ESRC and EPRSC grants with substantial contributions from industry, as well as multiple EU grants, several other grants, and funded projects. [31] V. A. Banks, A. Eriksson, J. O’Donoghue, and N. A. Stanton, “Is partially automated driving a bad idea? Observations from an on-road study,” Appl. Ergonom., vol. 68, pp. 138–145, Apr. 2018. [Online]. Available: http://www.sciencedirect.com/science/article/pii/ S0003687017302594 [32] F. Naujoks, D. Befelein, K. Wiedemann, and A. Neukum, “A review of non-driving-related tasks used in studies on automated driving,” in Proc. Int. Conf. Appl. Hum. Factors Ergonom., 2017, pp. 525–537. [33] T. Suzaki et al., “Steering behavior model of drivers on driving simulator through visual information,” in Proc. Asia–Pacific Signal Inf. Process. Assoc. Annu. Summit Conf. (APSIPA), Dec. 2016, pp. 1–4. grants, and funded projects.
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DIABETES RISK AND ASSOCIATIONS WITH DEMOGRAPHIC AND BEHAVIORAL FACTORS IN RUSSIAN POPULATION: DATA FROM THE ESSE-RF STUDY
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ОРИГИНАЛЬНЫЕ СТАТЬИ ОРИГИНАЛЬНЫЕ СТАТЬИ РИСК САХАРНОГО ДИАБЕТА И ЕГО АССОЦИАЦИИ С СОЦИАЛЬНО-ДЕМОГРАФИЧЕСКИМИ И ПОВЕДЕНЧЕСКИМИ ФАКТОРАМИ РИСКА В РОССИЙСКОЙ ПОПУЛЯЦИИ: ДАННЫЕ ИССЛЕДОВАНИЯ ЭССЕ-РФ Р., Топольская Н. В., Шабалин В. В.; Оренбург: Басы- рова И. Р., Лопина Е. А.; Самара: Гудкова С. А., Черепанова Н. А.; Санкт-Петер- бург: Баранова Е. И., Конради А. О., Шляхто Е. В.; Томск: Карпов Р. С., Кавеш- ников В. С., Серебрякова В. Н.; Тюмень: Медведева И. В., Сторожок М. А., Шалаев С. В.; Кемерово: Артамонова Г. В., Барбараш О. Л., Виниченко Т. А., Данильченко Я. В., Мулерова Т. А., Максимов С. А., Табакаев М. В. Участники исследования ЭССЕ-РФ, соавторы статьи: Москва: Баланова Ю. А., Гомыранова Н. В., Имаева А. Э., Капустина А. В., Кон- цевая А. В., Литинская О. А., Мамедов М. Н., Муромцева Г. А., Оганов Р. Г., Суво- рова Е. И., Худяков М. Б.; Владивосток: Мокшина М. В., Невзорова В. А., Родио­нова Л. В., Шестакова Н. В.; Волгоград: Ледяева А. А., Чумачек Е. В.; Вологда: Ильин В. А., Калашников К. Н., Калачикова О. Н., Попов А. В.; Воро- неж: Бабенко Н. И., Бондарцов Л. В., Фурменко Г. И.; Иваново: Романчук С. В., Шутемова Е. А.; Красноярск: Данилова Л. К., Евсюков А. А., Косинова А. А., Петрова М. М., Руф Р. Р., Топольская Н. В., Шабалин В. В.; Оренбург: Басы- рова И. Р., Лопина Е. А.; Самара: Гудкова С. А., Черепанова Н. А.; Санкт-Петер- бург: Баранова Е. И., Конради А. О., Шляхто Е. В.; Томск: Карпов Р. С., Кавеш- ников В. С., Серебрякова В. Н.; Тюмень: Медведева И. В., Сторожок М. А., Шалаев С. В.; Кемерово: Артамонова Г. В., Барбараш О. Л., Виниченко Т. А., Данильченко Я. В., Мулерова Т. А., Максимов С. А., Табакаев М. В. РИСК САХАРНОГО ДИАБЕТА И ЕГО АССОЦИАЦИИ С СОЦИАЛЬНО-ДЕМОГРАФИЧЕСКИМИ И ПОВЕДЕНЧЕСКИМИ ФАКТОРАМИ РИСКА В РОССИЙСКОЙ ПОПУЛЯЦИИ: ДАННЫЕ ИССЛЕДОВАНИЯ ЭССЕ-РФ Евстифеева С. Е. 1, Шальнова С. А. 1, Деев А. Д. 1, Белова О. А. 2, Гринштейн Ю. И. 3, Дупляков Д. В. 4, Ефанов А. Ю. 5, Жернакова Ю. В. 6, Индукаева Е. В. 7, Кулакова Н. В. 8, Либис Р. А. 9, Недогода С. В. 10, Ротарь О. П. 11, Толпаров Г. В. 12, Трубачева И. А. 13, Черных Т. М. 14, Шабунова А. А. 15, Бойцов С. А. 1,6 от имени участников исследования ЭССЕ-РФ Евстифеева С. Е.*  — к. м.н., с. н.с. отдела эпидемиологии хронических неин- фекционных заболеваний, Шальнова С. А. — д. м.н., профессор, руководитель отдела эпидемиологии хронических неинфекционных заболеваний, Деев А. Д. — к.ф-м.н, руководитель лаборатории медицинской биостатистики, Белова О. А. — главный врач, Гринштейн Ю. И. — д. м.н., профессор, зав. кафед­рой терапии ИПО, Дупляков Д. В. — д. м.н., профессор, заместитель главного врача, профессор кафедры кардиологии и  кардиохирургии ИПО, Ефанов А. Ю. — к. м.н., зам. директора по лечебной работе, Жернакова Ю. В. — д. м.н., с. н.с. отдела координации и мониторинга научных программ, Индука- ева Е. В. — к. м.н., с. н.с. лаборатории эпидемиологии, Кулакова Н. В. — к. м.н., доцент кафедры терапии и  инструментальной диагностики, Либис Р. А. — д. м.н., профессор, зав. кафедрой госпитальной терапии с  курсом клиниче- ской фармакологии, Недогода С. В. — д. м.н., профессор, зав. кафедрой терапии и эндокринологии ФУВ, проректор по лечебной работе, Ротарь О. П. — к. м.н., зав. лабораторией эпидемиологии артериальной гипертензии, Толпа- ров Г. В. — врач-кардиолог, Трубачева И. А. — д. м.н., руководитель отделения популяционной кардиологии с  группой научно-медицинской информации, патентоведения и международных связей, Черных Т. М. — д. м.н., профессор, зав. кафедрой госпитальной терапии и эндокринологии, Шабунова А. А. — д. экон.н., профессор, зам. директора, Бойцов С. А. — член-корр. РАН, профес- сор, д. м.н., и. о. директора, руководитель отдела клинической кардиологии и молекулярной генетики. Цель. Оценить 10-летний риск потенциального развития сахарного диабета 2 типа (СД2) в российской популяции с помощью шкалы FINDRISC и изучить его ассоциации с  социально-демографическими и  поведенческими факто- рами по данным эпидемиологического исследования ЭССЕ-РФ. Материал и  методы. В  работе использованы данные многоцентрового исследования (Эпидемиология сердечно-сосудистых заболеваний в различ- ных регионах Российской Федерации: ЭССЕ-РФ). Всего было обследовано 21923 человек 25-64 лет, из которых у 1045 человек (мужчин — 3,76% и жен- щин — 5,39%) отмечался СД в анамнезе. РИСК САХАРНОГО ДИАБЕТА И ЕГО АССОЦИАЦИИ С СОЦИАЛЬНО-ДЕМОГРАФИЧЕСКИМИ И ПОВЕДЕНЧЕСКИМИ ФАКТОРАМИ РИСКА В РОССИЙСКОЙ ПОПУЛЯЦИИ: ДАННЫЕ ИССЛЕДОВАНИЯ ЭССЕ-РФ В окончательный анализ включено 20878 лиц (8058 мужчин и  12820 женщин) без СД, которым определялся 10-летний риск развития СД2 с  помощью шкалы FINDRISC (The FINnish Diabetes RIsk SCore). Уровень риска и вероятность развития СД2 оценивался по полученной сумме баллов на основании ответов на вопросы. Кроме того, анализировались ассоциации с  образованием, семейным положением, местом жительства, достатком, а  также статусом курения и  потребления алкоголя. Материал и  методы. В  работе использованы данные многоцентрового исследования (Эпидемиология сердечно-сосудистых заболеваний в различ- ных регионах Российской Федерации: ЭССЕ-РФ). Всего было обследовано 21923 человек 25-64 лет, из которых у 1045 человек (мужчин — 3,76% и жен- щин — 5,39%) отмечался СД в анамнезе. В окончательный анализ включено 20878 лиц (8058 мужчин и  12820 женщин) без СД, которым определялся 10-летний риск развития СД2 с  помощью шкалы FINDRISC (The FINnish Diabetes RIsk SCore). Уровень риска и вероятность развития СД2 оценивался по полученной сумме баллов на основании ответов на вопросы. Кроме того, анализировались ассоциации с  образованием, семейным положением, местом жительства, достатком, а  также статусом курения и  потребления алкоголя. Результаты. Порог высокого риска СД2 для нашей популяции составил ≥12 баллов, площадь под кривой (AUC) — 0,76, что соответствует хорошему каче- ству модели. Распространенность высокого риска по российским критериям составила 20,4%. Многофакторный анализ, продемонстрировал, что после коррекции по возрасту и региону высокий риск СД2 у мужчин ассоциируется с  отказом от  курения (отношение шансов (ОШ) 1,34; 95% доверительный интервал (ДИ) 1,14-1,58; р=0,0004) и употреблением алкоголя (ОШ 2,01; 95% ДИ 1,48-2,71; р=0,0001), а у женщин с низким доходом, низким образователь- ным уровнем и у замужних. *Автор, ответственный за переписку (Corresponding author): SEvstifeeva@gnicpm.ru Заключение. Средний уровень баллов по FINDRISC составил для всей попу- ляции 6,5±0,03, а  абсолютный риск 5,3%. Выявлены ассоциации высокого риска СД2 (≥12 баллов) с поведенческими факторами у мужчин и с социально- демографическими показателями у женщин. Участники исследования ЭССЕ-РФ, соавторы статьи: Москва: Баланова Ю. А., Гомыранова Н. В., Имаева А. Э., Капустина А. В., Кон- цевая А. В., Литинская О. А., Мамедов М. Н., Муромцева Г. А., Оганов Р. Г., Суво- рова Е. И., Худяков М. Б.; Владивосток: Мокшина М. В., Невзорова В. А., Родио­нова Л. В., Шестакова Н. В.; Волгоград: Ледяева А. А., Чумачек Е. В.; Вологда: Ильин В. А., Калашников К. Н., Калачикова О. Н., Попов А. В.; Воро- неж: Бабенко Н. И., Бондарцов Л. В., Фурменко Г. И.; Иваново: Романчук С. В., Шутемова Е. А.; Красноярск: Данилова Л. К., Евсюков А. А., Косинова А. А., Петрова М. М., Руф Р. Russ J Cardiol 2017, 9 (149): 13–20 http://dx.doi.org/10.15829/1560-4071-2017-9-13-20 Aim. To evaluate 10-year risk of potential development of type 2 diabetes (DM) in Russian population with the FINDRISC score, and to assess its associations with social, demographic and behavioral factors by the data from epidemiological study ESSE-RF. Key words: ESSE-RF, prevalence, association, FINDRISC — The FINnish Diabetes RIsk SCore, behavioral factors, social and demographic factors. Material and methods. In the work, the data used, from multi-center study (Epidemiology of cardiovascular diseases in various regions of Russian Federation: ESSE-RF). Totally, 21923 persons investigated, age 25-64 y. o., of those 1045 (3,76% males, 5,39% females) had DM. To the final analysis 20878 persons included (8058 males, 12820 females) with no DM, for whom the 10-year risk was assessed with the FINDRISC (The FINnish Diabetes RIsk SCore). Level of risk and probability of DM onset were evaluated by the points summation. Also, associations were analyzed with education, marital status, place of inhabitance, income, smoking and alcohol status. 1National Research Center for Preventive Medicine  of the Ministry of Health, Moscow; 2Cardiovascular Dispensary, Ivanovo; 3V.F. Voino-Yasenetsky Krasnoyarsk State Medical University, Krasnoyarsk; 4Oblastnoy Clinical Cardiological Dispensary, Samara; 5Tyumen State Medical University of the Ministry of Health, Tyumen; 6Russian Cardiological Research-and-Production Complex of the Ministry of Health, Moscow; 7Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo; 8Pacific State Medical University, Vladivostok; 9Orenburg State Medical University, Orenburg; 10Volgograd State Medical University, Volgograd; 11Federal Almazov North-West Medical Research Centre of the Ministry of Health, Saint- Petersburg; 12North Ossetian State Medical Academy, Vladikavkaz, the Republic of North Ossetia-Alania; 13Tomsky National Research Medical Center of RAS, Scientific-Research Institute of Cardiology, Tomsk; 14N.N. Burdenko Voronezh State Medical University, Voronezh; 15Institute of Socio-Economic Development of Territories RAS, Vologda, Russia. Results. The threshold for high DM risk in Russian population was set at ≥12 level, with AUC 0,76, that represents good quality of model. The prevalence of the high risk by Russian criteria was 20,4%. Multifactorial analysis demonstrated that after correction for region and age, DM high risk was associated with smoking cessation (odds ratio (OR) 1,34; 95% confidence interval (CI) 1,14-1,58; р=0,0004) and alcohol consumption (OR 2,01; 95% CI 1,48-2,71; р=0,0001), and in women — with low income, low educational level and being married. Conclusion. Mean score by FINDRISC was 6,5±0,03, and absolute risk 5,3%. There were associations found of higher DM risk (≥12 points) with behavioral factors in males and social-demographic factors in women. Российский кардиологический журнал 2017, 9 (149): 13–20 http://dx.doi.org/10.15829/1560-4071-2017-9-13-20 Ключевые слова: ЭССЕ-РФ, распространенность, ассоциации, FINDRISC — The FINnish Diabetes RIsk SCore, поведенческие факторы, социально-демогра- фические показатели. 1ФГБУ Государственный научно-исследовательский центр профилактической медицины Минздрава России, Москва; 2ОБУЗ Кардиологический диспансер, Иваново; 3ФГБОУ ВО Красноярский государственный медицинский универси- тет им. проф. В. Ф. Войно-Ясенецкого Минздрава России, Красноярск; 4ГБУЗ Областной клинический кардиологический диспансер, Самара; 5ФГБОУ ВО Тюменский государственный медицинский университет Минздрава России, Тюмень; 6ФГБУ Российский кардиологический научно-производственный ком- плекс Минздрава России, Москва; 7ФГБНУ Научно-исследовательский инсти- тут комплексных проблем сердечно-сосудистых заболеваний, Кемерово; 8ФГБОУ ВО Тихоокеанский государственный медицинский университет Мин- здрава России, Владивосток; 9ФГБОУ ВО Оренбургский государственный медицинский университет Минздрава России, Оренбург; 10ФГБОУ ВО Волго­ градский государственный медицинский университет Минздрава России, Волгоград; 11ФГБУ Северо-Западный Федеральный медицинский исследова- тельский центр им. В. А. Алмазова Минздрава России, Санкт-Петербург; 12ФГБОУ ВО Северо-Осетинская государственная медицинская академия Минздрава России, Владикавказ; 13ФГБНУ Томский национальный исследова- тельский медицинский центр РАН, Научно-исследовательский институт кардио­логи, Томск; 14ФГБОУ ВО Воронежский государственный медицинский университет им. Н. Н. Бурденко Минздрава России, Воронеж; 15ФГБУН Инсти- тут социально-экономического развития территорий РАН, Вологда, Россия. ВОЗ — Всемирная Организация Здравоохранения, “Д” — диспансерный учет, ДИ — доверительный интервал, ESC — Европейского общества кардиологов (European Society of Cardiology), ИМТ — индекс массы тела, EASD — Европей- ской ассоциации по изучению сахарного диабета (European Association for the Study of Diabetes), IDF  — Международная диабетическая федерация (International Diabetes Federation), NATION  — Национальное всероссийское эпидемиологическое исследование распространенности СД 2 типа у взрос­ лого населения РФ, НИЗ — неинфекционные заболевания, ОШ — отношение шансов, ОР  — относительный риск, СД2  — сахарный диабет 2 тип, СД  — сахарный диабет, ССЗ — сердечно-сосудистые заболевания, FINDRISC — The FINnish Diabetes RIsk SCore, ФР — фактор риска, ЭССЕ-РФ — эпидемиология сердечно-сосудистых заболеваний в различных регионах Российской Феде- рации, б — баллы. Рукопись получена 06.07.2017 Рецензия получена 04.09.2017 Принята к публикации 08.09.2017 13 13 Российский кардиологический журнал № 9 (149) | 2017 DIABETES RISK AND ASSOCIATIONS WITH DEMOGRAPHIC AND BEHAVIORAL FACTORS IN RUSSIAN POPULATION: DATA FROM THE ESSE-RF STUDY Evstifeeva S. E. 1, Shalnova S. А. 1, Deev А. D. 1, Belova О. А. 2, Grinshtein Yu. I. 3, Duplyakov D. V. 4, Efanov А. Yu. 5, Zhernakova Yu. V. 6, Indukaeva Е. V. 7, Kulakova N. V. 8, Libis R. А. 9, Nedogoda S. V. 10, Rotar О. P. 11, Tolparov G. V. 12, Trubacheva I. А. 13, Chernykh Т. М. 14, Shabunova А. А. 15, Boytsov S. А. 1,6 on behalf of the ESSE-RF study workgroup Evstifeeva S. E. 1, Shalnova S. А. 1, Deev А. D. 1, Belova О. А. 2, Grinshtein Yu. I. 3, Duplyakov D. V. 4, Efanov А. Yu. 5, Zhernakova Yu. V. 6 Indukaeva Е. V. 7, Kulakova N. V. 8, Libis R. А. 9, Nedogoda S. V. 10, Rotar О. P. 11, Tolparov G. V. 12, Trubacheva I. А. 13, Chernykh Т. М. 14 Shabunova А. А. 15, Boytsov S. А. 1,6 on behalf of the ESSE-RF study workgroup Russ J Cardiol 2017, 9 (149): 13–20 http://dx.doi.org/10.15829/1560-4071-2017-9-13-20 Материал и методы определить размер бедствия, или реальную распро- страненность заболевания, зная которую, можно прогнозировать ситуацию, контролировать эффек- тивность профилактической и лечебно-диагностиче- ской помощи населению, и  разрабатывать адекват- ные меры, направленные на улучшение ситуации. Материал и методы Объектом многоцентрового эпидемиологического исследования (Эпидемиология сердечно-сосудистых заболеваний в различных регионах Российской Феде- рации — ЭССЕ-РФ) были представительные выборки из неорганизованного мужского и женского населе- ния в возрасте от 25-64 лет из 13 регионов РФ (Воро- нежская, Ивановская, Волгоградская, Вологодская, Кемеровская, Тюменская области, города Владивос- ток, Оренбург, Самара, Томск и  Санкт-Петербург, республика Северная Осетия-Алания, Красноярский край). Исследование было одобрено НЭК ФГБУ “ГНИЦПМ” Минздрава России, ФГБУ “РКНПК” Минздрава России, ФГБУ “ФМИЦ им. В. А. Алма- зова” Минздрава России и центров-соисполнителей. Все обследованные лица подписали добровольное информированное согласие на участие в нем. Отклик на обследование в целом составил около 80%. В большинстве работ показано, что длительный период доклинической стадии СД2, в течение кото- рого формируются диабетические осложнения, дает шанс предотвратить или отсрочить манифестацию СД2 при своевременном проведении профилактиче- ских мероприятий у  лиц, в  том числе с  высоким с  риском развития СД2. Исходя из  этого, в  разных странах мира были разработаны различные про- граммы по  профилактике СД, которые корректиру- ются по мере совершенствования медицинских тех- нологий. Для раннего выявления СД в мире создано множество инструментов-вопросников, которые прошли валидизацию и были оптимизированы с уче- том особенности страны, контингента обследуемых и  достоверности полученных результатов. Наиболее известным является разработанный Финской диабе- тической ассоциацией в рамках реализации государ- ственной программы по профилактике СД2 вопрос- ник FINDRISC — The FINnish Diabetes RIsk SCore, который был рекомендован (2007) к использованию рабочей группой Европейского общества кардиоло- гов (European Society of Cardiology, ESC) и Европей- ской ассоциации по  изучению сахарного диабета (European Association for the Study of Diabetes, EASD) и в настоящее время успешно используется во многих странах мира [7, 8]. Опрос проводился по стандартной анкете, разра- ботанной на основе адаптированных международных методик. Вопросник построен по  модульному типу и содержит информацию о социально-демографиче- ских характеристиках, поведенческих привычках, анамнестических данных, экономических условиях жизни и  т. д. Подробный протокол исследования ЭССЕ-РФ был опубликован в журнале “Профилак- тическая медицина” 2013г [10]. Анализировали возраст, пол, образование, семей- ное положение, место жительства и регион прожива- ния. Russ J Cardiol 2017, 9 (149): 13–20 http://dx.doi.org/10.15829/1560-4071-2017-9-13-20 В глобальном докладе ВОЗ (2016г) отмечается, что распространенность сахарного диабета (СД) во всем мире достигла масштабов эпидемии. В общей струк- туре диабета доля СД 2 типа (СД2) составляет 90-95% [1]. За период 1980-2014гг число лиц с СД возросло с 108 млн до 422 млн, а распространенность среди лиц старше 18 лет с  4,7% до  8,5%, соответственно. В 2012 году 1,5 млн случаев смерти были напрямую вызваны диабетом, а  еще 2,2 млн случаев были обусловлены высоким содержанием глюкозы крови. По прогнозам ВОЗ, в 2030г СД будет занимать седь- мое место среди причин смертности [2]. о России как для ВОЗ, так и IDF, вызывает большие сомнения, поскольку до  последнего времени были опубликованы единичные исследования, посвящен- ные распространенности СД2. Из ряда предыдущих исследований известно, что фактическая распространенность СД и его осложне- ний существенно отличается от  данных диспансер- ного учета (“Д”). Так, по данным Российского феде- рального регистра СД (конец 2016г) на “Д” состояло 4,35 млн (3%) населения, из них 92% (4 млн) с СД2. В  то  же время, ведущие российские специалисты в  области эндокринологии отмечают, что федераль- ный регистр не  отражает реальную распространен- ность СД, так как регистрируются лица по обращае- мости. ВОЗ и IDF также отмечают отсутствие досто- верной статистики распространенности СД, в  том числе в российской популяции [5]. По данным (2015г) Международной диабетиче- ской федерации (International Diabetes Federation, IDF), распространенность СД в мире составляет 8,8% (интервал неопределенности 7,4-11,2%) и 5 млн смер- тей зафиксировано вследствие СД (для сравнения — вследствие ВИЧ/СПИДа и  туберкулеза умирает по 1,5 млн взрослых) [3]. С целью получения достоверной статистики рас- пространенности СД в России, в 2013г было проведено Всероссийское эпидемиологическое исследование распространенности СД 2 типа у взрослого населения РФ — NATION. Исследование NATION показало, что истинная распространенность СД2 в 2 раза превышает зарегистрированную и  составляет 5,4%. Наибольшая распространенность СД2 была выявлена у лиц старше 45 лет, с ожирением и с ССЗ [6]. В Российской Федерации (2016г), по данным ВОЗ, распространенность СД в  целом составляла 9,3%, (мужчины — 8,0%, женщины — 10,3%), а вклад СД в общую смертность составляет около 1% случаев [4]. По данным IDF (2015г), стандартизованная по возрас- ту распространенность СД у  взрослых (20-79 лет) составляет 9,2% (интервал неопределенности — 4,7- 13,3%) и  около 186 тыс. взрослых погибли от  СД в России [3]. Следует отметить, что источник данных Необходимость проведения эпидемиологических исследований очевидна, поскольку они позволяют 14 14 ОРИГИНАЛЬНЫЕ СТАТЬИ Результаты у В таблице 2 представлена оценка риска развития СД2 в  баллах (б), полученная по  вопроснику FINDRISK в популяции российских мужчин и жен- щин 25-64 лет в  зависимости от  демографических характеристик. Среднее значение риска в популяции в  целом составило 6,5 балла, что соответствовало 5,3% средней вероятности развития СД2 в  течение 10-лет (у мужчин 6,0 и 4,4% и у женщин 6,7 и 5,7%, соответственно). Женщины имеют достоверно более высокие характеристики риска СД2, чем мужчины. Отмечено статистически значимое увеличение сред- него уровня риска с  возрастом, что отражает алго- ритм, заложенный в  вопроснике. Образовательный ценз мужчин и женщин значимо не влияет на уровень риска развития СД2, так же, как и проживание в селе или в городе. В то же время, средний риск достоверно ассоциируется с  семейным положением мужчин (5,6  б у  неженатых против 7,9  б у  вдовых, р=0,008) и  не  зависит от  семейного положения женщин. Напротив, риск развития СД2 не зависит от достатка мужчин, но  значительно выше у  малообеспеченных женщин (7,2 б у бедных и 7,0 б у малообеспеченных против 5,5 б у высоко обеспеченных, р=0,01 и р=0,03, соответственно). Рис. 1. Распределение 10-летнего риска вероятного развития СД2 у россий- ских мужчин и женщин (данные ЭССЕ-РФ). SAS (Statistical Analysis System, версия 6.12). Прово- дился расчет средних значений и  их стандартной ошибки (M±m), квинтилей и  ранговых статистик. Использовались методы аналитической статистики: дисперсионно-ковариационный анализ в версии про- цедур SAS PROC GLM (обобщенный линейный ана- лиз), метод логистической регрессии (PROC LOGISTIC), проводилась оценка β-коэффициента и использовался тест Вальда (Wx 2). Проводился ROC- анализ (Receiver Operator Characteristic) шкалы FIND- RISC для определения оптимального количества бал- лов (пороговое значение), распознающее “высокий” риск СД2. Качество шкалы оценивали с  помощью площади под кривой (AUC), которая оценивает модель следующим образом: 0,9-1,0  — отличное, 0,8-0,9  — очень хорошее, 0,7-0,8 — хорошее, 0,6-0,7 — среднее, 0,5-0,6 — неудовлетворительное. Проведен многофак- торный анализ ассоциаций социально-демографиче- ских факторов с  “высокой” шкалой FINDRISC ≥12 баллов у мужчин и женщин. Оценивались отношения шансов (ОШ) и 95% доверительные интервалы (95% ДИ) ассоциаций “высокого” риска по FINDRISC (≥12 баллов) с  социальными показателями и  поведенче- скими факторами риска (ФР). Применялась прямая стандартизация данных по  европейскому стандарту. Уровень статистической значимости отмечался как значимый при р<0,05. В регионах значения риска варьировали от  5,1  б в Самаре до 6,7 б в Томске у мужчин и от 6,2 б до 7,4 б у  женщин в  этих  же регионах. Таблица 1 Таблица 1 Алгоритм расчета риска СД2 по FINDRISC Общее количество баллов Уровень риска СД2 Вероятность развития СД2 <7 Низкий 1 из 100 или 1% 7-11 Незначительно повышенный 1 из 25 или 4% 12-14 Умеренный 1 из 6 или 17% 15-20 Высокий 1 из 3 или 33% >20 Очень высокий 1 из 2 или 50% Алгоритм расчета риска СД2 по FINDRISC Алгоритм расчета риска СД2 по FINDRISC Рис. 1. Распределение 10-летнего риска вероятного развития СД2 у россий- ских мужчин и женщин (данные ЭССЕ-РФ). 44,9 34,7 13,0 7,2 0,2 54,3 31,7 9,2 4,7 0,2 39,0 36,5 15,4 8,7 0,3 0 10 20 30 40 50 60 1% 4% 17% 33% 50% Все Мужчины Женщины % Рис. 1. Распределение 10-летнего риска вероятного развития СД2 у россий- ских мужчин и женщин (данные ЭССЕ-РФ). 44,9 34,7 13,0 7,2 0,2 54,3 31,7 9,2 4,7 0,2 39,0 36,5 15,4 8,7 0,3 0 10 20 30 40 50 60 1% 4% 17% 33% 50% Все Мужчины Женщины % Материал и методы Из  поведенческих привычек оценивали статус курения (курит, бросил, никогда не курил) и потре- бления алкоголя (не употреблял в  течение года, редко — реже, чем раз в месяц, мало — менее 84/42 мл чистого алкоголя в  неделю для мужчин и  женщин, соответственно, умеренно — менее 168/84 мл чистого алкоголя в  неделю, и  чрезмерно  — более 168/84 мл чистого алкоголя). Образование оценивали, как ниже среднего, среднее и выше среднего; семейное поло- жение: никогда не был женат/замужем, женат/заму- жем сейчас, разведен/разведена, вдовец/вдова; место жительства: город/село; уровень достатка; и  реги- оны-участники. СД диагностировался на основании положительного ответа на вопрос (модуль “Заболева- ния”): “Говорил ли Вам когда-нибудь врач, что у Вас имеется/имелся сахарный диабет 2 типа?”. Так, в России, Мустафина С. В. и др. (2016) пред- ставили данные валидизации вопросника FINDRISC на европеоидной сибирской популяции по результа- там проспективного исследования, полученный показатель был несколько ниже прогнозируемого на финской популяции. Определено пороговое зна- чение  — 11 баллов. Лица с  суммой баллов более 11 имели значительный риск развития СД2 [9]. Представляя собой современный вызов общест- венному здравоохранению, СД2 является одним из четырех приоритетных неинфекционных заболе- ваний (НИЗ), принятие мер в  отношении которых запланировано на  уровне мировых лидеров. Его высокая медико-социальная значимость послужила основанием для изучения нами риска возникнове- ния числа новых случаев СД2 за последующие десять лет в разных возрастных категориях жителей и реги- онах РФ. Всего было обследовано 21923 человек, из  кото- рых у  1045 человек (мужчин  — 3,76% и  женщин  — 5,39%) отмечался СД в  анамнезе. В  окончательный анализ включено 20878 лиц (8058 мужчин и  12820 женщин) без СД, которым определялся 10-летний риск развития СД2 с  помощью шкалы FINDRISC. Уровень риска и вероятность развития СД2 оценива- лись по полученной сумме баллов на основании отве- тов на вопросы (табл. 1). Цель: оценить 10-летний риск потенциального развития СД2 в  российской популяции с  помощью шкалы FINDRISC и  его ассоциацию с  социально- демографическими и  поведенческими факторами, по  данным эпидемиологического исследования ЭССЕ-РФ. Анализ данных был выполнен с помощью системы статистического анализа и извлечения информации — 15 15 Российский кардиологический журнал № 9 (149) | 2017 Таблица 1 Алгоритм расчета риска СД2 по FINDRISC Общее количество баллов Уровень риска СД2 Вероятность развития СД2 <7 Низкий 1 из 100 или 1% 7-11 Незначительно повышенный 1 из 25 или 4% 12-14 Умеренный 1 из 6 или 17% 15-20 Высокий 1 из 3 или 33% >20 Очень высокий 1 из 2 или 50% Таблица 1 Результаты Аналогично, вероят- ность развития СД2 была наибольшей в  Томске (5,3%), а наименьшей в Волгограде (3,3%) и в Самаре (3,4%) в  мужской когорте. В  женской когорте риск варьировал от 6,6% в Томске и Красноярске до 5,0% в Самаре и Тюмени. Курение и потребление алкоголя у  женщин не  ассоциировалось с  высоким риском СД2, тогда как бросившие курить мужчины чаще имели повышенный риск СД2 как по  сравнению с  некурящими, так и  по  сравнению с  курящими. С  последними статистически незначимо. Анало- гично, все употребляющие алкоголь ассоциируются с  высоким риском СД2, чрезмерно употребляющие имели самый высокий риск. В зависимости от  количества полученных бал- лов, популяция была ранжирована по  категориям 10-летнего риска развития СД2 (рис. 1). Результаты В катего- рии низкого риска преобладают мужчины, во всех 16 16 ОРИГИНАЛЬНЫЕ СТАТЬИ Таблица 2 Таблица FINDRISС, средний балл и риск развития СД2 типа в течение 10-лет и однофакторный анализ показателей после коррекции на возраст, в российской популяции (по данным ЭССЕ-РФ) после коррекции на возраст, в российской популяции (по данным ЭССЕ-РФ) Мужчины Женщины Средний балл (m) Средний % ОШ 95% ДИ Р Средний балл (m) Средний % ОШ 95% ДИ Р Все 6,0 (0,04) 4,4 6,7 (0,01) 5,7 Образование Ниже среднего 6,0 (0,2) 4,5 1 7,0 (0,2) 6,1 1 Среднее 5,9 (0,06) 4,2 0,89 0,67 1,19 0,4283 7,0 (0,05) 5,9 0,91 0,74 1,11 0,3599 Выше среднего 6,2 (0,06) 4,7 1,07 0,80 1,42 0,6662 6,4 (0,05) 5,4 0,71 0,58 0,88 0,0015 Место проживания Город 6,1 (0,05) 4,5 1 6,7 (0,04) 5,7 1 Село 5,8 (0,01) 4,3 1,03 0,86 1,23 0,7473 6,9 (0,08) 5,7 1,05 0,94 1,18 0,3645 Семейное положение Никогда не был женат/ замужем 5,6 (0,15) 3,8 1 6,6 (0,09) 5,6 1 Женат/замужем 6,1 (0,05) 4,5 1,33 1,02 1,74 0,0367 6,7 (0,05) 5,7 1,187 1,03 1,36 0,0150 Разведен/разведена 6,0 (0,15) 4,5 1,44 1,03 2,02 0,0334 6,7 (0,09) 5,5 1,007 0,85 1,19 0,9332 Вдовец/вдова 7,9 (0,86) 8,0 1,26 0,74 2,15 0,3875 6,6 (0,32) 5,5 1,167 0,98 1,38 0,0739 Социальное положение Высоко обеспеченные 6,0 (0,8) 3,2 1 5,5 (0,5) 3,9 1 Обеспеченные 6,1 (0,05) 4,6 3,03 0,39 23,72 0,2898 6,6 (0,05) 5,6 1,45 0,68 3,09 0,3345 Средний достаток 5,9 (0,09) 4,3 3,06 0,39 23,98 0,2862 6,8 (0,07) 5,7 1,51 0,71 3,22 0,2853 Малообеспеченные 5,9 (0,15) 4,2 2,96 0,37 23,27 0,3032 7,0 (0,1) 6,0 1,70 0,80 3,64 0,1701 Бедные 5,9 (0,31) 4,0 3,73 0,46 30,07 0,2158 7,2 (0,25) 6,7 2,38 1,10 5,18 0,0282 Статус курения Никогда не курили 5,8 (0,07) 4,2 1 6,6 (0,04) 5,7 1 Бросили курить 6,5 (0,08) 5,1 1,34 1,14 1,58 0,0004 6,8 (0,1) 5,6 0,98 0,84 1,14 0,7949 Курят сейчас 5,8 (0,1) 4,3 1,03 0,88 1,21 0,6896 7,0 (0,19) 6,0 0,98 0,84 1,14 0,7955 Потребление алкоголя Никогда в течение года 5,7 (0,09) 4,0 1 6,7 (0,07) 5,7 1 Редко 6,0 (0,07) 4.4 1,23 1,03 1,48 0,0233 6,7 (0,05) 5,7 0,98 0,89 1,09 0,7819 Мало 6,0 (0,09) 4,3 1,22 0,99 1,57 0,0574 6,6 (0,09) 5,7 0,98 0,84 1,14 0,8187 Умеренно 6,5 (0,14) 4,9 1,28 0,97 1,68 0,0804 7,0 (0,17) 6,2 1,05 0,83 1,34 0,6677 Чрезмерно 7,2 (0,18) 5,9 2,01 1,48 2,71 0,0001 7,6 (0,27) 6,8 1,31 0,91 1,88 0,1491 Регионы Оренбург 5,3 (0,13) 3,4 1 6,6 (0,13) 5,6 1 Владивосток 6,5 (0,14) 4,9 1,99 1,42 2,80 0,0001 6,4 (0,11) 4,8 0,75 0,60 0,94 0,0131 Красноярск 6,5 (0,16) 5,1 2,15 1,50 3,09 0,0001 7,3 (0,14) 6,6 1,41 1,11 1,78 0,0041 Волгоград 5,2 (0,16) 3,3 1,10 0,72 1,67 0,6618 6,5 (0,13) 5,6 1,04 0,82 1,30 0,7498 Вологда 5,9 (0,14) 4,0 1,33 0,92 1,92 0,1269 6,4 (0,13) 5,3 0,95 0,74 1,22 0,7092 Воронеж 6,2 (0,17) 4,9 1,95 1,36 2,79 0,0003 7,0 (0,16) 6,4 1,39 1,12 1,72 0,0030 Иваново 6,5 (0,15) 4,8 2,05 1,43 2,93 0,0001 6,8 (0,12) 6,1 1,31 1,06 1,63 0,0135 Кемерово 6,5 (0,15 5,1 2,16 1,53 3,07 0,0001 7,3 (0,14) 6,5 1,32 1,05 1,66 0,0161 Самара 5,1 (0,13) 3,4 1,16 0,78 1,71 0,4621 6,2 (0,13) 5,0 0,88 0,69 1,13 0,3248 Санкт-Петербург 6,2 (0,17) 4,8 1,93 1,34 2,79 0,0004 6,2 (0,13) 4,7 0,72 0,57 0,91 0,0054 Томск 6,7 (0,16) 5,3 2,16 1,52 3,08 0,0001 7,4 (0,13) 6,6 1,35 1,07 1,69 0,0109 Тюмень 5,3 (0,17) 3,6 1,12 0,74 1,68 0,5995 6,2 (0,11) 5,0 0,82 0,66 1,02 0,0824 Р.С.Осетия (Владикавказ) 6,3 (0,15 4,5 1,53 1,07 2,19 0,0203 6,9 (0,11) 6,0 1,07 0,86 1,31 0,5502 Примечание: FINDRISС — FINnish Diabetes Risk Score; % — средний риск развития СД 2 типа в течение 10-лет, Р. Результаты С. Осетия — Республика Северная Осетия. других  — женщины. Согласно финской модели, в Российской популяции 7,4% лиц имеющих высо- кий и очень высокий риск, в том числе 4,1% муж- чин и 7,5% женщин. При анализе соответствия пороговых значений финской модели для российской популяции оказа- лось, что для нашей страны порог высокого риска СД2 несколько ниже и составляет ≥12 баллов. Про- 17 17 17 17 Российский кардиологический журнал № 9 (149) | 2017 Российский ка верка надежности данного порога с помо анализа показала, что полученная площад Рис. 2. ROC-анализ высокой шкалы FINDRISC (≥12 баллов) по дования ЭССЕ-РФ. Сокращения: TP (True Positives) — верно классифицированн ные примеры (истинно положительные случаи), FP (False Posi тельные примеры, классифицированные как положительные тельные случаи) (ошибка II рода), AUC – Area Under the Curve кривой). Рис. 3. Показатели, предсказывающие высокий риск СД2 (≥1 сийских мужчин и женщин участников исследования ЭССЕ-РФ по FINDRISC. Сокращения: Н/сред  — образование ниже среднего, ОШ шансов. 0 25 50 75 100 0 25 50 75 Доля истинно положительных Доля ложноположительных FP1 TP AUC= Мужчины 0,8 0,9 1 1,1 1,2 1,3 1,4 1,5 1,6 Экс-курение Употребление алкоголя Женщ 0,8 0,9 1 1,1 1,2 1,3 1,4 1,5 1,6 Замужние Небог Рис. 2. ROC-анализ высокой шкалы FINDRISC (≥12 баллов) по данным иссле- дования ЭССЕ-РФ. 0 25 50 75 100 0 25 50 75 100 Доля истинно положительных Доля ложноположительных FP1 TP AUC=0,763 у  мужчин не  ассоциируется с  риском диабета, а  вот образованные женщины реже имеют повышенный риск. Женатые и  разведенные мужчины достоверно чаще имеют риск развития СД2, p<0,04. Замужние женщины также чаще имеют риск СД2, p<0,02, а также более чем в 2 раза чаще имеют риск СД 2 женщины с очень низким достатком, p<0,03. Бросившие курить мужчины имеют на  34,4% чаще высокий риск СД2. У мужчин, употребляющих алкоголь, достоверно чаще выявляется высокий риск СД2, особенно у  тех, кто пьет чрезмерно. Наиболее высокий риск СД2 зареги- стрирован у женщин Красноярска, Воронежа и Томс- ка; наиболее низкий  — во  Владивостоке, Самаре, Санкт-Петербурге и  Тюмени. У  мужчин риск СД2 выше в  Красноярске, Томске, Кемерово, Иваново, и ниже в Волгограде, Самаре и Тюмени. 0 25 50 75 100 0 25 50 75 100 Доля истинно положительных Доля ложноположительных FP1 TP AUC=0,763 Многофакторный анализ, представленный на  рисунке 3, продемонстрировал сохраняющуюся ассоциацию возраста у мужчин и женщин с высоким риском СД2, соответствующим российскому крите- рию ≥12 баллов. Результаты После коррекции по возрасту и реги- ону оказалось, что высокий риск СД2 у  мужчин не ассоциируется ни с одним из социальных показа- телей, а лишь с отказом от курения и употреблением алкоголя. Напротив, у женщин чаще выявлялся высо- кий риск СД2 при низком доходе, низком образова- тельном уровне и у замужних. Статус курения и упо- требления алкоголя у  женщин не  связан с  высоким риском СД2. Рис. 2. ROC-анализ высокой шкалы FINDRISC (≥12 баллов) по данным иссле- дования ЭССЕ-РФ. Сокращения: TP (True Positives) — верно классифицированные положитель- ные примеры (истинно положительные случаи), FP (False Positives) — отрица- тельные примеры, классифицированные как положительные (ложноположи- тельные случаи) (ошибка II рода), AUC – Area Under the Curve (площадь под кривой). Сокращения: TP (True Positives) — верно классифицированные положитель- ные примеры (истинно положительные случаи), FP (False Positives) — отрица- тельные примеры, классифицированные как положительные (ложноположи- тельные случаи) (ошибка II рода), AUC – Area Under the Curve (площадь под кривой). Рис. 3. Показатели, предсказывающие высокий риск СД2 (≥12 баллов) у рос- сийских мужчин и женщин участников исследования ЭССЕ-РФ, рассчитанный по FINDRISC. Сокращения: Н/сред  — образование ниже среднего, ОШ  — отношение шансов. Мужчины 0,8 0,9 1 1,1 1,2 1,3 1,4 1,5 1,6 Экс-курение Употребление алкоголя Женщины 0,8 0,9 1 1,1 1,2 1,3 1,4 1,5 1,6 Замужние Небогатые Н/сред Мужчины 0,8 0,9 1 1,1 1,2 1,3 1,4 1,5 1,6 Экс-курение Употребление алкоголя Женщины 0,8 0,9 1 1,1 1,2 1,3 1,4 1,5 1,6 Замужние Небогатые Н/сред Обсуждение и  2,4% лиц, соответственно [12], в  Кыргызстане высокий риск имели 9,6% и очень высокий — 1,0%, в  том числе мужчины и  женщины имели высокий риск  — 5,7% и  10,8%, и  очень высокий  — 0,2% и 1,2%, соответ­ственно [13], а в Казахстане высокий и  очень высокий риск развития СД2 отмечался у  16,3% и  был значительно выше среди женщин по сравнению с мужчинами (20,6% и 14,2%, р<0,001, соответственно) [14]. ИМТ и возраст были достоверно выше по сравнению с городской когортой [6]. В нашем исследовании многофакторный анализ продемонстрировал, что высокий риск СД2 у муж- чин не  ассоциируется ни  с  одним из  социальных показателей, а лишь с отказом от курения и употре- блением алкоголя. Повышение риска развития СД2 у  бросивших курить хорошо объясняется извест- ным фактом увеличения массы тела у тех, кто бро- сает курить, если этот процесс не сопровождается повышением физической активности и  соответ­ ствующей диетой [20]. Ожирение, в свою очередь, является важнейшим фактором риска развития диабета [21]. Высокое потребление алкоголя также может приводить к ожирению, учитывая калорий- ность алкоголя [22]. У  женщин, наоборот, чаще выявлялся высокий риск СД2 при низком доходе, низком образовательном уровне и у замужних. Ста- тус курения и  употребления алкоголя у  женщин не связан с высоким риском СД2, возможно, из-за низкой распространенности этих факторов в  рос- сийской популяции. Близкие данные получены в  исследовании, проведенном в  Финляндии, в котором женщины 45-64 лет с уровнем образова- ния ниже среднего имели более высокий риск СД2 по сравнению с женщинами с более высоким уров- нем образования и  после поправки на  ожирение и  ФР (курение, алкоголь, физическую активность и  диету) эта ассоциация с  высоким риском (≥15 баллов) сохранилась. Зависимость между уровнем дохода и риском СД2 у женщин отмечалась только при поправке на возраст (ОШ 1,87; 95% ДИ: 1,08- 3,22), а в полной модели эта связь исчезала (р>0,05). У  мужчин “высокий риск” по  FINDRISC также ассоциировался с уровнем образования ниже сред- него. Ассоциации риска СД2 с  уровнем дохода у мужчин не выявлена (р>0,05) [23]. В Европейских странах исследования с использо- ванием опросника FINDRISC показали существен- ные вариации показателей высокого и  очень высо- кого риска от  более чем 25% в  Германии до  15% в Финляндии [15-17]. Надежность шкалы FINDRISC в  российской популяции без СД2 (данные ЭССЕ-РФ), определен- ная с  помощью ROC-анализа, продемонстрировала хорошее качество модели. Полученная площадь под кривой (AUC), “высокой” шкалы FINDRISC ≥12 баллов, составила — 0,76. Обсуждение Схожие данные были полу- чены в Новосибирской когорте AUC — 0,73 (у муж- чин AUC — 0,73, у женщин AUC — 0,70), что важно, учитывая одинаковый порог высокого уровня риска в  наших популяциях. Интересно, что в  финской популяции (2002г) AUC составила 0,72 у  мужчин и  0,70 у  женщин, то  есть, для финской популяции порог 15 баллов является также достаточно надеж- ным, однако этот факт свидетельствует о  различии уровня риска в различных популяциях [8, 9]. Согласно полученным критериям, доля лиц высокого и очень высокого риска в  российской популяции сущест- венно возрастает, составляя 20,4% в  целом и  14,2% и 24,4%, что свидетельствует о неблагоприятном про- гнозе в отношении СД в нашей стране. В других зарубежных исследованиях рассчитыва- лась вероятность заболеваемости СД2 с  использова- нием модифицированных шкал, в которую вносились различные параметры признаков в  зависимости от  которых отмечался различный риск предполагае- мой заболеваемости СД2, например: США 17,7% (19,4 — мужчины и 18,6% — женщины) и 5,7% [18, 19]. Надо отметить, что ROC-анализ вышеперечисленных шкал демонстрировал хорошее, очень хорошее и отличное качество построенных моделей для иссле- дуемой популяции. Интересно, что при всем разно­ образии риск развития СД2 в разных странах не пре- вышал 10%, тогда как в США он составил 17,7%. Обсуждение Средний 10-летний риск развития СД2, рассчи- танный по  FINDRISC, в  российской популяции составил 5,3% в целом (4,4% у мужчин и 5,7% у жен- щин). К  российским расчетам риска по  шкале FINDRISC близкие показатели были получены в финском исследовании — 4,1% [7]. В голландском исследовании 5-летний диапазон заболеваемости СД2, рассчитанный с  помощью шкалы FINDRISC, составил 2,3-9,9% [11]. Рис. 3. Показатели, предсказывающие высокий риск СД2 (≥12 баллов) у рос- сийских мужчин и женщин участников исследования ЭССЕ-РФ, рассчитанный по FINDRISC. Сокращения: Н/сред  — образование ниже среднего, ОШ  — отношение шансов. Данные, полученные в  Сибирской популяции (n=8050, возраст 45-69 лет, мужчин — 45,6%, Новоси- бирский городской регистр 2003-2014гг) близки к данным исследования ЭССЕ-РФ. Так, незначитель- ный FINDRISC отмечался у  28,8% лиц, незначи- тельно повышенный у  39,5%, умеренный, высокий и очень высокий у 22,9%, 8,4% и 0,4% лиц, соответ­ ственно [9]. Однако, надо отметить, что в  Новоси- бирской популяции умеренный риск был вдвое выше, чем в  исследовании ЭССЕ-РФ, что, вероятно, свя- зано с более пожилой популяцией. верка надежности данного порога с помощью ROC- анализа показала, что полученная площадь под кри- вой (AUC) составила — 0,76, что соответствует хоро- шему качеству модели (рис. 2). Таким образом, доля лиц высокого и  очень высокого риска существенно возрастает, составляя 20,4% в целом и 14,2% и 24,4%, соответственно, у мужчин и женщин, что почти в три раза выше, чем с  использованием критериев Фин- ской модели. В таблице 2 представлены также результаты одно- факторного анализа логистической регрессии “высо- кого” риска по FINDRISC ≥12 баллов у мужчин и жен- щин в  зависимости от  социальных и  поведенческих факторов при коррекции на  возраст. Образование Сравнение результатов опроса по  FINDRISC в  постсоветских республиках и  странах Европы показало неоднородность данных. В  исследовании 2011г, проведенном в республике Беларусь, высокий и  очень высокий FINDRISC был отмечен у  13,1% 18 18 ОРИГИНАЛЬНЫЕ СТАТЬИ и  2,4% лиц, соответственно [12], в  Кыргызстане высокий риск имели 9,6% и очень высокий — 1,0%, в  том числе мужчины и  женщины имели высокий риск  — 5,7% и  10,8%, и  очень высокий  — 0,2% и 1,2%, соответ­ственно [13], а в Казахстане высокий и  очень высокий риск развития СД2 отмечался у  16,3% и  был значительно выше среди женщин по сравнению с мужчинами (20,6% и 14,2%, р<0,001, соответственно) [14]. Литература Алгоритмы специализированной медицинской помощи больным сахарным диабе- том. Сахарный диабет 2017; 20: 1S: 1-112). 16. Salinero-Forta MA, Carrillo-de Santa Paua E, Abanades-Herranza JC, et al. en nombre del Grupo MADIABETES. Riesgo basal de Diabetes Mellitus en Atencion Primaria segun cuestionario FINDRISC, factores asociados y evolucion clınica tras 18 meses de seguimiento. Rev Clin Esp 2010; 9: 448-53. 6. Dedov II, Shestakova MV, Galstyan GR. The prevalence of type 2 diabetes mellitus in the adult population of Russia (NATION study). Diabetes Care 2016; 19 (2): 104-12. Russian (Дедов И. И., Шестакова М. Д., Галстян Г. Р. Распространенность сахарного диабета 2 типа у  взрослого населения России (исследование NATION). Сахарный диабет 2016; 19 (2): 104-12). 17. Karolinska Institutet, Department of Biosciences and Nutrition. Evaluation of the German Diabetes Risk Score as a screening tool for undiagnosed diabetes Christin Heidemann, Gert Mensink, Birgitta Strandvik. Master Thesis in Applied Public Health Nutrition (30 ECTS). Master Course in Applied Public Health Nutrition (120 ECTS), 2012: 47. 18. Kahn HS, Cheng YJ, Thompson TJ, et al. Two risk-scoring systems for predicting incident diabetes mellitus in US adults age 45 to 64 years. Ann Intern Med 2009; 150: 741-51. 7. Lindström J, Tuomilehto J. The diabetes risk score: a practical tool to predict type 2 diabetes risk. Diabetes Care 2003; 26 (3): 725-31. 19. Kanaya AM, Wassel Fyr CL, de Rekeneire N, et al. Predicting the development of diabetes in older adults: the derivation and validation of a prediction rule. Diabetes Care 2005; 28: 404-8. 8. Saaristo T, Peltonen M, Lindström J, et al. Cross-sectional evaluation of the Finnish Diabetes Risk Score: a tool to identify undetected type 2 diabetes, abnormal glucose tolerance and metabolic syndrome. Diab Vasc Dis Res. 2005; 2: 67-72. 20. Melnichenko GA, Butrova SA, Larina AA. The effect of smoking on health and body weight. Obesity and Metabolism 2010; 1: 15-9. Russian (Мельниченко Г. А., Бутрова С. А., Ларина А. А. Влияние табакокурения на  здоровье и  массу тела. Ожирение и  мета- болизм 2010; 1: 15-9). 9. Mustafina SV, Rymar OD, Sazonova OV, et al. Validation of the Finnish diabetes risk score (FINDRISC) for the Caucasian population of Siberia. Epidemiology 2016; 19 (2): 113-8. Russian (Мустафина С. В., Рымар О. Д., Сазонова О. В., и др. Валидизация финской шкалы риска “FINDRISC” на европеоидной популяции Сибири. Эпидемиология 2016; 19 (2): 113-8). 21. Butrova SA. From the epidemic of obesity to the epidemic of diabetes. Литература 1. ESC Guidelines on diabetes, pre- diabetes, and cardiovascular diseases developed in collaboration with the EASD. Russ J Cardiol 2014; 3 (107): 7-61. Russian (Рекомендации по  диабету, преддиабету и  сердечно-сосудистым заболеваниям. EASD/ESC. Российский кардиологический журнал 2014; 3 (107): 7-61). 12. 12. Shepelkevich AP, Khoiodova EA, Salko OB, et al. Assessment of risk of type 2 diabetes mellitus development in the republic of Belarus. Healthcare 2012; 11: 4-8. Russian (Шепелькевич А. П., Холодова Е. А., Салко О. Б., и др. Оценка риска развития сахар- ного диабета 2-го типа в республике Беларусь. Здравоохранение 2012; 11: 4-8). 2. WHO data, Global Diabetes Report: Newsletter, November 2016. [Electronic source] WHO website: http://www.who.int WHO / Diabetes (date of circulation: 10.10.2017). (Данные ВОЗ, Глобальный доклад по  диабету: информационный бюллетень, ноябрь 2016. [Электронный источник] сайт ВОЗ: http://www.who.int WHO/Diabetes. Дата обраще- ния: 10.04.2017). 13. Knyazeva VG, Sultanalieva RB. Using the questionnaire FINDRISC as an effective tool for assessing the risk of violations of carbohydrate metabolism in Kyrgyzstan. International Scientific Journal The Symbol of Science 2016; 26: 158-61. Russian (Князева В. Г., Султаналиева Р. Б. Использование анкеты FINDRISC как эффективного инструмента оценки риска нарушений углеводного обмена в Кыргызстане. Международный науч- ный журнал Символ науки 2016; 26: 158-61). 3. Atlas of Diabetes, IDF Seventh Edition, 2015. [Electronic Source] http://www.idf.org International Diabetes Federation Diabetes Atlas. 7th ed., 2015 (date of circulation: 04.10.2017). (Атлас Диабета, IDF седьмое издание, 2015. [Электронный источник] http://www.idf.org International Diabetes Federation Diabetes Atlas. 7th ed., 2015. Дата обращения: 10.04.2017). 14. Sheryazdanova DN, Turmukhambetova AA, Laryushina YeM, et al. Type 2 diabetes mellitus ten-year risk estimation in Karaganda region: populational cross-sectional study. Medicine and ecology 2016; 2: 64-9. Russian (Шерьязданова Д. Н., Ларюшина Е. М., Турмухамбетова А. А., и др. Оценка десятилетнего риска развития сахарного диабе- та 2 типа в Карагандинской области: популяционное кросс-секционное исследова- ние. Медицина и экология 2016; 2: 64-9). 4. WHO data on the incidence of diabetes, 2016. [Electronic source] / http://www.who. int WHO/Diabetes (date of circulation: 10.04.2017). (Данные ВОЗ по заболеваемости диабетом, 2016. [Электронный источник] http://www.who.int WHO/Diabetes. Дата обращения: 10.04.2017). 15. Viitasalo K, Lindström J, Hemiö K, et al. Occupational health care identifies risk for type 2 diabetes and cardiovascular disease. Prim Care Diabetes 2012; 6: 95-102. diabetes and cardiovascular disease. Prim Care Diabetes 2012; 6: 95-102. 5. Dedov II, Shestakova MV, Mayrova AYu. Standards of specialized diabetes care. Diabetes mellitus 2017; 20: 1S: 1-112. Russian (Дедов И. И., Шестакова М. В., Майорова А. Ю. Заключение По данным исследования ЭССЕ-РФ, 10-летний риск развития СД2 в российской популяции по кри- териям FINDRISC составил 5,3%; 4,4% у  мужчин и 5,7% у женщин. В регионах РФ выявлена различная распростра- ненность 10-летнего риска СД2. Многофакторный анализ с поправкой на возраст и регионы прожива- ния продемонстрировал, что высокий риск СД2 по  российским критериям (FINDRISC ≥12 баллов) достоверно чаще выявляется у  малообеспеченных женщин, при низком образовательном уровне и у замужних. У мужчин после поправки на возраст и  регион проживания, ассоциированные с  высоким риском СД2 социально-демографические показатели не  выявлены, но  высокий риск чаще выявляется у бросивших курить и потребляющих алкоголь. В исследовании ЭССЕ-РФ средний риск развития СД2 у  городских жителей значимо не  различался от  сельских, но  достоверно варьировал в  регионах. В  российском исследовании NATION частота слу- чаев СД2 была значимо выше у  жителей сельских поселений по сравнению с городскими (6,7% против 5,0%, p<0,001). Авторы исследования объясняют дан- ные различия тем, что у  сельских жителей средний 19 19 Российский кардиологический журнал № 9 (149) | 2017 Литература International Endocrinology Journal 2013; 2 (50): 19-24. Russian (Бутрова С. А. От эпидемии ожире- ния к эпидемии сахарного диабета. Международный эндокринологический журнал 2013; 2 (50): 19-24). 10. Boytsov SA, Chazov EI, Shlyakhto EV, et al. Epidemiology of cardiovascular diseases in different regions of Russia (ESSE-RF). The rationale for and design of the study. Preventive medicine 2013; 6: 25-34. Russian (Бойцов С. А., Чазов Е. И., Шляхто Е. В., Шальнова С. А., Конради А. О. и др. Эпидемиология сердечно-сосудистых заболева- ний в различных регионах России (ЭССЕ-РФ). Обоснование и дизайин исследова- ний. Журнал Профилактическая медицина 2013; 6: 25-34). 22. Sun K, Ren M, Liu D, et al. Alcohol consumption and risk of metabolic syndrome: a meta- analysis of prospective studies. Clin Nutr 2014; 33 (4): 596-602. 23. Wikström K, Lindström J, Tuomilehto J, et al. Socio-economic differences in dysglycemia and lifestyle-related risk factors in the Finnish middle-aged population. Eur J Public Health 2011; 21 (6): 768-74. 23. Wikström K, Lindström J, Tuomilehto J, et al. Socio-economic d 11. Alssema M, Vistisen D, Heymans MW, et al. The Evaluation of Screening and Early Detection Strategies for Type 2 Diabetes and Impaired Glucose Tolerance (DETECT-2) update of the Finnish diabetes risk score for prediction of incident type 2 diabetes. Diabetologia 2011; 54: 1004-12. 20 20
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Analysis of High Force Voice Coil Motors for Magnetic Levitation
Actuators
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Received: 29 September 2020; Accepted: 5 December 2020; Published: 9 December 2020 Abstract: A voice coil motor is a simple and linear electromagnetic actuator. Since it has a non-contact force and very low stiffness, it is widely used for precision positioning devices including magnetic levitation systems. During magnetic levitation, high force of a voice coil motor is required to compensate for the weight of the device and ensure a fast dynamic response. In this paper, two types of voice coil motors were analyzed by their volumetric change. The change of the generated force according to the volumetric change was inspected by finite element simulation models. The enhancement of force was dependent on which type of the voice coil motor is used, which component is enlarged, and which direction is the voice coil motor expands in. Based on the analysis results, two voice coil motors were optimally designed for a magnetic levitation positioning device. As a result of the design, it was confirmed that different types of voice coil motor generate different forces even if they have the same volume. For the two types of voice coil motors, the force differed by up to 40%. Keywords: voice coil motor; high force; magnetic levitation; positioning device Actuators 2020, 9, 133; doi:10.3390/act9040133 Article Jae-Yeol Kim and Dahoon Ahn * Department of Mechanical Engineering, Kongju National University, Cheonan 31080, Korea; jaeyeol@smail.kongju.ac.kr * Correspondence: dhahn@kongju.ac.kr; Tel.: +82-41-521-9263 Jae-Yeol Kim and Dahoon Ahn * Department of Mechanical Engineering, Kongju National University, Cheonan 31080, Korea; jaeyeol@smail.kongju.ac.kr * Correspondence: dhahn@kongju.ac.kr; Tel.: +82-41-521-9263 www.mdpi.com/journal/actuators actuators Article Analysis of High Force Voice Coil Motors for Magnetic Levitation Jae-Yeol Kim and Dahoon Ahn * Department of Mechanical Engineering, Kongju National University, Cheonan 31080, Korea; jaeyeol@smail.kongju.ac.kr * Correspondence: dhahn@kongju.ac.kr; Tel.: +82-41-521-9263 Received: 29 September 2020; Accepted: 5 December 2020; Published: 9 December 2020   Abstract: A voice coil motor is a simple and linear electromagnetic actuator. Since it has a non-con force and very low stiffness, it is widely used for precision positioning devices including magn levitation systems. During magnetic levitation, high force of a voice coil motor is required to compens for the weight of the device and ensure a fast dynamic response. In this paper, two types of vo coil motors were analyzed by their volumetric change. The change of the generated force accord to the volumetric change was inspected by finite element simulation models. The enhancemen force was dependent on which type of the voice coil motor is used, which component is enlarg and which direction is the voice coil motor expands in. Based on the analysis results, two voice motors were optimally designed for a magnetic levitation positioning device. As a result of the des it was confirmed that different types of voice coil motor generate different forces even if they have same volume. For the two types of voice coil motors, the force differed by up to 40%. actuators Article Analysis of High Force Voice Coil Motors for Magnetic Levitation Jae-Yeol Kim and Dahoon Ahn * Department of Mechanical Engineering, Kongju National University, Cheonan 31080, Korea; jaeyeol@smail.kongju.ac.kr * Correspondence: dhahn@kongju.ac.kr; Tel.: +82-41-521-9263 Received: 29 September 2020; Accepted: 5 December 2020; Published: 9 December 2020   Abstract: A voice coil motor is a simple and linear electromagnetic actuator. Since it has a non-con force and very low stiffness, it is widely used for precision positioning devices including magn levitation systems. During magnetic levitation, high force of a voice coil motor is required to compens for the weight of the device and ensure a fast dynamic response. In this paper, two types of vo coil motors were analyzed by their volumetric change. The change of the generated force accord to the volumetric change was inspected by finite element simulation models. The enhancemen force was dependent on which type of the voice coil motor is used, which component is enlarg and which direction is the voice coil motor expands in. Based on the analysis results, two voice motors were optimally designed for a magnetic levitation positioning device. As a result of the des it was confirmed that different types of voice coil motor generate different forces even if they have same volume. For the two types of voice coil motors, the force differed by up to 40%. ice Coil Motors for onal University, Cheonan 31080, Korea; -521-9263 2020; Published: 9 December 2020   r electromagnetic actuator. Since it has a non-contact r precision positioning devices including magnetic gh force of a voice coil motor is required to compensate ynamic response. In this paper, two types of voice hange. The change of the generated force according e element simulation models. The enhancement of e coil motor is used, which component is enlarged, nds in. Based on the analysis results, two voice coil vitation positioning device. As a result of the design, motor generate different forces even if they have the tors, the force differed by up to 40%. ic levitation; positioning device as become an essential requirement for positioning ated industries. An air bearing guide, which is a neration and backlash, and thus can implement a ing friction guides because there is no friction [1–3]. positioning devices for fabrication and inspection rs and displays, in particular, as they require high uum environment and additional components are e complex and large. actuators Article Analysis of High Force Voice Coil Motors for Magnetic Levitation Jae-Yeol Kim and Dahoon Ahn * Department of Mechanical Engineering, Kongju National University, Cheonan 31080, Korea; jaeyeol@smail.kongju.ac.kr * Correspondence: dhahn@kongju.ac.kr; Tel.: +82-41-521-9263 Received: 29 September 2020; Accepted: 5 December 2020; Published: 9 December 2020   Abstract: A voice coil motor is a simple and linear electromagnetic actuator. Since it has a non-con force and very low stiffness, it is widely used for precision positioning devices including magn levitation systems. During magnetic levitation, high force of a voice coil motor is required to compens for the weight of the device and ensure a fast dynamic response. In this paper, two types of vo coil motors were analyzed by their volumetric change. The change of the generated force accord to the volumetric change was inspected by finite element simulation models. The enhancemen force was dependent on which type of the voice coil motor is used, which component is enlarg and which direction is the voice coil motor expands in. Based on the analysis results, two voice motors were optimally designed for a magnetic levitation positioning device. As a result of the des it was confirmed that different types of voice coil motor generate different forces even if they have same volume. For the two types of voice coil motors, the force differed by up to 40%. In addition, it has a limitation vibration as it has a multi-body structure in which eries to allow a multi-degree of freedom (DOF) [4]. ce, a moving component is a single body of light and ermore, there is no connection unit, such as a guide, d, making it suitable for achieving a high degree of em, and many studies have been conducted on it in ely used as an actuator in a maglev device in order to oke [8–14]. Since the VCM has a very simple structure over and the stator, it does not suffer from friction or actuators actuators Article Analysis of High Force Voice Coil Motors for Magnetic Levitation Jae-Yeol Kim and Dahoon Ahn * Department of Mechanical Engineering, Kongju National University, Cheonan 31080, Korea; jaeyeol@smail.kongju.ac.kr * Correspondence: dhahn@kongju.ac.kr; Tel.: +82-41-521-9263 1. Introduction In recent years, a high degree of precision has become an essential requirement for positioning systems in line with the development of the related industries. An air bearing guide, which is a contactless guide, has no problems with dust generation and backlash, and thus can implement a positioning system more precisely than any existing friction guides because there is no friction [1–3]. Thus, the air bearing guide is widely used in positioning devices for fabrication and inspection processes in the manufacturing of semiconductors and displays, in particular, as they require high precision. However, its use is limited in a vacuum environment and additional components are required to supply air, which makes the structure complex and large. In addition, it has a limitation in removing environmental disturbances such as vibration as it has a multi-body structure in which multiple moving components are connected in series to allow a multi-degree of freedom (DOF) [4]. Otherwise, in a magnetic levitation (maglev) device, a moving component is a single body of light and simple form, and its actuator is contactless. Furthermore, there is no connection unit, such as a guide, between the moving components and the ground, making it suitable for achieving a high degree of precision. Thus, a maglev device is the latest system, and many studies have been conducted on it in the last 30 years [4–14]. In particular, a voice coil motor (VCM) is widely used as an actuator in a maglev device in order to implement nanometer-level precision in a short stroke [8–14]. Since the VCM has a very simple structure and there is no mechanical contact between the mover and the stator, it does not suffer from friction or backlash, resulting in high precision and a rapid response. Moreover, the generated magnetic force is proportional to the current that flows along the coil, which is suitable for precision control. However, the VCM has the drawback of weaker force compared to its volume than contact-type actuators such www.mdpi.com/journal/actuators 2 of 16 Actuators 2020, 9, 133 as piezoelectric, magnetostrictive and electrostrictive actuators [15,16]. A moving component should always be levitated in a maglev positioning system, so the actuators should generate greater force than the weight of the moving component. To that end, several studies have been conducted to increase the force of the VCM. Hollis et al. 1. Introduction proposed a VCM of two sets of magnet pairs facing to each other to implement a maglev wrist using a robot arm application [8]. Kim et al. also studied various forms of VCM using a permanent magnet and coil for maglev device applications [9,10]. Zhang and Meng studied a VCM composed of a double-sided magnet pair for a maglev device [11]. Choi et al. [17] and Ahn et al. [12] proposed a VCM with an axial symmetric rectangular structure that can produce considerable force, studied the optimal design method, and tested its mechanical and electrical characteristics. Lee et al. [18] optimized the shape of the permanent magnet and yoke by considering the magnetic saturation effect in order to maximize the force of a VCM with a steel and permanent magnet. Furthermore, other researchers aimed to improve the force of the VCM using a Halbach array. Choi et al. [13,19] utilized a permanent magnet for the weight compensation of a maglev device in a bid to propose a new VCM structure using a Halbach array. Kim et al. [14,20] designed a high-force VCM using a Halbach array and applied it to a six-DOF active vibration isolator and a six-DOF maglev positioning system. Dong et al. [21] used a Halbach magnet array by comparing three different VCM structures whose double-sided magnet array was different for large force and small force ripples. Jansen et al. [22] proposed a method of modeling the force and electrical characteristics of the VCM using a Halbach array. Although various studies have been conducted by changing the shape and layout of the magnet and coil in a VCM, the resulting increase of VCM force was insignificant, thus showing a limitation with regard to requirements such as fast response and large weight compensation of the levitated component. This was because most researchers attempted structural changes in order to generate greater force in a small volume while considering the applicability of the VCM. Thus, this study aimed to analyze changes in force when making a VCM large to generate greater force, which contrasted with existing studies that attempted to change the arrangement of the components such as a coil winding, permanent magnets, and yokes. 1. Introduction To do this, two types of VCM were selected, and a model for changing the volume of the VCMs, accordingly the size of the magnet and coil, was established in order to analyze the change in the generated force using the finite element method (FEM). Finally, two types of VCM were optimally designed for a maglev positioning device to prove the analysis results. 2. Structure of VCMs A VCM consists of a permanent magnet that generates a magnetic flux, a yoke that concentrates the magnetic flux in an air gap by manipulating the magnetic flux path, and a coil that generates force by flowing current. Figure 1 shows the basic structure of the two types of VCM analyzed in this study. Figure 1a shows the structure of a typical VCM. Two pairs of magnets are arranged on each side of the coil to form a strong magnetic flux in the perpendicular direction (z) to the air gap, and force is generated in the horizontal direction (x) by the current flowing along the coil in the perpendicular direction (y) to the magnetic flux. Thus, we call this type of VCM “horizontal force voice coil motor” (HVCM). Here, a yoke is attached to the upper and lower sides to concentrate the magnetic flux in the air gap. Figure 1b shows the rectangular type axis-symmetric VCM developed by Choi et al. [17], in which four pairs of magnets are arranged in an external yoke, while an internal yoke that is sufficiently thick to prevent magnetic saturation is positioned in the center. Two upper and lower coils are wrapped around the internal yoke. Here, a magnetic flux is formed in the horizontal direction (x and y) in the air gap between the permanent magnet and the center yoke, and force is generated in the vertical direction (z) as the current flows along the coil in the perpendicular direction (y and x) to the magnetic flux. Thus, we call this type of VCM “vertical force voice coil motor” (VVCM). In this research, how to increase the force of the VCM was studied as a measure for overcoming insufficient force, which is the 3 of 16 Actuators 2020, 9, 133 drawback of general VCMs. The force of the VCM is Lorentz force described by Equation (1), and the magnitude of the force is calculated by Equation (2). → F = i Z coil (d → l × → B ) = i Z coil Bdl sin θ (1) → F = |i| Z coil → B → dl sin θ (2) (1) → F = |i| Z coil → B → dl sin θ (2) (2) Figure 1. (a) Structure of a typical voice coil motor (VCM) with double-sided magnets, (b) Structure of a square column type VCM. 2. Structure of VCMs Figure 1 (a) Structure of a typical voice coil motor (VCM) with double-sided magnets (b) Structure Figure 1. (a) Structure of a typical voice coil motor (VCM) with double-sided magnets, (b) Structure of a square column type VCM. In Equations (1) and (2), i refers to the current, l refers to the length of the coil, B refers to the magnetic flux density in the coil, and θ refers to the angle between the flow direction of the current in the coil and the magnetic flux density. Force vector F can be calculated by integrating a cross product of the infinitesimal length vector multiplied by current and the magnetic flux density in each portion of the coil through the whole coil volume. From Equation (2), it is found that the force of the VCM can be improved by several methods. First, the generated force is proportional to the current in Equation (2). Thus, an increase in force can be obtained in proportion to the increase in the input current, regardless of the VCM’s structure or size. However, an increase in the current incurs increased heat dissipation due to the electric resistance of the coil, thereby inducing a temperature rise in the coil and the surrounding components. Thus, the possible input current can be determined according to the allowable temperature limit of the magnets, coils and others. Since the temperature rise and allowable temperature vary even in the same VCM depending on the operation conditions - such as the cooling 4 of 16 Actuators 2020, 9, 133 method, connection of the VCM to other structure, and the required force trajectory - the effect of an increase in current is excluded from this study. The force can increase when the value of integration of Equation (2) increases. In order to make the sine function term have the maximum value of 1, the flow direction of the current in the coil and the magnetic flux density should be perpendicular, which is implemented by the structure of the magnet and coil in the two types of VCMs, as shown in Figure 1. To increase the integration value in Equation (2), the volume of the coil (i.e. the integration volume) or the magnetic flux density in the volume occupied by the coil should increase. 2. Structure of VCMs Accordingly, either the number of turns of the coil winding, length, and width should be increased, or a larger magnet should be used. That is, the overall volume of the VCM should be increased. Therefore, this study analyzes a change in force according to the degree of volume change and the volume change method. 3.1. Design Parameters Expansion Direction Case Parameters Description Normalized Dimension (mm) Variation (mm) x(width) a mw Magnet width 52 52–104 b cw Coil width 50 10–100 mw Magnet width 52 10.4–104 y(length) c cl Coil length 50 10–100 ml Magnet length 50 10–100 z(height) d mh Magnet height 20 4–40 e ch Coil height 20 4–40 Table 2. Design Parameters of the VVCM. Expansion Direction Case Parameters Description Normalized Dimension (mm) Variation (mm) x, y(width) a mt Magnet thickness 20 4–40 b ct Coil thickness 20 4–40 c mw Magnet width 20 4–40 cw Coil width 20 4–40 z(height) d mh Magnet height 52 52–104 e ch Coil height 24 4–49 mh Magnet height 52 52–104 Table 1. Design Parameters of the HVCM. Expansion Direction Case Parameters Description Normalized Dimension (mm) Variation (mm) x(width) a mw Magnet width 52 52–104 b cw Coil width 50 10–100 mw Magnet width 52 10.4–104 y(length) c cl Coil length 50 10–100 ml Magnet length 50 10–100 z(height) d mh Magnet height 20 4–40 e ch Coil height 20 4–40 Table 2. Design Parameters of the VVCM. Table 2. Design Parameters of the VVCM. Expansion Direction Case Parameters Description Normalized Dimension (mm) Variation (mm) x, y(width) a mt Magnet thickness 20 4–40 b ct Coil thickness 20 4–40 c mw Magnet width 20 4–40 cw Coil width 20 4–40 z(height) d mh Magnet height 52 52–104 e ch Coil height 24 4–49 mh Magnet height 52 52–104 Figure 2 displays the method of changing the HVCM shape parameters as cross-sectional diagrams. Figure 2a–e corresponds to case (a)–(e), as shown in Table 1. Figure 2a,b refers to cases (a) and (b), where the HVCM volume changes in the x-direction, which is the width direction. Thus, the sizes of both magnets and yokes change in the direction perpendicular to the magnetization direction, and the change in the value is indicated by the change in the magnet’s width, as shown in Table 1. The coil’s width increases and decreases at the same rate as the change in the magnet’s width in case (b), whereas the coil’s width is fixed to the nominal size and each coil bundle is moved to be positioned in the center of each magnet in case (a). 3.1. Design Parameters As discussed in Section 2, when the volume of the VCM increases, the magnetic flux density increases, or the coil volume that is affected by the magnetic flux increases, expecting the increase in the force generated by the VCM. For a quantitative analysis of force using FEM, the parameters for changing the volume of the HVCM and the VVCM, and their directional shape change model, were established. Figures 2 and 3 show the method of changing their shape in detail, while Tables 1 and 2 present a detailed description of the parameters. The shape parameters can change from 20% to 200% based on the nominal size. Figure 2. Cont. Figure 2. Cont. 5 of 16 Actuators 2020, 9, 133 Figure 2. Variations of the horizontal force voice coil motor (HVCM)’s structure according to changes in the design parameters: (a) case (a), change of magnet width; (b) case (b), change of VCM width; (c) case (c) change of VCM length; (d) case (d) change of magnet height; (e) case (e) change of coil height. Figure 2. Variations of the horizontal force voice coil motor (HVCM)’s structure according to changes in the design parameters: (a) case (a), change of magnet width; (b) case (b), change of VCM width; (c) case (c) change of VCM length; (d) case (d) change of magnet height; (e) case (e) change of coil height. Figure 3. Cont. Figure 3. Cont. Figure 3. Cont. 6 of 16 Actuators 2020, 9, 133 Figure 3. Variations of the vertical force voice coil motor (VVCM)’s structure according to changes in the design parameters: (a) case (a), change of magnet thickness; (b) case (b), change of coil thickness; (c) case (c), change of VCM width; (d) case (d) change of magnet height; (e) case (e) change of VCM height. Figure 3. Variations of the vertical force voice coil motor (VVCM)’s structure according to changes in the design parameters: (a) case (a), change of magnet thickness; (b) case (b), change of coil thickness; (c) case (c), change of VCM width; (d) case (d) change of magnet height; (e) case (e) change of VCM height. Table 1. Design Parameters of the HVCM. Table 1. Design Parameters of the HVCM. 3.1. Design Parameters This is to consider the case of the largest force occurrence because the magnetic flux density by the permanent magnet is strongest in the point aligned with the center of the magnet. Here, if the magnet width is smaller than the nominal value of the coil width, part of the coil is placed outside the magnetic field of the permanent magnet, which does not generate force. Thus, such a case is excluded from the analysis. As a result, only the range in which the magnet width is larger than the coil width is checked in case (a). Figure 2c shows case (c), where the HVCM volume changes in the y-direction, which is the length direction. Thus, the sizes of both magnet and yoke 7 of 16 Actuators 2020, 9, 133 change in the direction perpendicular to the magnetization direction, and the coil is also interlocked, so its length also increases. If the coil’s length is not interlocked, so that the magnet becomes longer than the coil, force is generated in an unintended direction by the round part of both ends of the coil. Conversely, if the magnet is shorter than the coil, part of the coil may not generate force. Figure 2d,e show cases (d) and (e), where the HVCM volume changes in the z-direction, which is the height direction. In case (d), the magnet height increases, and the coil’s height is fixed to the nominal size. On the other hand, in case (e), the coil’s height increases, and the magnet’s height is fixed to the nominal size. Thus, the parts of the magnet or yoke marked with a dotted line move without any changes in size, maintaining the air gap with the coil. g g p Figure 3 shows the method of changing the VVCM’s shape parameters as cross-sectional diagrams. Figure 3a–e corresponds to case (a)–(e) as shown in Table 2. Figure 3a–c refer to case (a)–(c), where the VVCM volume changes in the x and y directions, that is, the width direction. In contrast with the HVCM, since the VVCM is symmetrical around the z-axis, the volume increases in the x and y directions simultaneously. For case (a), the size of the four pairs of magnets, whereby each pair is located up and down along the inner surface of the external yoke, change in the magnetization direction. 3.1. Design Parameters That is, the magnet’s thickness changes, which changes the location and size of the external yoke, thereby changing the overall size of the VVCM in the width direction. In contrast, in case (b), the coil’s thickness changes, and the four pairs of magnets move without any change in their size, maintaining the air gap with the coil. Accordingly, the location and size of the external yoke change, thereby changing the overall size of the VVCM in the width direction. Case (c) is a method in which the widths of both magnet and coil change together. Thus, the coil’s total length increases in case (c)m whereas the number of coil turns increases in case (b). Moreover, the size of the magnet changes in the direction perpendicular to the magnetization direction, in contrast with case (a). Accordingly, the location and size of the external yoke change, thereby changing the overall size of the VVCM in the width direction. Figure 3d,e refer to cases (d) and (e), where the VVCM volume changes in the z-direction, which is the height direction. Thus, the dimension of the magnet changes in the height direction perpendicular to the magnetization direction. However, the coil moves to be positioned in the point aligned with the center of each magnet, without any change in size in case (d), and the dimension changes at the same rate as that of the magnet in case (e). Similarly, to the previous case (b) of the HVCM, when the magnet height is smaller than the coil height, part of the coil is not affected by the magnetic field, which was excluded from the analysis. Figure 4 shows two extreme cases of HVCM design with the same volume. One in Figure 4a is expanded lengthwise and one in Figure 4b is expanded widthwise while both are keeping the same volume. As noticed from the two extreme cases, it is expected that the volume increase does not guarantee the force increase. The quantitative study by FEM models with the defined design parameters is followed. 3.2. The FEM Model Based on the design parameters shown in Tables 1 and 2, three-dimensional simulation models of two types of VCMs were established. For the FEM analysis, Maxwell V14.0 (Ansys, USA) was used. The current density in the coil cross-section was set to be constant to present the increase of coil turns according to the increase of the coil cross-sectional area. In addition, all the analyses were performed by designating an N-45 grade NdFeB magnet, and its characteristics are shown in Table 3. Figure 5 shows an example of a meshed model of two VCMs. The force applied to the coil and the magnetic flux density at the coil were calculated by the analysis. The analysis region was set to have 1000% offset size of the analyzed VCM model, and the medium was air. Each analysis was terminated when the energy error converged to less than 0.1%, and the maximum number of iterations was limited to 30. 8 of 16 Actuators 2020, 9, 133 Figure 4. Extreme variations of HVCM with the same volume, (a) Lengthwise expansion, (b) Widthwise expansion. Figure 4. Extreme variations of HVCM with the same volume, (a) Lengthwise expansion, (b) Widthwise expansion. Table 3. Properties of the NdFeB magnet. Table 3. Properties of the NdFeB magnet. Grade Remanence (T) Coercive Force (kA/m) N-45M 1.35 876 Figure 5. (a) Meshed simulation model of HVCM with nominal dimensions, (b) Meshed simulation model of VVCM with nominal dimensions. Figure 5. (a) Meshed simulation model of HVCM with nominal dimensions, (b) Meshed simulation model of VVCM with nominal dimensions. 3.3. Results Figure 6a shows the change in force according to the change in size of the HVCM. In all shape change methods, as the VCM volume increases, the force increases. However, there is a difference in force change according to the direction of the volume change. The increase in force in case (a)–(c), in which the HVCM increases in the horizontal direction, i.e., in the width and lengthwise direction, is significantly larger than that of cases (d) and (e), in which the HVCM increases in the height direction, i.e., the vertical direction. In particular, the increase in force in cases (b) and (c), where the volume of both coil and magnet increases, is more effective than case (a), where only the magnet’s volume increases. Case (a) is somewhat limited in terms of increasing the force as it has a saturation form after a certain level of increase. This directional effect of an increase in force can be explained more clearly through the change in the mean magnetic flux density according to the increase in the HVCM volume, as shown in Figure 6b. The increase in the magnetic flux density in case (a)–(c), in which the HVCM increases in the horizontal direction, that is, the width and lengthwise direction, is also significantly larger than that of cases (d) and (e), in which the HVCM increases in the height direction, i.e., in the vertical direction. Here, the increase in the mean magnetic flux density according to the 9 of 16 Actuators 2020, 9, 133 increase in the magnet size is the largest in case (a), and smaller in cases (b) and (c). As presented in Equation (2), the force of the VCM is affected by increases in both magnetic flux density and coil length, but the increase in coil volume is more effective than the increase in magnetic flux density, as shown in Figure 6a,b. In particular, the magnetic flux density increases in line with the increase in the magnet’s volume, but it becomes saturated beyond a certain level. Thus, when a large-scale HVCM is available, an increase in coil volume leads to a substantial increase in force, and an increase in a magnet size raises the magnetic flux, which only plays a role in maintaining the magnetic flux density at a certain level even if the coil’s volume increases. This trend is also revealed in cases (d) and (e). 3.3. Results In case (d), the magnetic flux density increases to some extent, and is then saturated as the magnet’s height increases, whereas in case (e), the magnetic flux density decreases as the distance between the magnets increases. Nonetheless, the force increases more in case (e) than in case (d), which indicates that the effect of a change in the coil volume is more significant than the effect of a change in the magnet volume. Figure 6. Cont. Figure 6. Cont. Figure 6. Cont. 10 of 16 Actuators 2020, 9, 133 Figure 6. Results of analysis of the HVCM, (a) Force, (b) Average magnetic flux density, (c) force increase per volume increase. 6. Results of analysis of the HVCM, (a) Force, (b) Average magnetic flux density, (c) force per volume increase. Figure 6. Results of analysis of the HVCM, (a) Force, (b) Average magnetic flux density, (c) force increase per volume increase. Finally, force change to volume change was presented regarding the change in the shape parameters to verify the efficiency of the increase in the volume for the increase in the force of the HVCM. The absolute value of the volume change was used to represent the increase and decrease in force using signs. As shown in Figure 6c, case (c) is most noticeable. That is, the increase in volume in the lengthwise directions of the magnet and coil is the most efficient method of increasing the force. As Figure 6a verified that the effect of the increase in volume in the height direction (cases (d) and (e)) was limited, Figure 6c also shows that the effect by height increase diminishes according to volume increase. Thus, the height of the coil and magnet is fixed to an appropriate value rather than increasing it, and the volume in the direction perpendicular to the height is desirable in order to increase the force of the HVCM. One more noticeable result is the case (b). When the volume increases in the width direction, the number of coil turns increases, thereby increasing the part of the coil that is not involved in the generation of force (as shown in Figure 7). It can cause distortion of the coil shape overall and generation of Lorentz force in the undesired direction. Thus, it is necessary to maintain a width to length ratio below a certain value. 3.3. Results Moreover, the difference between the results of the case (a) and (b) tells that the increase in the widths of the magnet and coil by interlocking them (cases (b)) is more efficient than increasing the width of the magnet only (case (a)). Thus, it is preferable to fix the magnet width to be no larger than necessary to motion compared to the coil width when designing the HVCM. Figure 8a shows the change in force according to the change in the VVCM size. Except for case (b), all cases showed that the generated force increased as the volume of the VVCM increased. The trend of increase in force differed depending on which part and direction of the VVCM increased in volume. The increase in force was larger in cases (c) and (e) of the VVCM, where the volume of both magnet and coil increased than in cases (a) and (d), where the volume of either the magnet or coil only increased. In addition, the increase in force was larger in case (e) than in case (c), and in case (d) than in case (a), respectively. In other words, force is larger in the cases where the volume increased in the vertical height direction than the cases where the volume increased in the horizontal width direction. In relation to the change in the mean magnetic flux density according to the increase in the VVCM volume, as shown in Figure 8b, only case (b) showed a rapid reduction of the magnetic flux density. Since the size of the magnet remained the same, while the coil’s volume increased, thereby increasing the air gap. Thus, the magnetic flux density decreased, accordingly the force decreased, as shown in Figure 8a, despite the increase in the coil volume. In both cases (a) and (c), the volume increased in the horizontal 11 of 16 Actuators 2020, 9, 133 width direction. The magnetic flux density increased in case (c), where the magnet dimension increased in the direction perpendicular to the magnetization direction, more than in case (a), where the magnet dimension increased in the direction parallel to the magnetization direction. Furthermore, the volume of coil also increased in case (c), thereby further increasing the force significantly. In both cases (d) and (e), the volume increased in the vertical height direction. 3.3. Results Evidently, case (d), in which the magnet size increased but the coil volume did not change, showed a higher magnetic flux density. However, the increase in force was larger in case (e), i.e., the same as the HVCM result, which indicates that the change in the coil volume influenced more in the change of force in the VVCM as well. Figure 7. Usable part of VCM coils for force generation, an example of normal winding (top) and an example of abnormally enlarged winding in the width direction (bottom). Figure 7. Usable part of VCM coils for force generation, an example of normal winding (top) and an example of abnormally enlarged winding in the width direction (bottom). Next, Figure 8c shows force change to volume change regarding the change in the shape parameters. The absolute value of the volume change was used to represent the increase and decrease in force using signs. The force is efficiently increased when the respective size of the coil and magnet increased simultaneously in width direction (case (c)) and height direction (cased (e)). Note that the value of case (c) is higher than the value of case (e), which contrasted with the results of force. This is because the increase in the VVCM’s overall volume according to the increase in the magnet’s width in case (c) was significantly smaller than the increase in the VVCM’s overall volume according to the increase in the magnet’s height in case (e). On the other hand, although case (d), in which only the magnet’s height increased, could obtain an increase in force, the force increase with respect to volume increase was diminished demonstrating significantly low efficiency of the volume increase. Therefore, when designing the VVCM, it is recommended that the height of the magnet is not set larger than necessary compared to the height of the coil. In case (a), a small change of volume affects largely on force change, but its effect rapidly diminishes. As a result, the generated force barely changes as shown in Figure 8a. Thus, selecting the proper thickness of the magnet and coil followed by increasing the VVCM volume in the width and height directions (case (c) and (e)) was found to be effective in increasing the force of the VVCM. 12 of 16 Actuators 2020, 9, 133 ults of analysis of the VVCM, (a) Force, (b) Average magnetic flux density, (c Figure 8. 4. VCM Designs for a Maglev Positioning Device Based on the results of the analysis presented in Section 3, two types of VCMs were designed for a precision maglev precision positioning device. Since a maglev device performs positioning while floating in the air and a VCM generates uni-directional force, for six-DOF motion control, it requires more than 6 VCMs. In this paper, eight VCMs were arranged as shown in Figure 9a by considering the symmetry. VCMs for a vertical motion were arranged at the four vertices, and VCMs for a horizontal motion were arranged along the four edges. Utilizing the VCMs at vertices, the maglev positioning device can achieve the out-of-plane motion, that is, motion in the z, θx, and θy directions. Similarly, utilizing the VCMs at edges, the maglev positioning device can achieve the in-plane motion, that is, motion in the x, y, and θz directions. In the maglev device, not only a space for VCMs but also a space in which to arrange the displacement sensor and the mechanical stopper for homing was also needed, as shown in Figure 9a,b. The mover of the maglev positioning device has a size of 250 mm × 250 mm × 40 mm. Accordingly, the given space for VCMs is limited as presented in Table 4. Because of this space arrangement and assembly, a type of VVCM that was suitable to be increased in the vertical direction was selected for a vertical motion, while a type of HVCM that was suitable to increase in the length direction was selected for a horizontal motion. The design of the VCM started with setting the outer size. Each VCM was set so that its outer size occupies the whole given volume to generate the maximum force. The gap between the coil and the magnet and the difference in width between the coil and magnet was set to 1mm in consideration of the motion range of the maglev positioning device. In the case of HVCM, the maximum force was obtained through the FEM analysis by adjusting the height of the coil and magnet in units of 1 mm through. In the case of VVCM, the maximum force was obtained through the FEM analysis by adjusting the thickness of the coil and magnet in units of 1 mm through FEM analysis. The final design parameters and specifications are shown in Table 4 and Figure 10. 3.3. Results Results of analysis of the VVCM, (a) Force, (b) Average magnetic flux density, (c) force density. 13 of 16 Actuators 2020, 9, 133 13 of 16 In summary, with the increase in the size of the HVCM and the VVCM, the magnetic flux density and coil volume increase, which in turn increases the force. In particular, the increase in the coil volume significantly influenced the increase in force, and the increase in the magnet volume raised the magnetic flux, thereby maintaining the magnetic flux density at a similar rate even when the coil’s volume increased. Thus, the magnet size must increase in link with the coil size. For the HVCM, only the method which increases both the size of the magnet and the coil in the lengthwise directions is found to be valid in terms of the manufacturability and the efficiency of the increase in force. Here, an increase in the coil volume should lead to an increase in the total length. For the VVCM, an improvement of force can be attempted by increasing the volume in the vertical or horizontal direction. Here, an increase in the coil volume in the vertical direction should lead to an increase in the number of accumulated turns by heightening the coil height, while an increase in the horizontal direction should lead to an increase in the total length by widening the coil width rather than the thickness. 5. Conclusions In this paper, two types of VCMs were selected and a shape change model was established. One of them had a typical structure and comprised a coil positioned between two pairs of magnets. The other had a rectangular shape with an external yoke consisting of four pairs of magnets and a rectangular inner yoke. An efficient method of improving the force was analyzed through the increase in VCM volume by employing the shape change model analysis of the two selected types of VCMs. 1. An increase in force can be attempted by increasing the VCM volume. However, the efficiency of an increase in force differs depending on the method and direction of the volume increase and the type of VCM. 1. An increase in force can be attempted by increasing the VCM volume. However, the efficiency of an increase in force differs depending on the method and direction of the volume increase and the type of VCM. 2. The HVCM’s force increased significantly when the volume increased in the width and the length direction. However, the most efficient method of enhancing force consisted of increasing the volume in the lengthwise direction while maintaining a certain ratio between the width and length in consideration of the coil fabrication. 3. The VVCM’s force increased significantly when the volume increased in the width and height directions. The total length of the coil increases when the VVCM width increases, and the number of coil turns increases when the VVCM height increases. 4. Two VCMs could efficiently increase the volume with appropriate direction, after selecting an appropriate thickness for the magnet and coil and setting the magnet’s size to be no larger than that of the coil. 4. Two VCMs could efficiently increase the volume with appropriate direction, after selecting an appropriate thickness for the magnet and coil and setting the magnet’s size to be no larger than that of the coil. Based on the results of the VCM analysis, two types of VCMs were designed. The maglev device should always maintain a levitation force that corresponds to its own weight for levitation of the moving component, as well as generating an inertial force, in order to achieve a fast motion. Accordingly, the HVCM and VVCM were designed to have high force for the given space limitation. 4. VCM Designs for a Maglev Positioning Device In addition to this, in order to show the effectiveness of the study, the force maximization of VVCM and HVCM was carried out assuming that the spaces given to each were changed. The comparison of the results is presented in Table 5. As can be seen from the Table 5, the results are very different depending on which type of VCM is optimized for the same volume of space. The incorrectly selected VCM only shows a force constant of about 60%. This confirms that the efficiency of increasing the force according to the volume expansion of the VCM is different for each direction of the volume expansion. Table 4. Space limitation and specifications of the fabricated HVCM and VVCM. Table 4. Space limitation and specifications of the fabricated HVCM and VVCM. HVCM VVCM Given space for VCM design (mm × mm × mm) 90 × 50 × 40 55 × 55 × 40 Force constant (N/A) 17.71 19.31 Electrical resistance (Ω) 4.50 2.73 Coil diameter without sheath (mm) 0.5 0.5 Number of coil turns 368 420 Moving mass (g) 471 530 14 of 16 14 of 16 Actuators 2020, 9, 133 Figure 9. (a) Placement of the VCMs on the mover of the maglev device (inverted in the picture), (b) Placement of the displacement sensors and a mechanical stopper for homing. Figure 9. (a) Placement of the VCMs on the mover of the maglev device (inverted in the picture), (b) Placement of the displacement sensors and a mechanical stopper for homing. Figure 10. The optimized design parameters (a) HVCM, (b) VVCM. Figure 10. The optimized design parameters (a) HVCM, (b) VVCM. 15 of 16 Actuators 2020, 9, 133 Table 5. Comparison of force constant for each optimized design result. Table 5. Comparison of force constant for each optimized design result. Table 5. Comparison of force constant for each optimized design result. Horizontal Force Generation Vertical Force Generation Given Space for VCM Design (mm × mm × mm) 90 × 50 × 40 55 × 55 × 40 HVCM (N/A) 17.71 5.98 VVCM (N/A) 10.23 9.65 Horizontal Force Generation Vertical Force Generation Given Space for VCM Design (mm × mm × mm) 90 × 50 × 40 55 × 55 × 40 HVCM (N/A) 17.71 5.98 VVCM (N/A) 10.23 9.65 5. Conclusions In this paper, we analyzed the force increase according to the VCM volume increase, and it is expected that the data presented here can be useful when designing the VCM. It can provide a starting point for the design, such as what type of VCM to select according to the constraints such as a given space, and how much the initial values of magnets and coils should be. It can also be used as a reference when deciding in which direction the size should be increased during design refinement. Author Contributions: Formal analysis and original draft preparation, J.-Y.K.; Conceptualization, methodology and software, and review and editing, D.A. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2019R1G1A1099736). Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 3. Ro, S.K.; Park, J.K. A compact ultra-precision air bearing stage with 3-DOF planar motions using electromagnetic motors. Int. J. Precis. Eng. Manuf. 2011, 12, 115–119. [CrossRef] 2. Ro, S.K.; Kim, S.; Kwak, Y.; Park, C.H. A linear air bearing stage with active magnetic preloads for ultraprecise. Precis. Eng. 2010, 34, 186–194. [CrossRef] References 1. Gao, W.; Arai, Y.; Shibuya, A.; Kiyono, S.; Park, C.H. 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In Proceedings of the 2016 IEEE 11th Conference on Industrial Electronics and Applications, Hefei, China, 5–7 June 2016. 22. Janssen, J.L.G.; Paulides, J.J.H.; Encica, L.; Lomonova, E. High-performance moving-coil actuators with double-sided PM arrays: A design comparison. In Proceedings of the International Conference on Electrical Machines and Systems, Incheon, Korea, 10–13 October 2010. 22. Janssen, J.L.G.; Paulides, J.J.H.; Encica, L.; Lomonova, E. High-performance moving-coil actuators with double-sided PM arrays: A design comparison. In Proceedings of the International Conference on Electrical Machines and Systems, Incheon, Korea, 10–13 October 2010. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. 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The importance of a comparative thermophysiological study in female jeans To cite this article: I Braga et al 2018 IOP Conf. Ser.: Mater. Sci. Eng. 459 012034 View the article online for updates and enhancements. This content was downloaded from IP address 193.137.92.39 on 14/12/2018 at 14:27 IOP Conference Series: Materials Science and Engineering IOP Conference Series: Materials Science and Engineering PAPER • OPEN ACCESS This content was downloaded from IP address 193.137.92.39 on 14/12/2018 at 14:27 Aegean International Textile and Advanced Engineering Conference (AITAE 2018) IOP Conf. Series: Materials Science and Engineering459 (2019) 012034 IOP Publishing doi:10.1088/1757-899X/459/1/012034 Aegean International Textile and Advanced Engineering Conference (AITAE 2018) IOP Conf. Series: Materials Science and Engineering459 (2019) 012034 doi:10.1 Aegean International Textile and Advanced Engineering Conference (AITAE 2018) IOP Conf. Series: Materials Science and Engineering459 (2019) 012034 IOP Publishing doi:10.1088/1757-899X/459/1/012034 The importance of a comparative thermophysiological study in female jeans I Braga1, 2, M J Abreu2 and M Oliveira3 1Federal University of Piauí, Curse de Bach .in Fashion, Design and Stylish , Campus Universitário Ministro Petrônio Portella, Bairro Ininga, Teresina /PI, Brazil 22C2T-Center for Textile Science and Technology, University of Minho, Campus de Azurém, Guimarães, Portugal 3University of Minho, Campus Gualtar, Braga, Portugal I Braga1, 2, M J Abreu2 and M Oliveira3 1Federal University of Piauí, Curse de Bach .in Fashion, Design and Stylish , Campus Universitário Ministro Petrônio Portella, Bairro Ininga, Teresina /PI, Brazil 22C2T-Center for Textile Science and Technology, University of Minho, Campus de Azurém, Guimarães, Portugal 3University of Minho, Campus Gualtar, Braga, Portugal iarabraga@yahoo.com.br I Braga1, 2, M J Abreu2 and M Oliveira3 1Federal University of Piauí, Curse de Bach .in Fashion, Design and Stylish , Campus Universitário Ministro Petrônio Portella, Bairro Ininga, Teresina /PI, Brazil 22C2T-Center for Textile Science and Technology, University of Minho, Campus de Azurém, Guimarães, Portugal 3University of Minho, Campus Gualtar, Braga, Portugal iarabraga@yahoo.com.br Abstract. The comfort of clothing is essential as to performance relative to the wearer and is considered a quality factor in the choice of a particular garment. That is why the science of comfort is used to evaluate jeans, a symbol of popular fashion and the consumption of the poor classes of Brazil [1]. Therefore, the present paper consists of the presentation of the experimental phase of a PhD research on the study of the comfort of Brazilian female jeans, specifically referring to thermal comfort tests through the use of equipment such as Permetest and Thermal Manikin for measuring thermophysiological comfort properties. 1. Introduction The popular jeans in question refers to jeans created, produced and consumed by the popular poor class of the Northeast region of Brazil, with the main focus being the items sold at fairs and popular markets in Fortaleza, Ceará state, where fashion popular jeans have stood out due to economic incomes, job creation and income for the poor populations of the peripheral and rural urban areas of the cities of Ceará [2]. The aim of this research is to compare five different models of jeans used by consumers in order to carry out an analysis of the products offered in popular markets, taking into account thermo- physiological aspects, such as air permeability, thermal resistance and moisture behavior, in order to establish an understanding of the comfort characteristics that the female target population using this specific product choose. The relevance of this research lies in the analysis of this type of jeans that are being offered in the popular markets, determining the most demanded jeans regarding comfort conditions in the scope of combine the study of science of comfort and fashion concepts in the Brazilian popular fashion market and even worldwide. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 3. Results Results Results The washing The Popular jeans were initially analy washing Popular jeans were initially analy washing Popular jeans were initially analy types Popular jeans were initially analy types Popular jeans were initially analy types are pres Popular jeans were initially analy are pres Popular jeans were initially analy are presented in table 1. Popular jeans were initially analy ented in table 1. Popular jeans were initially analy ented in table 1. Popular jeans were initially analy ented in table 1. Popular jeans were initially analy ented in table 1. Popular jeans were initially analys ented in table 1. sed for fabric composition, structure ed for fabric composition, structure ed for fabric composition, structure ed for fabric composition, structure ed for fabric composition, structure ed for fabric composition, structure ed for fabric composition, structure ed for fabric composition, structure ed for fabric composition, structure ed for fabric composition, structure ed for fabric composition, structure ed for fabric composition, structure, , count threats ount threats ount threats ount threats ount threats and industrial and industrial and industrial and industrial and industrial and industrial Industrial wash Jeans Models Composition Industrial wash Structure Count threats (yarns/cm) Jeans Models Image Composition Industrial wash Structure Count threats (yarns/cm) Jeans Models Image Composition Industrial wash Structure Count threats (yarns/cm) Jeans Models Composition Industrial wash Structure Count threats (yarns/cm) Jeans Models Industrial wash Count threats 98%contton 2%spandex Twill Jeans 98%contton 2%spandex Acid destroyed hand scraping Twill 29 x 21 Jeans 98%contton 2%spandex Acid wash and destroyed hand scraping Twill (2/1 1) 29 x 21 Jeans 1 98%contton 2%spandex wash and destroyed hand scraping (2/1 1) 29 x 21 98%contton 2%spandex wash destroyed - (2/1 1) 21%polyester Stone wash and (permanganate) Tabl Jeans 2 77%contton 21%polyester 2% spandex Stone wash and acid wash (permanganate) Twill 37 x 23 Tabl Jeans 2 77%contton 21%polyester 2% spandex Stone wash and acid wash (permanganate) Twill (2/1 1) 37 x 23 Table Jeans 2 77%contton 21%polyester 2% spandex Stone wash and acid wash (permanganate) (2/1 1) 37 x 23 1. 3. Results Results Results J Jeans 2 77%contton 21%polyester 2% spandex Stone wash and acid wash (permanganate) (2/1 1) 37 x 23 Jeans description 21%polyester Stone wash and (permanganate) eans description 98%contton 2% spandex Dark water jet overall Twill (3/1 1) eans description Jeans 3 98%contton 2% spandex Dark water jet wash and verall (resin) Twill (3/1 1) 39 x 22 eans description Jeans 3 98%contton 2% spandex Dark water jet wash and verall c (resin) Twill (3/1 1) 39 x 22 eans description Jeans 3 98%contton 2% spandex Dark water jet wash and crinkle (resin) Twill (3/1 1) 39 x 22 eans description 98%contton 2% spandex Dark water jet wash and rinkle Twill (3/1 1) 96,5%contton 3,5% spandex Dark Twill (3/1 1) Jeans 4 96,5%contton 3,5% spandex Dark dyeing Twill (3/1 1) 40 x 26 Jeans 4 96,5%contton 3,5% spandex Dark sul dyeing Twill (3/1 1) 40 x 26 Jeans 4 96,5%contton 3,5% spandex ulfur dyeing Twill (3/1 1) 40 x 26 96,5%contton 3,5% spandex ur Twill (3/1 1) 21%polyester 2% spandex Rinse wash Twill (3/1 1) Jeans 5 77%contton 21%polyester 2% spandex Rinse wash softener Twill (3/1 1) 38 x 23 Jeans 5 77%contton 21%polyester 2% spandex Rinse wash oftener Twill (3/1 1) 38 x 23 Jeans 5 77%contton 21%polyester 2% spandex Rinse wash oftener Twill (3/1 1) 38 x 23 77%contton 21%polyester 2% spandex Rinse wash and Twill (3/1 1) and and thus comparing the values of the characterization measurements materials and of the data of the jeans hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of the treatments performed. physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 After that, t and thus comparing the values of the characterization measurements materials and of the data of the jeans hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of the treatments performed. physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. 3. Results Results Results The comparison of the means in homogeneous subsets, with harmonic mean = 10 After that, t and thus comparing the values of the characterization measurements materials and of the data of the jeans thermophysiological tests. hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of the treatments performed. physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 After that, t and thus comparing the values of the characterization measurements materials and of the data of the thermophysiological tests. hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of the treatments performed. physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 After that, t and thus comparing the values of the characterization measurements materials and of the data of the thermophysiological tests. hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of the treatments performed. physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 After that, the results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the thermophysiological tests. hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of the treatments performed. physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the thermophysiological tests. 3. Results Results Results hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of the treatments performed. physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the thermophysiological tests. hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of the treatments performed. physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the thermophysiological tests. hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of the treatments performed. physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the thermophysiological tests. hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of the treatments performed. To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the thermophysiological tests. 3. Results Results Results hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of varianc hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of varianc hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of varianc hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. 3. Results Results Results The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of varianc hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of varianc hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of varianc hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. 3. Results Results Results The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of varianc hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of varianc hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of varianc hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. 3. Results Results Results The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of varianc hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of varianc hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of varianc hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. 3. Results Results Results The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of varianc hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of varianc hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the With the test of homogeneity of variances, the Levene test has as hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. 3. Results Results Results The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the es, the Levene test has as hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the es, the Levene test has as hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10 he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the es, the Levene test has as hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey test. The comparison of the means in homogeneous subsets, with harmonic mean = 10,000. 3. Results Results Results he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the es, the Levene test has as hypothesis H0 that there is homogeneity of variances between the different treatments, when the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey ,000. he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the es, the Levene test has as hypothesis H0 that there is homogeneity of variances between the different treatments, when analysing the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey ,000. 3. Results Results Results he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the es, the Levene test has as analysing the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the es, the Levene test has as analysing the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the es, the Levene test has as analysing the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey he results of the ANOVA variance analysis were calculated using the models of jeans and thus comparing the values of the characterization measurements materials and of the data of the es, the Levene test has as the data, it was observed the existence of both homogeneity and heterogeneity between the averages of To better emphasize the relationship between the different jeans as the physical characteristics the analysis of multiple comparisons of the means was done through the Tukey table The analysis of the comparison of the measures table The analysis of the comparison of the measures table 2 and the comparison of the homogeneous groups are shown in figure 1. The analysis of the comparison of the measures and the comparison of the homogeneous groups are shown in figure 1. The analysis of the comparison of the measures and the comparison of the homogeneous groups are shown in figure 1. 3. Results Results Results The analysis of the comparison of the measures and the comparison of the homogeneous groups are shown in figure 1. The analysis of the comparison of the measures and the comparison of the homogeneous groups are shown in figure 1. The analysis of the comparison of the measures and the comparison of the homogeneous groups are shown in figure 1. The analysis of the comparison of the measures and the comparison of the homogeneous groups are shown in figure 1. The analysis of the comparison of the measures and the comparison of the homogeneous groups are shown in figure 1. The analysis of the comparison of the measures and the comparison of the homogeneous groups are shown in figure 1. The analysis of the comparison of the measures and the comparison of the homogeneous groups are shown in figure 1. The analysis of the comparison of the measures and the comparison of the homogeneous groups are shown in figure 1. The analysis of the comparison of the measures and the comparison of the homogeneous groups are shown in figure 1. The analysis of the comparison of the measures and the comparison of the homogeneous groups are shown in figure 1. The analysis of the comparison of the measures and the comparison of the homogeneous groups are shown in figure 1. The analysis of the comparison of the measures and the comparison of the homogeneous groups are shown in figure 1. The analysis of the comparison of the measures means and the comparison of the homogeneous groups are shown in figure 1. means and the comparison of the homogeneous groups are shown in figure 1. means of the mass per unit area are expressed in and the comparison of the homogeneous groups are shown in figure 1. of the mass per unit area are expressed in and the comparison of the homogeneous groups are shown in figure 1. of the mass per unit area are expressed in and the comparison of the homogeneous groups are shown in figure 1. of the mass per unit area are expressed in and the comparison of the homogeneous groups are shown in figure 1. of the mass per unit area are expressed in and the comparison of the homogeneous groups are shown in figure 1. 2. Materials and methods were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body were performed at 37ºC corresponding to the internal temperature of the human body for the Social Sciences), Tukey tests, with significance level of 0.05. 2. Materials and methods The data were analy for the Social Sciences), Tukey tests, with significance level of 0.05. The data were analy for the Social Sciences), Tukey tests, with significance level of 0.05. The data were analy for the Social Sciences), Tukey tests, with significance level of 0.05. The data were analy for the Social Sciences), Tukey tests, with significance level of 0.05. The data were analy for the Social Sciences), Tukey tests, with significance level of 0.05. The data were analy for the Social Sciences), Tukey tests, with significance level of 0.05. The data were analy for the Social Sciences), Tukey tests, with significance level of 0.05. The data were analysed statistically using Excel and SPSS software version 23 (Statistical Package for the Social Sciences), using the SPSS in ANOVA analysis of variance, applying the later tests, Tukey tests, with significance level of 0.05. ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, Tukey tests, with significance level of 0.05. ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, Tukey tests, with significance level of 0.05. ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, Tukey tests, with significance level of 0.05. ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, Tukey tests, with significance level of 0.05. ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, Tukey tests, with significance level of 0.05. ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, Tukey tests, with significance level of 0.05. 2. Materials and methods ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, ed statistically using Excel and SPSS software version 23 (Statistical Package using the SPSS in ANOVA analysis of variance, applying the later tests, 2. Materials and methods The study is based on the methodology of ISO 11092: 2014 [3]. The methodology of evaluation of the physiological evaluation of clothing is organized in five levels. For this stage of research, we applied the techniques level 1, the physical-mechanical analysis of the materials (using testing equipment) and level 2, the biophysical analysis of the garment (using thermal manikin). Five models of female's jeans were purchased in the popular shopping centers of Fortaleza, Buraco da Gia and Beco da Poeira in Forteleza/Brazil. In order to analyse the thermal properties of these fabrics, the relative water vapour permeability (RWVP) and the absolute water vapour permeability (AWVP) were measured using PERMETEST[4], and the air permeability was measured on a Textest FX 3300 instrument [5] and the measurement of thermal insulation with the use of Thermal Manikin[6] Maria of the University of Minho PT-Teknik made in Denmark, in a thermal chamber with controlled environment with temperature and humidity between 28ºC with a variation of ± 2ºC and 65%, with variation of ± 2%. The thermal resistance tests 1 Aegean International Textile and Advanced Engineering Conference (AITAE 2018) IOP Conf. Series: Materials Science and Engineering459 (2019) 012034 IOP Publishing doi:10.1088/1757-899X/459/1/012034 Aegean International Textile and Advanced Engineering Conference (AITAE 2018) IOP Conf. Series: Materials Science and Engineering459 (2019) 012034 IOP Publishing doi:10.1088/1757-899X/459/1/012034 Aegean International Textile and Advanced Engineering Conference (AITAE 2018) IOP Conf. Series: Materials Science and Engineering459 (2019) 012034 IOP Publishing doi:10.1088/1757-899X/459/1/012034 were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. 3. Results Results Results of the mass per unit area are expressed in and the comparison of the homogeneous groups are shown in figure 1. of the mass per unit area are expressed in and the comparison of the homogeneous groups are shown in figure 1. of the mass per unit area are expressed in of the mass per unit area are expressed in of the mass per unit area are expressed in of the mass per unit area are expressed in of the mass per unit area are expressed in of the mass per unit area are expressed in of the mass per unit area are expressed in of the mass per unit area are expressed in of the mass per unit area are expressed in Figure Figure Figure 11. mass/unit area mass/unit area mass/unit area mass/unit area mass/unit area mass/unit area(g/m (g/m (g/m2) homogeneous homogeneous homogeneous homogeneous homogeneous homogeneous groups groups groups Figure Figure Figure 11. mass/unit area mass/unit area mass/unit area mass/unit area mass/unit area mass/unit area(g/m (g/m (g/m2) homogeneous homogeneous homogeneous homogeneous homogeneous homogeneous groups groups groups Tukey HSD Tukey HSD Models Jeans 1 Jeans 4 Jeans 3 Jeans 5 Jeans 2 Sig. Tab Tukey HSD Models Jeans 1 Jeans 4 Jeans 3 Jeans 5 Jeans 2 Sig. Table Tukey HSD Models Jeans 1 Jeans 4 Jeans 3 Jeans 5 Jeans 2 Sig. le 2. Tukey HSDa Mass 281,43 289,08 Mass /unit area (g/m Mass Subset for alpha= 0.05 1 281,43 289,08 0,052 /unit area (g/m Mass /unit area (g/m Subset for alpha= 0.05 281,43 289,08 0,052 /unit area (g/m /unit area (g/m Subset for alpha= 0.05 321,93 323,52 /unit area (g/m /unit area (g/m Subset for alpha= 0.05 2 321,93 323,52 0,976 /unit area (g/m /unit area (g/m Subset for alpha= 0.05 321,93 323,52 0,976 /unit area (g/m2) /unit area (g/m Subset for alpha= 0.05 332,08 ) /unit area (g/m2) Subset for alpha= 0.05 3 332,08 1,000 332,08 1,000 Figure Figure Figure 11. mass/unit area mass/unit area mass/unit area mass/unit area mass/unit area mass/unit area(g/m (g/m (g/m2) homogeneous homogeneous homogeneous homogeneous homogeneous homogeneous groups groups groups Tukey HSD Tukey HSD Models Jeans 1 Jeans 4 Jeans 3 Jeans 5 Jeans 2 Sig. Tab Tukey HSD Models Jeans 1 Jeans 4 Jeans 3 Jeans 5 Jeans 2 Sig. Table Tukey HSD Models Jeans 1 Jeans 4 Jeans 3 Jeans 5 Jeans 2 Sig. le 2. 2. Materials and methods were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. were performed at 37ºC corresponding to the internal temperature of the human body. 3. Results Results Results Tukey HSDa Mass 281,43 289,08 Mass /unit area (g/m Mass Subset for alpha= 0.05 1 281,43 289,08 0,052 /unit area (g/m Mass /unit area (g/m Subset for alpha= 0.05 281,43 289,08 0,052 /unit area (g/m /unit area (g/m Subset for alpha= 0.05 321,93 323,52 /unit area (g/m /unit area (g/m Subset for alpha= 0.05 2 321,93 323,52 0,976 /unit area (g/m /unit area (g/m Subset for alpha= 0.05 321,93 323,52 0,976 /unit area (g/m2) /unit area (g/m Subset for alpha= 0.05 332,08 ) /unit area (g/m2) Subset for alpha= 0.05 3 332,08 1,000 332,08 1,000 Figure Figure Figure 11. mass/unit area mass/unit area mass/unit area mass/unit area mass/unit area mass/unit area(g/m (g/m (g/m2) homogeneous homogeneous homogeneous homogeneous homogeneous homogeneous groups groups groups 2 Aegean International Textile and Advanced Engineering Conference (AITAE 2018) IOP Conf. Series: Materials Science and Engineering459 (2019) 012034 IOP Publishing doi:10.1088/1757-899X/459/1/012034 Aegean International Textile and Advanced Engineering Conference (AITAE 2018) IOP Conf. Series: Materials Science and Engineering459 (2019) 012034 IOP Publishing doi:10.1088/1757-899X/459/1/012034 Evaluating the mass/unit area mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed values of mass gre mass/unit area (g/m Evaluating the mass/unit area mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed values of mass gre mass/unit area (g/m Evaluating the mass/unit area mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed values of mass gre mass/unit area (g/m Evaluating the mass/unit area mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed values of mass gre mass/unit area (g/m Evaluating the mass/unit area mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed values of mass gre mass/unit area (g/m Evaluating the mass/unit area mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. 3. Results Results Results evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed values of mass gre mass/unit area (g/m Evaluating the mass/unit area mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed values of mass greater than group 1 and smaller than group 3. mass/unit area (g/m2). Evaluating the mass/unit area mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. ). Evaluating the mass/unit area mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. Evaluating the mass/unit area mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. Evaluating the mass/unit area values mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. values mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. values of the mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. of the mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. of the mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. 3. Results Results Results evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. of the 5 jeans mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. jeans mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. jeans models, mass values, although jeans 4 presented 7.65 (g/m statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. models, mass values, although jeans 4 presented 7.65 (g/m2) higher than jeans 1, they are considered statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. models, ) higher than jeans 1, they are considered statistically homogeneous the value of significance = 0.052. evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. models, group 1 ) higher than jeans 1, they are considered statistically homogeneous the value of significance = 0.052. Group 2 evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. group 1 ) higher than jeans 1, they are considered Group 2 evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. group 1 ) higher than jeans 1, they are considered Group 2 evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed ater than group 1 and smaller than group 3. 3. Results Results Results jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous jeans 1 0.6970 mm, smaller jeans 3 and 5 are also evaluated as homogeneous smaller jeans 3 and 5 are also evaluated as homogeneous with smaller with smaller with permeability (AWVP) The permeability (AWVP) The permeability (AWVP) comparison of the means data of the tests thermophysiological properties analysis as permeability (AWVP) are comparison of the means data of the tests thermophysiological properties analysis as permeability, r are evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as relative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as elative water vapour permeability (RWVP) and evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as and a evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as absolute water vapour evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as bsolute water vapour evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as bsolute water vapour evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as bsolute water vapour evaluated in homogeneous subsets, with harmonic mean = 10,000 comparison of the means data of the tests thermophysiological properties analysis as bsolute water vapour evaluated in homogeneous subsets, with harmonic mean = 10,000. 3. Results Results Results Thickness(mm) , the jeans 3 and 5 are also evaluated as homogeneous with bigger Thickness(mm) the group 1 jeans 3 and 5 are also evaluated as homogeneous bigger Thickness(mm) group 1 jeans 3 and 5 are also evaluated as homogeneous bigger value Thickness(mm) group 1 jeans 3 and 5 are also evaluated as homogeneous value Thickness(mm) group 1 - jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous value Thickness(mm) homogeneous groups jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous homogeneous groups jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous homogeneous groups jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous homogeneous groups jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous homogeneous groups jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous homogeneous groups jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous homogeneous groups jeans 1 0.6970 mm, smalle jeans 3 and 5 are also evaluated as homogeneous homogeneous groups smalle jeans 3 and 5 are also evaluated as homogeneous with smalle with smalle with Figure Figure Figure Figure 2.. 3. Results Results Results Thickness(mm) Thickness(mm) Thickness(mm) Thickness(mm) Thickness(mm) Thickness(mm) homogeneous groups homogeneous groups homogeneous groups homogeneous groups homogeneous groups homogeneous groups homogeneous groups homogeneous groups value significance = 0.973 Evaluating the comparison of the fabric thickness value significance = 0.973 Evaluating the comparison of the fabric thickness value; group 2 significance = 0.973 Evaluating the comparison of the fabric thickness group 2 significance = 0.973 Evaluating the comparison of the fabric thickness group 2 significance = 0.973 Evaluating the comparison of the fabric thickness group 2 - jeans 4 significance = 0.973 Evaluating the comparison of the fabric thickness jeans 4 significance = 0.973; Evaluating the comparison of the fabric thickness jeans 4 ; and Evaluating the comparison of the fabric thickness jeans 4, 0.7170 mm and group 4 Evaluating the comparison of the fabric thickness 0.7170 mm group 4 Evaluating the comparison of the fabric thickness 0.7170 mm group 4 Evaluating the comparison of the fabric thickness 0.7170 mm group 4 - jeans 2 Evaluating the comparison of the fabric thickness 0.7170 mm; group 3 jeans 2 Evaluating the comparison of the fabric thickness group 3 jeans 2 Evaluating the comparison of the fabric thickness group 3 jeans 2 .0, Evaluating the comparison of the fabric thickness group 3 - 0,8340 mm, Evaluating the comparison of the fabric thickness jeans 3 and 5 are also evaluated as homogeneous 8340 mm, Evaluating the comparison of the fabric thickness values jeans 3 and 5 are also evaluated as homogeneous 8340 mm, values jeans 3 and 5 are also evaluated as homogeneous 8340 mm, with values, jeans 3 and 5 are also evaluated as homogeneous with , the jeans 3 and 5 are also evaluated as homogeneous with bigger the group 1 jeans 3 and 5 are also evaluated as homogeneous bigger group 1 jeans 3 and 5 are also evaluated as homogeneous bigger value group 1 jeans 3 and 5 are also evaluated as homogeneous value group 1 - jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous value. 3. Results Results Results Group 3 group 1- jeans 1 and jeans 4 had lower ) higher than jeans 1, they are considered Group 2 - evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed roup 3 jeans 1 and jeans 4 had lower ) higher than jeans 1, they are considered - jeans 3 and evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed roup 3 jeans 1 and jeans 4 had lower ) higher than jeans 1, they are considered eans 3 and evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed roup 3 - j jeans 1 and jeans 4 had lower ) higher than jeans 1, they are considered eans 3 and evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed jeans jeans 1 and jeans 4 had lower ) higher than jeans 1, they are considered eans 3 and evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed eans 2 jeans 1 and jeans 4 had lower ) higher than jeans 1, they are considered eans 3 and jeans evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed 2 presents higher jeans 1 and jeans 4 had lower ) higher than jeans 1, they are considered jeans evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed presents higher jeans 1 and jeans 4 had lower ) higher than jeans 1, they are considered jeans 5 are also evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed presents higher jeans 1 and jeans 4 had lower ) higher than jeans 1, they are considered 5 are also evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed presents higher jeans 1 and jeans 4 had lower ) higher than jeans 1, they are considered 5 are also evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed presents higher jeans 1 and jeans 4 had lower ) higher than jeans 1, they are considered 5 are also evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed presents higher jeans 1 and jeans 4 had lower ) higher than jeans 1, they are considered 5 are also evaluated as homogeneous with significance = 0.976, when purchased with the other jeans showed presents higher the data shown in figure 2. 3. Results Results Results The analysis of the comparison of the averages as the thickness measures are expressed in table the data shown in figure 2. The analysis of the comparison of the averages as the thickness measures are expressed in table the data shown in figure 2. The analysis of the comparison of the averages as the thickness measures are expressed in table the data shown in figure 2. The analysis of the comparison of the averages as the thickness measures are expressed in table the data shown in figure 2. The analysis of the comparison of the averages as the thickness measures are expressed in table the data shown in figure 2. The analysis of the comparison of the averages as the thickness measures are expressed in table the data shown in figure 2. The analysis of the comparison of the averages as the thickness measures are expressed in table the data shown in figure 2. The analysis of the comparison of the averages as the thickness measures are expressed in table the data shown in figure 2. The analysis of the comparison of the averages as the thickness measures are expressed in table the data shown in figure 2. 3. Results Results Results air permeability groups: group 1, with lower values /s), jeans 4 (49.62 air permeability groups: group 1, with lower values ), jeans 4 (49.62 air permeability groups: group 1, with lower values ), jeans 4 (49.62 air permeability groups: group 1, with lower values ), jeans 4 (49.62 air permeability groups: group 1, with lower values ), jeans 4 (49.62 air permeability values groups: group 1, with lower values - jeans 5 (37,19 ), jeans 4 (49.62 l/m values jeans 5 (37,19 l/m values, we observed homogeneity of the values divided into two jeans 5 (37,19 l/m2/s) and jeans 3 , we observed homogeneity of the values divided into two jeans 5 (37,19 ) and jeans 3 , we observed homogeneity of the values divided into two jeans 5 (37,19 ) and jeans 3 , we observed homogeneity of the values divided into two jeans 5 (37,19 ) and jeans 3 , we observed homogeneity of the values divided into two l/m ) and jeans 3 , we observed homogeneity of the values divided into two l/m2/s ) and jeans 3 (49.89 , we observed homogeneity of the values divided into two /s) and jeans 1 (38,23 (49.89 , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 (49.89 l/m , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 l/m2 , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 2/s), the latter showing higher air , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 ), the latter showing higher air , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 ), the latter showing higher air , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 ), the latter showing higher air , we observed homogeneity of the values divided into two l/m ), the latter showing higher air , we observed homogeneity of the values divided into two l/m2/s ), the latter showing higher air , we observed homogeneity of the values divided into two /s) and group 2: ), the latter showing higher air , we observed homogeneity of the values divided into two ) and group 2: ), the latter showing higher air , we observed homogeneity of the values divided into two ) and group 2: ), the latter showing higher air , we observed homogeneity of the values divided into two ) and group 2: ), the latter showing higher air , we observed homogeneity of the values divided into two ) and group 2: ), the latter showing higher air , we observed homogeneity of the values divided into two ) and group 2: ), the latter showing higher air groups: group 1, with lower values jeans 2 (48.69 permeability value. 3. Results Results Results Compari groups: group 1, with lower values jeans 2 (48.69 permeability value. Compari groups: group 1, with lower values jeans 2 (48.69 permeability value. Compari groups: group 1, with lower values jeans 2 (48.69 permeability value. Comparing the groups: group 1, with lower values jeans 2 (48.69 permeability value. ng the groups: group 1, with lower values jeans 2 (48.69 l/m permeability value. ng the air permeability groups: group 1, with lower values l/m2/s permeability value. 3. Results Results Results comparison of the means data of the tests thermophysiological properties analysis as bsolute water vapour comparison of the means data of the tests thermophysiological properties analysis as bsolute water vapour comparison of the means data of the tests thermophysiological properties analysis as bsolute water vapour permeability comparison of the means data of the tests thermophysiological properties analysis as permeability comparison of the means data of the tests thermophysiological properties analysis as permeability comparison of the means data of the tests thermophysiological properties analysis as permeability air permeability air permeability in figure 3. The analysis of the means measurements air permeability are shown in table in figure 3. The analysis of the means measurements air permeability are shown in table in figure 3. The analysis of the means measurements air permeability are shown in table in figure 3. 3. Results Results Results values, jeans 3 and 5 are also evaluated as homogeneous with . Thickness(mm) , the jeans 3 and 5 are also evaluated as homogeneous with bigger Thickness(mm) the group 1 jeans 3 and 5 are also evaluated as homogeneous bigger Thickness(mm) group 1 jeans 3 and 5 are also evaluated as homogeneous bigger value Thickness(mm) group 1 jeans 3 and 5 are also evaluated as homogeneous value Thickness(mm) group 1 - jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous value Thickness(mm) homogeneous groups jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous homogeneous groups jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous homogeneous groups jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous homogeneous groups jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous homogeneous groups jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous homogeneous groups jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous homogeneous groups jeans 1 0.6970 mm, smalle jeans 3 and 5 are also evaluated as homogeneous homogeneous groups smalle jeans 3 and 5 are also evaluated as homogeneous wit smalle wit smalle wit Tukey HSD value significance = 0 973 Tukey HSD Models Jeans Jeans 4 Jeans 3 Jeans 5 Jeans 2 Sig. Evaluating the comparison of the fabric thickness value significance = 0 973 Tukey HSD Models Jeans Jeans 4 Jeans 3 Jeans 5 Jeans 2 Sig. Evaluating the comparison of the fabric thickness value; group 2 significance = 0 973 Tabl Tukey HSD Models Jeans 1 Jeans 4 Jeans 3 Jeans 5 Jeans 2 Sig. Evaluating the comparison of the fabric thickness group 2 significance = 0 973 Tabl Tukey HSDa Evaluating the comparison of the fabric thickness group 2 significance = 0 973 Table ,6970 1,000 Evaluating the comparison of the fabric thickness group 2 - jeans 4 significance = 0 973 3. 3. Results Results Results Thickness Subset for alpha= 0.05 1 ,6970 1,000 Evaluating the comparison of the fabric thickness jeans 4 significance = 0 973; Thickness Thickness (mm) Subset for alpha= 0.05 ,6970 1,000 Evaluating the comparison of the fabric thickness jeans 4 ; and Thickness Thickness (mm) Subset for alpha= 0.05 2 ,7170 1,000 Evaluating the comparison of the fabric thickness jeans 4, 0.7170 mm and group 4 Thickness Thickness (mm) Subset for alpha= 0.05 2 ,7170 1,000 Evaluating the comparison of the fabric thickness 0.7170 mm group 4 Thickness(mm) Thickness (mm) Subset for alpha= 0.05 ,7170 1,000 Evaluating the comparison of the fabric thickness 0.7170 mm group 4 (mm) Thickness (mm) Subset for alpha= 0.05 3 ,7490 ,7530 ,973 Evaluating the comparison of the fabric thickness 0.7170 mm group 4 jeans 2 Thickness (mm) Subset for alpha= 0.05 ,7490 ,7530 ,973 Evaluating the comparison of the fabric thickness 0.7170 mm; group 3 jeans 2 Subset for alpha= 0.05 ,8340 1,000 Evaluating the comparison of the fabric thickness group 3 jeans 2 4 ,8340 1,000 Evaluating the comparison of the fabric thickness group 3 jeans 2 0 ,8340 1,000 Evaluating the comparison of the fabric thickness group 3 - 0 8340 mm Figure Evaluating the comparison of the fabric thickness jeans 3 and 5 are also evaluated as homogeneous 8340 mm Figure Evaluating the comparison of the fabric thickness values jeans 3 and 5 are also evaluated as homogeneous 8340 mm Figure values jeans 3 and 5 are also evaluated as homogeneous 8340 mm with Figure 2. values, jeans 3 and 5 are also evaluated as homogeneous with . 3. Results Results Results The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table The analysis of the comparison of the averages as the thickness measures are expressed in table 3, , Figure Figure Figure Figure 2.. 3. Results Results Results The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table The analysis of the means measurements air permeability are shown in table 4, with the data shown , with the data shown , with the data shown , with the data shown , with the data shown , with the data shown , with the data shown , with the data shown , with the data shown Figure groups Figure groups Figure 3 groups 3. Air Pemeability (l/m Air Pemeability (l/m Air Pemeability (l/m Air Pemeability (l/m Air Pemeability (l/m Air Pemeability (l/m Air Pemeability (l/m Air Pemeability (l/m2/s) /s) homogeneous homogeneous homogeneous homogeneous homogeneous homogeneous Tukey HSD groups: group 1, with lower values jeans 2 (48.69 permeability value. shown in figure Tukey HSD Models Jeans 5 Jeans 1 Jeans 2 Jeans 4 Jeans 3 Sig. 3. Results Results Results Compari groups: group 1, with lower values jeans 2 (48.69 permeability value. The analysis of the shown in figure Table Tukey HSD Models Jeans 5 Jeans 1 Jeans 2 Jeans 4 Jeans 3 Sig. Compari groups: group 1, with lower values jeans 2 (48.69 permeability value. The analysis of the shown in figure Table Tukey HSD Models Jeans 5 Jeans 1 Jeans 2 Jeans 4 Jeans 3 Sig. Compari groups: group 1, with lower values jeans 2 (48.69 permeability value. The analysis of the shown in figure Table 4 Tukey HSDa Comparing the groups: group 1, with lower values jeans 2 (48.69 permeability value. The analysis of the shown in figure - Air Pemeability (l/m Air Pemeability (l/m Subset for alpha = 0.05 37,19 38,23 ng the groups: group 1, with lower values jeans 2 (48.69 l/m permeability value. The analysis of the shown in figure 4 Air Pemeability (l/m Air Pemeability (l/m Subset for alpha = 0.05 1 37,19 38,23 ,897 ng the air permeability groups: group 1, with lower values l/m2/s permeability value. The analysis of the 4. 3. Results Results Results Air Pemeability (l/m Air Pemeability (l/m Subset for alpha = 0.05 37,19 38,23 ,897 air permeability groups: group 1, with lower values /s), jeans 4 (49.62 The analysis of the Air Pemeability (l/m Air Pemeability (l/m Subset for alpha = 0.05 air permeability groups: group 1, with lower values ), jeans 4 (49.62 The analysis of the relative water vapor permeability Air Pemeability (l/m Air Pemeability (l/m Subset for alpha = 0.05 air permeability groups: group 1, with lower values ), jeans 4 (49.62 relative water vapor permeability Air Pemeability (l/m Air Pemeability (l/m Subset for alpha = 0.05 48,69 49,62 49,89 ,838 air permeability groups: group 1, with lower values ), jeans 4 (49.62 relative water vapor permeability Air Pemeability (l/m Air Pemeability (l/m Subset for alpha = 0.05 2 48,69 49,62 49,89 ,838 air permeability groups: group 1, with lower values ), jeans 4 (49.62 relative water vapor permeability Air Pemeability (l/m2/s) Air Pemeability (l/m2/s) Subset for alpha = 0.05 48,69 49,62 49,89 ,838 air permeability values groups: group 1, with lower values - jeans 5 (37,19 ), jeans 4 (49.62 l/m relative water vapor permeability /s) /s) values jeans 5 (37,19 l/m relative water vapor permeability values, we observed homogeneity of the values divided into tw jeans 5 (37,19 l/m2/s) and jeans 3 relative water vapor permeability , we observed homogeneity of the values divided into tw jeans 5 (37,19 ) and jeans 3 relative water vapor permeability Figure groups , we observed homogeneity of the values divided into tw jeans 5 (37,19 ) and jeans 3 relative water vapor permeability Figure groups , we observed homogeneity of the values divided into tw jeans 5 (37,19 ) and jeans 3 relative water vapor permeability Figure 3 groups , we observed homogeneity of the values divided into tw l/m ) and jeans 3 relative water vapor permeability 3. 3. Results Results Results Air Pemeability (l/m , we observed homogeneity of the values divided into two l/m2/s ) and jeans 3 (49.89 relative water vapor permeability values Air Pemeability (l/m , we observed homogeneity of the values divided into two /s) and jeans 1 (38,23 (49.89 values Air Pemeability (l/m , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 (49.89 l/m values Air Pemeability (l/m , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 l/m2 are shown in table Air Pemeability (l/m , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 2/s), the latter showing higher ai are shown in table Air Pemeability (l/m , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 ), the latter showing higher ai are shown in table Air Pemeability (l/m , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 ), the latter showing higher air are shown in table Air Pemeability (l/m2/s) , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 ), the latter showing higher air are shown in table /s) homogeneous , we observed homogeneity of the values divided into two l/m ), the latter showing higher ai are shown in table homogeneous , we observed homogeneity of the values divided into two l/m2/s ), the latter showing higher ai are shown in table 5 homogeneous , we observed homogeneity of the values divided into two /s) and group 2 ), the latter showing higher ai 5, with the data homogeneous , we observed homogeneity of the values divided into two ) and group 2 ), the latter showing higher air , with the data homogeneous , we observed homogeneity of the values divided into two ) and group 2 ), the latter showing higher air , with the data homogeneous , we observed homogeneity of the values divided into two ) and group 2 ), the latter showing higher air , with the data , we observed homogeneity of the values divided into two ) and group 2 ), the latter showing higher air , with the data , we observed homogeneity of the values divided into two ) and group 2 ), the latter showing higher air , with the data Figure groups Figure groups Figure 3 groups 3. 3. Results Results Results Air Pemeability (l/m , we observed homogeneity of the values divided into tw l/m2/s ) and jeans 3 (49.89 relative water vapor permeability values Air Pemeability (l/m , we observed homogeneity of the values divided into tw /s) and jeans 1 (38,23 (49.89 values Air Pemeability (l/m , we observed homogeneity of the values divided into tw ) and jeans 1 (38,23 (49.89 l/m values Air Pemeability (l/m , we observed homogeneity of the values divided into tw ) and jeans 1 (38,23 l/m2 are shown in table Air Pemeability (l/m , we observed homogeneity of the values divided into tw ) and jeans 1 (38,23 2/s), the latter showing higher a are shown in table Air Pemeability (l/m , we observed homogeneity of the values divided into tw ) and jeans 1 (38,23 ), the latter showing higher a are shown in table Air Pemeability (l/m , we observed homogeneity of the values divided into tw ) and jeans 1 (38,23 ), the latter showing higher a are shown in table Air Pemeability (l/m2/s) , we observed homogeneity of the values divided into tw ) and jeans 1 (38,23 ), the latter showing higher a are shown in table /s) homogeneous , we observed homogeneity of the values divided into tw l/m ), the latter showing higher a are shown in table homogeneous , we observed homogeneity of the values divided into tw l/m2/s ), the latter showing higher a are shown in table 5 homogeneous , we observed homogeneity of the values divided into tw /s) and group ), the latter showing higher a 5, with the da homogeneous , we observed homogeneity of the values divided into tw ) and group ), the latter showing higher a , with the da homogeneous , we observed homogeneity of the values divided into tw ) and group ), the latter showing higher a , with the da homogeneous , we observed homogeneity of the values divided into tw ) and group ), the latter showing higher a , with the da , we observed homogeneity of the values divided into tw ) and group ), the latter showing higher a , with the da , we observed homogeneity of the values divided into tw ) and group ), the latter showing higher a , with the da Tukey HSD groups: group 1, with lower values jeans 2 (48.69 permeability value. 3. Results Results Results shown in figure Tukey HSD Models Jeans 5 Jeans 1 Jeans 2 Jeans 4 Jeans 3 Sig. Compari groups: group 1, with lower values jeans 2 (48.69 permeability value. The analysis of the shown in figure Table Tukey HSD Models Jeans 5 Jeans 1 Jeans 2 Jeans 4 Jeans 3 Sig. Compari groups: group 1, with lower values jeans 2 (48.69 permeability value. The analysis of the shown in figure Table Tukey HSD Models Jeans 5 Jeans 1 Jeans 2 Jeans 4 Jeans 3 Sig. Compari groups: group 1, with lower values jeans 2 (48.69 permeability value. The analysis of the shown in figure Table 4 Tukey HSDa Comparing the groups: group 1, with lower values jeans 2 (48.69 permeability value. The analysis of the shown in figure - Air Pemeability (l/m Air Pemeability (l/m Subset for alpha = 0.05 37,19 38,23 ng the groups: group 1, with lower values jeans 2 (48.69 l/m permeability value. The analysis of the shown in figure 4 Air Pemeability (l/m Air Pemeability (l/m Subset for alpha = 0.05 1 37,19 38,23 ,897 ng the air permeability groups: group 1, with lower values l/m2/s permeability value. The analysis of the 4. 3. Results Results Results Air Pemeability (l/m Air Pemeability (l/m Subset for alpha = 0.05 37,19 38,23 ,897 air permeability groups: group 1, with lower values /s), jeans 4 (49.62 The analysis of the Air Pemeability (l/m Air Pemeability (l/m Subset for alpha = 0.05 air permeability groups: group 1, with lower values ), jeans 4 (49.62 The analysis of the relative water vapor permeability Air Pemeability (l/m Air Pemeability (l/m Subset for alpha = 0.05 air permeability groups: group 1, with lower values ), jeans 4 (49.62 relative water vapor permeability Air Pemeability (l/m Air Pemeability (l/m Subset for alpha = 0.05 48,69 49,62 49,89 ,838 air permeability groups: group 1, with lower values ), jeans 4 (49.62 relative water vapor permeability Air Pemeability (l/m Air Pemeability (l/m Subset for alpha = 0.05 2 48,69 49,62 49,89 ,838 air permeability groups: group 1, with lower values ), jeans 4 (49.62 relative water vapor permeability Air Pemeability (l/m2/s) Air Pemeability (l/m2/s) Subset for alpha = 0.05 48,69 49,62 49,89 ,838 air permeability values groups: group 1, with lower values - jeans 5 (37,19 ), jeans 4 (49.62 l/m relative water vapor permeability /s) /s) values jeans 5 (37,19 l/m relative water vapor permeability values, we observed homogeneity of the values divided into two jeans 5 (37,19 l/m2/s) and jeans 3 relative water vapor permeability , we observed homogeneity of the values divided into two jeans 5 (37,19 ) and jeans 3 relative water vapor permeability Figure groups , we observed homogeneity of the values divided into two jeans 5 (37,19 ) and jeans 3 relative water vapor permeability Figure groups , we observed homogeneity of the values divided into two jeans 5 (37,19 ) and jeans 3 relative water vapor permeability Figure 3 groups , we observed homogeneity of the values divided into two l/m ) and jeans 3 relative water vapor permeability 3. 3. Results Results Results Thickness(mm) Thickness(mm) Thickness(mm) Thickness(mm) Thickness(mm) Thickness(mm) homogeneous groups homogeneous groups homogeneous groups homogeneous groups homogeneous groups homogeneous groups homogeneous groups homogeneous groups Tukey HSD value significance = 0 973 Tukey HSD Models Jeans Jeans 4 Jeans 3 Jeans 5 Jeans 2 Sig. Evaluating the comparison of the fabric thickness value significance = 0 973 Tukey HSD Models Jeans Jeans 4 Jeans 3 Jeans 5 Jeans 2 Sig. Evaluating the comparison of the fabric thickness value; group 2 significance = 0 973 Tabl Tukey HSD Models Jeans 1 Jeans 4 Jeans 3 Jeans 5 Jeans 2 Sig. Evaluating the comparison of the fabric thickness group 2 significance = 0 973 Tabl Tukey HSDa Evaluating the comparison of the fabric thickness group 2 significance = 0 973 Table ,6970 1,000 Evaluating the comparison of the fabric thickness group 2 - jeans 4 significance = 0 973 3. Thickness Subset for alpha= 0.05 1 ,6970 1,000 Evaluating the comparison of the fabric thickness jeans 4 significance = 0 973; Thickness Thickness (mm) Subset for alpha= 0.05 ,6970 1,000 Evaluating the comparison of the fabric thickness jeans 4 ; and Thickness Thickness (mm) Subset for alpha= 0.05 2 ,7170 1,000 Evaluating the comparison of the fabric thickness jeans 4, 0.7170 mm and group 4 Thickness Thickness (mm) Subset for alpha= 0.05 2 ,7170 1,000 Evaluating the comparison of the fabric thickness 0.7170 mm group 4 Thickness(mm) Thickness (mm) Subset for alpha= 0.05 ,7170 1,000 Evaluating the comparison of the fabric thickness 0.7170 mm group 4 (mm) Thickness (mm) Subset for alpha= 0.05 3 ,7490 ,7530 ,973 Evaluating the comparison of the fabric thickness 0.7170 mm group 4 - jeans 2 Thickness (mm) Subset for alpha= 0.05 ,7490 ,7530 ,973 Evaluating the comparison of the fabric thickness 0.7170 mm; group 3 jeans 2 Subset for alpha= 0.05 ,8340 1,000 Evaluating the comparison of the fabric thickness group 3 jeans 2 4 ,8340 1,000 Evaluating the comparison of the fabric thickness group 3 jeans 2 0 ,8340 1,000 Evaluating the comparison of the fabric thickness group 3 - 0 8340 mm Figure Evaluating the comparison of the fabric thickness jeans 3 and 5 are also evaluated as homogeneous 8340 mm Figure Evaluating the comparison of the fabric thickness values jeans 3 and 5 are also evaluated as homogeneous 8340 mm Figure values jeans 3 and 5 are also evaluated as homogeneous 8340 mm with Figure 2. 3. Results Results Results jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous jeans 1 0.6970 mm, smaller jeans 3 and 5 are also evaluated as homogeneous smaller jeans 3 and 5 are also evaluated as homogeneous with smaller with smaller with value significance = 0.973 Evaluating the comparison of the fabric thickness value significance = 0.973 Evaluating the comparison of the fabric thickness value; group 2 significance = 0.973 Evaluating the comparison of the fabric thickness group 2 significance = 0.973 Evaluating the comparison of the fabric thickness group 2 significance = 0.973 Evaluating the comparison of the fabric thickness group 2 - jeans 4 significance = 0.973 Evaluating the comparison of the fabric thickness jeans 4 significance = 0.973; Evaluating the comparison of the fabric thickness jeans 4 ; and Evaluating the comparison of the fabric thickness jeans 4, 0.7170 mm and group 4 Evaluating the comparison of the fabric thickness 0.7170 mm group 4 Evaluating the comparison of the fabric thickness 0.7170 mm group 4 Evaluating the comparison of the fabric thickness 0.7170 mm group 4 - jeans 2 Evaluating the comparison of the fabric thickness 0.7170 mm; group 3 jeans 2 Evaluating the comparison of the fabric thickness group 3 jeans 2 Evaluating the comparison of the fabric thickness group 3 jeans 2 .0, Evaluating the comparison of the fabric thickness group 3 - 0,8340 mm, Evaluating the comparison of the fabric thickness jeans 3 and 5 are also evaluated as homogeneous 8340 mm, Evaluating the comparison of the fabric thickness values jeans 3 and 5 are also evaluated as homogeneous 8340 mm, values jeans 3 and 5 are also evaluated as homogeneous 8340 mm, with values, jeans 3 and 5 are also evaluated as homogeneous with , the jeans 3 and 5 are also evaluated as homogeneous with bigger the group 1 jeans 3 and 5 are also evaluated as homogeneous bigger group 1 jeans 3 and 5 are also evaluated as homogeneous bigger value group 1 jeans 3 and 5 are also evaluated as homogeneous value group 1 - jeans 1 0.6970 mm jeans 3 and 5 are also evaluated as homogeneous value. 3. Results Results Results air permeability groups: group 1, with lower values /s), jeans 4 (49.62 air permeability groups: group 1, with lower values ), jeans 4 (49.62 air permeability groups: group 1, with lower values ), jeans 4 (49.62 air permeability groups: group 1, with lower values ), jeans 4 (49.62 air permeability groups: group 1, with lower values ), jeans 4 (49.62 air permeability values groups: group 1, with lower values - jeans 5 (37,19 ), jeans 4 (49.62 l/m values jeans 5 (37,19 l/m values, we observed homogeneity of the values divided into two jeans 5 (37,19 l/m2/s) and jeans 3 , we observed homogeneity of the values divided into two jeans 5 (37,19 ) and jeans 3 , we observed homogeneity of the values divided into two jeans 5 (37,19 ) and jeans 3 , we observed homogeneity of the values divided into two jeans 5 (37,19 ) and jeans 3 , we observed homogeneity of the values divided into two l/m ) and jeans 3 , we observed homogeneity of the values divided into two l/m2/s ) and jeans 3 (49.89 , we observed homogeneity of the values divided into two /s) and jeans 1 (38,23 (49.89 , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 (49.89 l/m , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 l/m2 , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 2/s), the latter showing higher air , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 ), the latter showing higher air , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 ), the latter showing higher air , we observed homogeneity of the values divided into two ) and jeans 1 (38,23 ), the latter showing higher air , we observed homogeneity of the values divided into two l/m ), the latter showing higher air , we observed homogeneity of the values divided into two l/m2/s ), the latter showing higher air , we observed homogeneity of the values divided into two /s) and group 2: ), the latter showing higher air , we observed homogeneity of the values divided into two ) and group 2: ), the latter showing higher air , we observed homogeneity of the values divided into two ) and group 2: ), the latter showing higher air , we observed homogeneity of the values divided into two ) and group 2: ), the latter showing higher air , we observed homogeneity of the values divided into two ) and group 2: ), the latter showing higher air , we observed homogeneity of the values divided into two ) and group 2: ), the latter showing higher air p y shown in figure p y The analysis of the shown in figure p y The analysis of the shown in figure p y The analysis of the shown in figure p y The analysis of the shown in figure p y The analysis of the shown in figure 4 p y The analysis of the 4. 3. Results Results Results Air Pemeability (l/m Air Pemeability (l/m Air Pemeability (l/m Air Pemeability (l/m Air Pemeability (l/m Air Pemeability (l/m Air Pemeability (l/m Air Pemeability (l/m2/s) /s) homogeneous homogeneous homogeneous homogeneous homogeneous homogeneous groups: group 1, with lower values jeans 2 (48.69 permeability value. Compari groups: group 1, with lower values jeans 2 (48.69 permeability value. Compari groups: group 1, with lower values jeans 2 (48.69 permeability value. Compari groups: group 1, with lower values jeans 2 (48.69 permeability value. Comparing the groups: group 1, with lower values jeans 2 (48.69 permeability value. ng the groups: group 1, with lower values jeans 2 (48.69 l/m permeability value. ng the air permeability groups: group 1, with lower values l/m2/s permeability value. 3. Results Results Results Absolute (RWVP) (Pa/m Tukey Models Absolute (Pa/m Permeability 1,000 Absolute (Pa/m2/W Absolute Permeability 1 3,3 1,000 Absolute Water /W-1 Absolute Permeability Subset for alpha= 0.05 3 1,000 Water 1) Absolute Permeability Subset for alpha= 0.05 Water V Absolute Water Permeability (RWVP) Subset for alpha= 0.05 2 4,0 4,4 ,271 Vapour Permeability Water (RWVP) Subset for alpha= 0.05 ,0 4 ,271 apour Permeability Water (RWVP) Subset for alpha= 0.05 apour Permeability Water Vapour (RWVP) (Pa/m Subset for alpha= 0.05 1,000 apour Permeability apour (Pa/m Subset for alpha= 0.05 3 5,5 5,5 1,000 apour Permeability apour (Pa/m2/W Subset for alpha= 0.05 5,5 5,5 1,000 apour Permeability /W-1) apour Permeability ) Tab (RWVP) Table (RWVP) le 6. (RWVP) . Absolute (RWVP) (Pa/m Absolute (Pa/m Absolute (Pa/m2/W Absolute Water /W-1Water 1) Water VVapour Permeability apour Permeability apour Permeability apour Permeability apour Permeability apour Permeability apour Permeability Figure (RWVP) Figure (RWVP) Figure (RWVP) Figure 5. (RWVP) (Pa/m Absolute (Pa/m Absolute (Pa/m2/W Absolute /W- Absolute Water -1) homogeneous Water homogeneous Water V homogeneous Vapour Permeability homogeneous apour Permeability homogeneous apour Permeability homogeneous groups apour Permeability groups apour Permeability groups apour Permeability apour Permeability observed in 3 different groups. Group 1 3 (4 Pa / m Evaluating the comparison of the observed in 3 different groups. Group 1 3 (4,0 Pa / m Evaluating the comparison of the observed in 3 different groups. Group 1 0 Pa / m Pa / m2 / W Evaluating the comparison of the observed in 3 different groups. Group 1 Pa / m / W Evaluating the comparison of the observed in 3 different groups. Group 1 Pa / m2 / W-1)) with bigger value of absolute water vapor permeability. Evaluating the comparison of the observed in 3 different groups. Group 1 / W ) with bigger value of absolute water vapor permeability. Evaluating the comparison of the observed in 3 different groups. Group 1 / W-1) ) with bigger value of absolute water vapor permeability. Evaluating the comparison of the observed in 3 different groups. Group 1 ) and jeans 5 (4 ) with bigger value of absolute water vapor permeability. Evaluating the comparison of the observed in 3 different groups. Group 1 and jeans 5 (4 ) with bigger value of absolute water vapor permeability. Evaluating the comparison of the observed in 3 different groups. 3. Results Results Results Group 1 and jeans 5 (4 ) with bigger value of absolute water vapor permeability. Evaluating the comparison of the observed in 3 different groups. Group 1 and jeans 5 (4 ) with bigger value of absolute water vapor permeability. Evaluating the comparison of the observed in 3 different groups. Group 1 and jeans 5 (4, ) with bigger value of absolute water vapor permeability. Evaluating the comparison of the observed in 3 different groups. Group 1 ,4 Pa / m ) with bigger value of absolute water vapor permeability. Evaluating the comparison of the absolute observed in 3 different groups. Group 1 - Pa / m ) with bigger value of absolute water vapor permeability. absolute jeans 4 (3 Pa / m2 ) with bigger value of absolute water vapor permeability. absolute jeans 4 (3 2 / W ) with bigger value of absolute water vapor permeability. absolute water vapour permeability values jeans 4 (3 / W-1) ) with bigger value of absolute water vapor permeability. water vapour permeability values jeans 4 (3,3 Pa / m ) the group 3 ) with bigger value of absolute water vapor permeability. water vapour permeability values 3 Pa / m the group 3 ) with bigger value of absolute water vapor permeability. water vapour permeability values 3 Pa / m the group 3 ) with bigger value of absolute water vapor permeability. water vapour permeability values 3 Pa / m2 / W the group 3 ) with bigger value of absolute water vapor permeability. water vapour permeability values / W the group 3 - jeans 1 and jeans 2 equal values (5 ) with bigger value of absolute water vapor permeability. water vapour permeability values / W-1) with lower value; group 2 jeans 1 and jeans 2 equal values (5 ) with bigger value of absolute water vapor permeability. water vapour permeability values ) with lower value; group 2 jeans 1 and jeans 2 equal values (5 ) with bigger value of absolute water vapor permeability. 3. Results Results Results (RWVP) (% Relative %) homogeneous groups Relative homogeneous groups Relative homogeneous groups Relative Water homogeneous groups Water homogeneous groups Water V homogeneous groups Vapour Permeability homogeneous groups apour Permeability homogeneous groups apour Permeability apour Permeability apour Permeability apour Permeability apour Permeability Figure (RWVP) Figure (RWVP) Figure (RWVP) Figure 4. (RWVP) (% Relative %) homogeneous groups Relative homogeneous groups Relative homogeneous groups Relative Water homogeneous groups Water homogeneous groups Water V homogeneous groups Vapour Permeability homogeneous groups apour Permeability homogeneous groups apour Permeability apour Permeability apour Permeability apour Permeability apour Permeability Figure (RWVP) Figure (RWVP) Figure (RWVP) Figure 4. (RWVP) (% Relative %) homogeneous groups Relative homogeneous groups Relative homogeneous groups Relative Water homogeneous groups Water homogeneous groups Water V homogeneous groups Vapour Permeability homogeneous groups apour Permeability homogeneous groups apour Permeability apour Permeability apour Permeability apour Permeability apour Permeability observed in 4 different groups. Group 1 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 (63.76%) and group 4 Evaluating the comparison of the relative water vapor permeability observed in 4 different groups. Group 1 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 (63.76%) and group 4 Evaluating the comparison of the relative water vapor permeability observed in 4 different groups. Group 1 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 (63.76%) and group 4 Evaluating the comparison of the relative water vapor permeability observed in 4 different groups. Group 1 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 (63.76%) and group 4 Evaluating the comparison of the relative water vapor permeability observed in 4 different groups. Group 1 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 (63.76%) and group 4 Evaluating the comparison of the relative water vapor permeability observed in 4 different groups. Group 1 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 (63.76%) and group 4 Evaluating the comparison of the relative water vapor permeability observed in 4 different groups. 3. Results Results Results Group 1 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 (63.76%) and group 4 Evaluating the comparison of the relative water vapor permeability observed in 4 different groups. Group 1 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 (63.76%) and group 4 - Evaluating the comparison of the relative water vapor permeability observed in 4 different groups. Group 1 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability observed in 4 different groups. Group 1 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability observed in 4 different groups. Group 1 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability observed in 4 different groups. Group 1 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability observed in 4 different groups. Group 1 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability observed in 4 different groups. Group 1 - and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability jeans 2 (56.28%) with lower and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. 3. Results Results Results Evaluating the comparison of the relative water vapor permeability jeans 2 (56.28%) with lower and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability jeans 2 (56.28%) with lower and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability jeans 2 (56.28%) with lower and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability jeans 2 (56.28%) with lower and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability jeans 2 (56.28%) with lower and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability jeans 2 (56.28%) with lower and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability jeans 2 (56.28%) with lower and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability jeans 2 (56.28%) with lower and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability jeans 2 (56.28%) with lower value; group 2 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. 3. Results Results Results Relative Tukey a Models Relative Relative (RWVP) Permeability 1 56,28 1,000 Relative Water (RWVP) Relative Permeability Subset for alpha= 0.05 1 56,28 1,000 Water (RWVP) Relative Permeability Subset for alpha= 0.05 56,28 1,000 Water V (RWVP) ( Relative Water Permeability Subset for alpha= 0.05 2 61,66 ,434 Vapour Permeability (%) Water Permeability (RWVP) Subset for alpha= 0.05 2 60 61,66 ,434 apour Permeability ) Water (RWVP) Subset for alpha= 0.05 60 61,66 ,434 apour Permeability Water Vapour (RWVP) Subset for alpha= 0.05 3 61,66 63,76 ,209 apour Permeability apour (RWVP) ( Subset for alpha= 0.05 61,66 63,76 209 apour Permeability apour (%) Subset for alpha= 0.05 68,98 1,000 apour Permeability ) 4 68,98 1,000 apour Permeability 68,98 1,000 Figure (RWVP) Figure (RWVP) Figure (RWVP) Figure 4. (RWVP) (% Relative %) homogeneous groups Relative homogeneous groups Relative homogeneous groups Relative Water homogeneous groups Water homogeneous groups Water V homogeneous groups Vapour Permeability homogeneous groups apour Permeability homogeneous groups apour Permeability apour Permeability apour Permeability apour Permeability apour Permeability Figure (RWVP) Figure (RWVP) Figure (RWVP) Figure 4. (RWVP) (% Relative %) homogeneous groups Relative homogeneous groups Relative homogeneous groups Relative Water homogeneous groups Water homogeneous groups Water V homogeneous groups Vapour Permeability homogeneous groups apour Permeability homogeneous groups apour Permeability apour Permeability apour Permeability apour Permeability apour Permeability Tab Jeans 2 Jeans 1 Jeans 5 Jeans Jeans 4 Table Tukey HSD Models Jeans 2 Jeans 1 Jeans 5 Jeans Jeans 4 Sig. le 5 Tukey HSDa Models Jeans 2 Jeans 1 Jeans 5 3 Jeans 4 Sig. 5. Relative Tukey a Models Relative Relative (RWVP) Permeability 1 56,28 1,000 Relative Water (RWVP) Relative Permeability Subset for alpha= 0.05 1 56,28 1,000 Water (RWVP) Relative Permeability Subset for alpha= 0.05 56,28 1,000 Water V (RWVP) ( Relative Water Permeability Subset for alpha= 0.05 2 61,66 ,434 Vapour Permeability (%) Water Permeability (RWVP) Subset for alpha= 0.05 2 60 61,66 ,434 apour Permeability ) Water (RWVP) Subset for alpha= 0.05 60 61,66 ,434 apour Permeability Water Vapour (RWVP) Subset for alpha= 0.05 3 61,66 63,76 ,209 apour Permeability apour (RWVP) ( Subset for alpha= 0.05 61,66 63,76 209 apour Permeability apour (%) Subset for alpha= 0.05 68,98 1,000 apour Permeability ) 4 68,98 1,000 apour Permeability 68,98 1,000 Figure (RWVP) Figure (RWVP) Figure (RWVP) Figure 4. 3. Results Results Results Evaluating the comparison of the relative water vapor permeability value; group 2 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. Evaluating the comparison of the relative water vapor permeability values value; group 2 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. values value; group 2 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. values, homogeneity was value; group 2 and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. , homogeneity was value; group 2 - and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. , homogeneity was - jeans 1 (60%) and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. , homogeneity was jeans 1 (60%) and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 jeans 4 (68.98%) with higher relative water vapor permeability value. 3. Results Results Results water vapour permeability values ) with lower value; group 2 jeans 1 and jeans 2 equal values (5 water vapour permeability values ) with lower value; group 2 jeans 1 and jeans 2 equal values (5 water vapour permeability values ) with lower value; group 2 jeans 1 and jeans 2 equal values (5 water vapour permeability values ) with lower value; group 2 jeans 1 and jeans 2 equal values (5 water vapour permeability values, homogeneity is ) with lower value; group 2 jeans 1 and jeans 2 equal values (5 homogeneity is ) with lower value; group 2 jeans 1 and jeans 2 equal values (5 homogeneity is ) with lower value; group 2 jeans 1 and jeans 2 equal values (5 homogeneity is ) with lower value; group 2 - jeans 1 and jeans 2 equal values (5 homogeneity is - jeans jeans 1 and jeans 2 equal values (5 homogeneity is jeans jeans 1 and jeans 2 equal values (5,5 homogeneity is jeans 5 are expressed in figure The analysis of the comparison of the heat loss are expressed in figure The analysis of the comparison of the heat loss are expressed in figure The analysis of the comparison of the heat loss are expressed in figure )) gg p p y The analysis of the comparison of the heat loss are expressed in figure ) gg p p y The analysis of the comparison of the heat loss are expressed in figure ) gg p p y The analysis of the comparison of the heat loss are expressed in figure ) gg p p y The analysis of the comparison of the heat loss are expressed in figure 4 ) gg p p y The analysis of the comparison of the heat loss 4. 3. Results Results Results The analysis of the The analysis of the relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability values values values are shown in table are shown in table are shown in table are shown in table are shown in table are shown in table are shown in table 55, with the data , with the data , with the data , with the data , with the data , with the data p y shown in figure p y The analysis of the shown in figure p y The analysis of the shown in figure p y The analysis of the shown in figure p y The analysis of the shown in figure p y The analysis of the shown in figure 4 p y The analysis of the 4. The analysis of the The analysis of the relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability relative water vapor permeability values values values are shown in table are shown in table are shown in table are shown in table are shown in table are shown in table are shown in table 55, with the data , with the data , with the data , with the data , with the data , with the data 3 Aegean International Textile and Advanced Engineering Conference (AITAE 2018) IOP Conf. Series: Materials Science and Engineering459 (2019) 012034 IOP Publishing doi:10.1088/1757-899X/459/1/012034 Aegean International Textile and Advanced Engineering Conference (AITAE 2018) IOP Conf. Series: Materials Science and Engineering459 (2019) 012034 IOP Publishing doi:10.1088/1757-899X/459/1/012034 Tab Jeans 2 Jeans 1 Jeans 5 Jeans Jeans 4 Table Tukey HSD Models Jeans 2 Jeans 1 Jeans 5 Jeans Jeans 4 Sig. le 5 Tukey HSDa Models Jeans 2 Jeans 1 Jeans 5 3 Jeans 4 Sig. 5. 3. Results Results Results , homogeneity was jeans 1 (60%) and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 , homogeneity was jeans 1 (60%) and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 , homogeneity was jeans 1 (60%) and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 , homogeneity was jeans 1 (60%) and jeans 5 (61.66%), jeans 5 also belonging to group 3 due to being statistically very close to jeans 3 shown in figure The analysis of the shown in figure The analysis of the shown in figure The analysis of the shown in figure The analysis of the shown in figure The analysis of the shown in figure 4 The analysis of the 4. The analysis of the The analysis of the relative water vapor permeability value relative water vapor permeability value relative water vapor permeability value relative water vapor permeability value relative water vapor permeability value relative water vapor permeability value relative water vapor permeability value relative water vapor permeability value relative water vapor permeability value relative water vapor permeability value relative water vapor permeability value relative water vapor permeability value relative water vapor permeability value relative water vapor permeability values are shown in table are shown in table are shown in table are shown in table are shown in table are shown in table are shown in table 66, with the data , with the data , with the data , with the data , with the data , with the data Figure (RWVP) Figure (RWVP) Figure (RWVP) Figure 5. (RWVP) (Pa/m Absolute (Pa/m Absolute (Pa/m2/W Absolute /W- Absolute Water -1) homogeneous Water homogeneous Water V homogeneous Vapour Permeability homogeneous apour Permeability homogeneous apour Permeability homogeneous groups apour Permeability groups apour Permeability groups apour Permeability apour Permeability Tab (RWVP) Jeans 4 Jeans Jeans 5 Jeans 1 Jeans 2 Sig. Table (RWVP) Tukey HSD Models Jeans 4 Jeans Jeans 5 Jeans 1 Jeans 2 Sig. le 6. (RWVP) Tukey HSDa Models Jeans 4 Jeans 3 Jeans 5 Jeans 1 Jeans 2 . 3. Results Results Results Absolute (RWVP) (Pa/m Tukey Models Absolute (Pa/m Permeability 1,000 Absolute (Pa/m2/W Absolute Permeability 1 3,3 1,000 Absolute Water /W-1 Absolute Permeability Subset for alpha= 0.05 3 1,000 Water 1) Absolute Permeability Subset for alpha= 0.05 Water V Absolute Water Permeability (RWVP) Subset for alpha= 0.05 2 4,0 4,4 ,271 Vapour Permeability Water (RWVP) Subset for alpha= 0.05 ,0 4 ,271 apour Permeability Water (RWVP) Subset for alpha= 0.05 apour Permeability Water Vapour (RWVP) (Pa/m Subset for alpha= 0.05 1,000 apour Permeability apour (Pa/m Subset for alpha= 0.05 3 5,5 5,5 1,000 apour Permeability apour (Pa/m2/W Subset for alpha= 0.05 5,5 5,5 1,000 apour Permeability /W-1) apour Permeability ) Figure (RWVP) Figure (RWVP) Figure (RWVP) Figure 5. (RWVP) (Pa/m Absolute (Pa/m Absolute (Pa/m2/W Absolute /W- Absolute Water -1) homogeneous Water homogeneous Water V homogeneous Vapour Permeability homogeneous apour Permeability homogeneous apour Permeability homogeneous groups apour Permeability groups apour Permeability groups apour Permeability apour Permeability Tab (RWVP) Jeans 4 Jeans Jeans 5 Jeans 1 Jeans 2 Sig. Table (RWVP) Tukey HSD Models Jeans 4 Jeans Jeans 5 Jeans 1 Jeans 2 Sig. le 6. (RWVP) Tukey HSDa Models Jeans 4 Jeans 3 Jeans 5 Jeans 1 Jeans 2 . Absolute (RWVP) (Pa/m Tukey Models Absolute (Pa/m Permeability 1,000 Absolute (Pa/m2/W Absolute Permeability 1 3,3 1,000 Absolute Water /W-1 Absolute Permeability Subset for alpha= 0.05 3 1,000 Water 1) Absolute Permeability Subset for alpha= 0.05 Water V Absolute Water Permeability (RWVP) Subset for alpha= 0.05 2 4,0 4,4 ,271 Vapour Permeability Water (RWVP) Subset for alpha= 0.05 ,0 4 ,271 apour Permeability Water (RWVP) Subset for alpha= 0.05 apour Permeability Water Vapour (RWVP) (Pa/m Subset for alpha= 0.05 1,000 apour Permeability apour (Pa/m Subset for alpha= 0.05 3 5,5 5,5 1,000 apour Permeability apour (Pa/m2/W Subset for alpha= 0.05 5,5 5,5 1,000 apour Permeability /W-1) apour Permeability ) Figure (RWVP) Figure (RWVP) Figure (RWVP) Figure 5. (RWVP) (Pa/m Absolute (Pa/m Absolute (Pa/m2/W Absolute /W- Absolute Water -1) homogeneous Water homogeneous Water V homogeneous Vapour Permeability homogeneous apour Permeability homogeneous apour Permeability homogeneous groups apour Permeability groups apour Permeability groups apour Permeability apour Permeability Tab (RWVP) Jeans 4 Jeans Jeans 5 Jeans 1 Jeans 2 Sig. Table (RWVP) Tukey HSD Models Jeans 4 Jeans Jeans 5 Jeans 1 Jeans 2 Sig. le 6. (RWVP) Tukey HSDa Models Jeans 4 Jeans 3 Jeans 5 Jeans 1 Jeans 2 . 3. Results Results Results Heat loss Heat loss Heat loss Heat loss from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models values of heat loss jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. Evaluating the comparison of the heat loss values of heat loss jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. Evaluating the comparison of the heat loss values of heat loss jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. Evaluating the comparison of the heat loss values of heat loss jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. Evaluating the comparison of the heat loss values of heat loss jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. 3. Results Results Results Evaluating the comparison of the heat loss values of heat loss jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. Evaluating the comparison of the heat loss values of heat loss - jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. Evaluating the comparison of the heat loss jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. Evaluating the comparison of the heat loss jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. Evaluating the comparison of the heat loss jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. Evaluating the comparison of the heat loss jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. Evaluating the comparison of the heat loss jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. Evaluating the comparison of the heat loss jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. Evaluating the comparison of the heat loss jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. 3. Results Results Results Evaluating the comparison of the heat loss jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. Evaluating the comparison of the heat loss values jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. values jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. values, jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. , we jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. we can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. 3. Results Results Results can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. 3. Results Results Results ) gg p p y The analysis of the comparison of the heat loss ) gg p p y The analysis of the comparison of the heat loss ) gg p p y The analysis of the comparison of the heat loss ) gg p p y The analysis of the comparison of the heat loss ) gg p p y The analysis of the comparison of the heat loss ) gg p p y The analysis of the comparison of the heat loss ) gg p p y The analysis of the comparison of the heat loss ) gg p p y The analysis of the comparison of the heat loss ) gg p p y The analysis of the comparison of the heat loss values ) gg p p y values ) gg p p y values referring to the different parts of the body ) gg p p y referring to the different parts of the body ) gg p p y referring to the different parts of the body ) gg p p y referring to the different parts of the body ) gg p p y referring to the different parts of the body referring to the different parts of the body referring to the different parts of the body referring to the different parts of the body referring to the different parts of the body referring to the different parts of the body referring to the different parts of the body referring to the different parts of the body referring to the different parts of the body referring to the different parts of the body referring to the different parts of the body referring to the different parts of the body 4 4 Aegean International Textile and Advanced Engineering Conference (AITAE 2018) IOP Conf. Series: Materials Science and Engineering459 (2019) 012034 IOP Publishing doi:10.1088/1757-899X/459/1/012034 Figure Figure Figure Figure 6. 3. Results Results Results can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans 5 and finally the jeans 4 standing out with greater value of loss of heat in all parts of the body. can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then can observe values homogeneity, with lower jeans 1, jeans 2 and jeans 3, being the order from the lowest to the highest, then jeans model are expressed in figure The analysis of the comparison of the total jeans model are expressed in figure The analysis of the comparison of the total jeans model are expressed in figure The analysis of the comparison of the total jeans model are expressed in figure The analysis of the comparison of the total jeans model are expressed in figure The analysis of the comparison of the total jeans model are expressed in figure The analysis of the comparison of the total jeans model are expressed in figure The analysis of the comparison of the total jeans model are expressed in figure The analysis of the comparison of the total jeans model are expressed in figure The analysis of the comparison of the total jeans model are expressed in figure The analysis of the comparison of the total jeans model are expressed in figure The analysis of the comparison of the total jeans model are expressed in figure The analysis of the comparison of the total jeans model are expressed in figure 5. The analysis of the comparison of the total The analysis of the comparison of the total The analysis of the comparison of the total (It) (It) and and and effective effective effective effective (Icle) (Icle) (Icle) thermal insulation thermal insulation thermal insulation thermal insulation thermal insulation thermal insulation thermal insulation values values values of each of each of each of each Figure Figure Figure Figure 7. 3. Results Results Results Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of five jeans five jeans five jeans five jeans models models models Figure Figure Figure Figure 7. 3. Results Results Results Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of Total thermal insulation and effective clothing insulation of five jeans five jeans five jeans five jeans models models models m2 It thermal insulation with presented, showing the lowest total and effective thermal insulation In the comparison of the 2.°C/W It (0,132 thermal insulation with It presented, showing the lowest total and effective thermal insulation In the comparison of the .°C/W (0,132 thermal insulation It (0,126) and presented, showing the lowest total and effective thermal insulation In the comparison of the .°C/W) and (0,132 m thermal insulation (0,126) and presented, showing the lowest total and effective thermal insulation In the comparison of the ) and m2.°C/W thermal insulation (0,126) and presented, showing the lowest total and effective thermal insulation In the comparison of the ) and Icle .°C/W thermal insulation (0,126) and presented, showing the lowest total and effective thermal insulation In the comparison of the Icle (0,100 .°C/W) and thermal insulation It, and also effective thermal insulation (0,126) and Icle presented, showing the lowest total and effective thermal insulation In the comparison of the (0,100 ) and , and also effective thermal insulation Icle presented, showing the lowest total and effective thermal insulation In the comparison of the (0,100 m ) and Icle , and also effective thermal insulation (0,081 presented, showing the lowest total and effective thermal insulation In the comparison of the total and effective insulation m2.°C/W Icle (0.087 , and also effective thermal insulation (0,081 presented, showing the lowest total and effective thermal insulation total and effective insulation .°C/W (0.087 , and also effective thermal insulation (0,081 m presented, showing the lowest total and effective thermal insulation total and effective insulation .°C/W), jeans 2 (0.087 , and also effective thermal insulation m2.°C/W presented, showing the lowest total and effective thermal insulation total and effective insulation ), jeans 2 (0.087 m , and also effective thermal insulation .°C/W presented, showing the lowest total and effective thermal insulation total and effective insulation ), jeans 2 m2.°C/W , and also effective thermal insulation .°C/W) and jeans 4 presented, showing the lowest total and effective thermal insulation total and effective insulation ), jeans 2 .°C/W , and also effective thermal insulation ) and jeans 4 presented, showing the lowest total and effective thermal insulation total and effective insulation ), jeans 2 It (0,139 .°C/W) this tree models , and also effective thermal insulation ) and jeans 4 presented, showing the lowest total and effective thermal insulation total and effective insulation (0,139 this tree models , and also effective thermal insulation ) and jeans 4 presented, showing the lowest total and effective thermal insulation total and effective insulation (0,139 this tree models , and also effective thermal insulation ) and jeans 4 It presented, showing the lowest total and effective thermal insulation total and effective insulation m2.°C/W this tree models , and also effective thermal insulation It (0,114 presented, showing the lowest total and effective thermal insulation total and effective insulation values, i .°C/W this tree models , and also effective thermal insulation Icle (0,114 presented, showing the lowest total and effective thermal insulation values, i .°C/W this tree models Icle. 4. Conclusions Conclusions Conclusions Conclusions Conclusions Through the evaluations of the parameters described above it is possible to highlight the following conclusions: Through the evaluations of the parameters described above it is possible to highlight the following conclusions: Through the evaluations of the parameters described above it is possible to highlight the following conclusions: Through the evaluations of the parameters described above it is possible to highlight the following conclusions: Through the evaluations of the parameters described above it is possible to highlight the following conclusions: Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following conclusions: Through the evaluations of the parameters described above it is possible to highlight the following conclusions: Through the evaluations of the parameters described above it is possible to highlight the following conclusions: Through the evaluations of the parameters described above it is possible to highlight the following conclusions: Through the evaluations of the parameters described above it is possible to highlight the following conclusions: Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following Through the evaluations of the parameters described above it is possible to highlight the following • The jeans 1, although presenting lower permeability the highest absolute permeability value to water vapor and effective • The jeans 1, although presenting lower permeability the highest absolute permeability value to water vapor and effective • The jeans 1, although presenting lower permeability the highest absolute permeability value to water vapor and effective • The jeans 1, although presenting lower permeability the highest absolute permeability value to water vapor and effective • The jeans 1, although presenting lower permeability. 4. Conclusions Conclusions Conclusions Conclusions Conclusions The the highest absolute permeability value to water vapor and effective Icle. • The jeans 1, although presenting lower . The the highest absolute permeability value to water vapor Icle. • The jeans 1, although presenting lower . The jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor Icle. 3. Results Results Results Following in descending order, jeans 5 (0,114 m presented, showing the lowest total and effective thermal insulation values, i .°C/W) and this tree models presented the highest values for total . Following in descending order, jeans 5 m2.°C/W presented, showing the lowest total and effective thermal insulation values, it is observed that jeans 1 ) and Icle presented the highest values for total . Following in descending order, jeans 5 .°C/W presented, showing the lowest total and effective thermal insulation values t is observed that jeans 1 Icle presented the highest values for total . Following in descending order, jeans 5 .°C/W) and values t is observed that jeans 1 Icle (0,094 presented the highest values for total . Following in descending order, jeans 5 ) and values. t is observed that jeans 1 (0,094 presented the highest values for total . Following in descending order, jeans 5 ) and Icle t is observed that jeans 1 (0,094 m presented the highest values for total . Following in descending order, jeans 5 Icle t is observed that jeans 1 m2.°C/W presented the highest values for total . Following in descending order, jeans 5 (0,070 t is observed that jeans 1 .°C/W presented the highest values for total . Following in descending order, jeans 5 (0,070 t is observed that jeans 1 .°C/W), and presented the highest values for total . Following in descending order, jeans 5 (0,070 m t is observed that jeans 1 ), and presented the highest values for total . Following in descending order, jeans 5 m2.°C/W t is observed that jeans 1 It (0,145 ), and jeans 3 presented the highest values for total . Following in descending order, jeans 5 .°C/W (0,145 eans 3 presented the highest values for total . Following in descending order, jeans 5 .°C/W) are (0,145 eans 3 presented the highest values for total . Following in descending order, jeans 5 ) are eans 3 presented the highest values for total . Following in descending order, jeans 5 ) are 4. Conclusions Conclusions Conclusions Conclusions Conclusions • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation It values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and It • The jeans 1, although presenting lower permeability the highest absolute permeability value to water vapor and effective • The jeans 1, although presenting lower permeability the highest absolute permeability value to water vapor and effective • The jeans 1, although presenting lower permeability the highest absolute permeability value to water vapor and effective • The jeans 1, although presenting lower permeability the highest absolute permeability value to water vapor and effective • The jeans 1, although presenting lower permeability. 4. Conclusions Conclusions Conclusions Conclusions Conclusions The the highest absolute permeability value to water vapor and effective Icle. • The jeans 1, although presenting lower . The the highest absolute permeability value to water vapor Icle. • The jeans 1, although presenting lower . The jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor Icle. 3. Results Results Results Heat loss Heat loss Heat loss Heat loss from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models Figure Figure Figure Figure 6. 3. Results Results Results Heat loss Heat loss Heat loss Heat loss from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models from body section of manikin dressed with five jeans models Figure Figure Figure Figure 6. 4. Conclusions Conclusions Conclusions Conclusions Conclusions • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor • The jeans 1, although presenting lower values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and the highest absolute permeability value to water vapor and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and and higher values of total thermal insulation It values of mass and thickness and low capacity of air jeans 1 presents the second lowest value of relative water vapor permeability and It ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, ns 2 presented higher values of mass and thickness, the the air permeability presented higher valu air permeability presented higher valu air permeability presented higher valu air permeability presented higher valu air permeability presented higher valu air permeability presented higher valu air permeability presented higher valu air permeability presented higher valu air permeability presented higher valu air permeability presented higher valu air permeability presented higher valu air permeability presented higher valu air permeability presented higher valu air permeability presented higher valu air permeability presented higher valu • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, • Jeans 2 presented higher values of mass and thickness, the the air permeability presented higher value air permeability presented higher value air permeability presented higher value air permeability presented higher value air permeability presented higher value air permeability presented higher value air permeability presented higher value air permeability presented higher value air permeability presented higher value air permeability presented higher value air permeability presented higher value air permeability presented higher value air permeability presented higher value air permeability presented higher value air permeability presented higher value 5 Aegean International Textile and Advanced Engineering Conference (AITAE 2018) IOP Conf. Future perspectives In further studies we will compare the results obtained through objective evaluation with data obtained through evaluation using a inquiry and scales. This study is also important for the producers and traders of Fortaleza of this type of product in order to contribute to the development of this huge market. Acknowledgments This work is financed by FEDER funds through the Competitivity Factors Operational Programme - COMPETE and by national funds through FCT – Foundation for Science and Technology within the scope of the project POCI-01-0145-FEDER-007136” and Federal University of Piauí, Curse de Bach. in Fashion, Design and Stylish. 4. Conclusions Conclusions Conclusions Conclusions Conclusions Series: Materials Science and Engineering459 (2019) 012034 IOP Publishing doi:10.1088/1757-899X/459/1/012034 than jeans 1. Jeans 2 presents lower value of relative water vapor permeability and as for the value of absolute permeability water vapour jeans 2 presents values equal to jeans 1 with greater permeability to absolute water vapor. When analysed the values for heat loss and total thermal insulation and presented data close to the values of jeans 1. than jeans 1. Jeans 2 presents lower value of relative water vapor permeability and as for the value of absolute permeability water vapour jeans 2 presents values equal to jeans 1 with greater permeability to absolute water vapor. When analysed the values for heat loss and total thermal insulation and presented data close to the values of jeans 1. • Jeans 3 and jeans 5 presented statistically equal values for the mass and thickness of the fabrics. The jeans 3 and jeans 5 present statistically very close values regarding the relative water vapor permeability value and absolute water vapor permeability. The heat loss and total and effective thermal insulation. higher value and jeans 5 the lowest value. • The jeans 4 for the values of mass and thickness presented low values, slightly larger than the jeans 1 and smaller values when compared with the other models. The air permeability values, presented the second lower value, with data similar to jeans 2. The jeans 4 presented highest relative water vapor permeability value and the lowest absolute water vapor permeability value. It stood out with higher values of heat loss in all parts of the body and when compared to all models of jeans. It presented lower value of total and effective thermal insulation, when comparing the values with the other models. The results show that the raw material, the structures, the construction of the fabrics, as well as the type of industrial washing influence the thermophysiological properties of the different tested models of jeans. It was possible to conclude that the objective evaluation of the comfort of popular clothing and specifically of jeans designed, produced and sold in Brazilian popular markets is unprecedented and that there is much to research, study and to learn from the popular fashion universe of the Brazilian Northeast. [6] Abreu I, Ribeiro P, Abreu M. The issue of thermal confort of medical clothing in the operating room. DYNA 2014;81:1–2. Aegean International Textile and Advanced Engineering Conference (AITAE 2018) IOP Conf. Series: Materials Science and Engineering459 (2019) 012034 IOP Publishing doi:10.1088/1757-899X/459/1/012034 References [1] Braga I, Abreu MJ. Social aspects in denim production. 2017. [ ] g p p [2] Braga I, Abreu MJ. The jeans in the popular Brazilian panorama. In: TIWC 2016. Poznan: 2016. page 534–40. [2] Braga I, Abreu MJ. The jeans in the popular Brazilian panorama. In: TIWC 2016. Poznan: 2016. page 534–40. [3] ISO. ISO 11092:2014 - Textiles – Physiological effects – Measurement of thermal and water- vapour resistance under steady-state conditions (sweating guarded-hotplate test). 2014. [4] Bogusławska-Bączek M, Hes L. Thermophysiological Properties of Dry and Wet Functional Sportswear Made of Synthetic Fibres. [Internet]. In: Tekstilec, 2017. 2017 [cited 2018 Jul 5]. page 331–8.Available from: http://www.tekstilec.si/wp-content/uploads/2017/12/331- 338.pdf p [5] Mangat A, Bajzik V, Lubos H, Buyuk F. Impact of surface profile of polyester knitted rib structure on its thermal properties. Ind. Textila [Internet] 2016 [cited 2017 Jun 20];67:103–8. Available from: http://media.proquest.com/media/pq/classic/doc/4060570821/fmt/pi/rep/NONE?cit%3 Aauth=Mangat%2C+Asif%3BHes%2C+Lubos%3BBajzik%2C+Vladimir%3BBuyuk%2C+ Funda&cit%3Atitle=Impact+of+surface+profile+of+polyester+knitted+rib+structure+on+its +...&cit%3Apub=Industri 6 Aegean International Textile and Advanced Engineering Conference (AITAE 2018) IOP Conf. Series: Materials Science and Engineering459 (2019) 012034 IOP Publishing doi:10.1088/1757-899X/459/1/012034 [6] Abreu I, Ribeiro P, Abreu M. The issue of thermal confort of medical clothing in the operating room. DYNA 2014;81:1–2. [6] Abreu I, Ribeiro P, Abreu M. The issue of thermal confort of medical clothing in the operating room. DYNA 2014;81:1–2. 7 7
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Supplementary Figure from Famitinib with Camrelizumab and Nab-Paclitaxel for Advanced Immunomodulatory Triple-Negative Breast Cancer (FUTURE-C-Plus): An Open-Label, Single-Arm, Phase II Trial
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Supplementary Figure 3. Association between CD8 and sTILs in patients. Spearman's rank correlation. 0 20 40 60 0 20 40 60 80 CD8 score (%) sTILs (%) P = 0.004 R= 0.488 Supplementary Figure 3. Association between CD8 and sTILs in patients. Spearman's rank correlation. 0 20 40 60 0 20 40 60 80 CD8 score (%) sTILs (%) P = 0.004 R= 0.488 Supplementary Figure 3. Association between CD8 and sTILs in patients. Spearman's rank correlation.
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ALTERED MERISTEM PROGRAM 1 Is involved in Development of Seed Dormancy in Arabidopsis
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Introduction alter the regulation of, and the response to, important hormones in dormancy and germination such as ABA and gibberellins. One upstream factor regulating this class of genes is LEAFY COTYLEDONS 1 (LEC1) [6]. Understanding how LEC1 and other genes regulate the AFL B3 network could be very important for manipulating many important traits that arise during seed development such as seed dormancy. Dormancy is an adaptive trait that is defined by the temporal inability of a seed to germinate under favourable conditions. Dormancy is initiated during seed development. The importance of the timing of seed germination in plant life cycles has resulted in a range of dormancy mechanisms to enhance survival in various environments. The mechanisms involved in blocking embryo growth in dormant seeds and the acquisition of embryo growth potential in non-dormant seeds are regulated by complex interactions between genetic and environmental factors. Under- standing the general mechanisms controlling seed dormancy is important, as is uncovering species-specific factors that could facilitate the manipulation of seed dormancy in some plants, but not in others. These species-specific factors could be crucial for manipulating seed dormancy in different crops. The VIVIPAROUS 8 (VP8) gene from maize has been shown to be a regulator of LEC1 and the AFL B3 factors [7]. vp8 mutant grains display low dormancy and show a viviparous phenotype. Mutations in the orthologous gene PLASTOCHRON 3/GOLIATH (PLA3/GO) in rice also produce viviparous grains [8]. In addition to loss of dormancy, mutations in VP8 and PLA3 produce plants with pleiotropic phenotypes including abnormal leaf morphology, altered shoot apical meristem organisation, shortened plastochron and abnormal hormone homeostasis. The viviparous phenotypes observed are correlated with reduced ABA content in rice seedlings and developing maize grains, and with increased expression of an ABA catabolic gene in maize [7,8]. VP8 and PLA3 encode proteins related to mammalian glutamate carboxy- peptidase IIs (GPCII) which process small peptides involved in metabolic and signalling pathways [9]. Members of this family are known to be involved in the removal of glutamates from neuropeptides and poly-gamma glutamated folate. The plant putative GPCIIs have a high degree of conservation with the animal GPCII’s, possessing the N-terminal membrane spanning domain, conserved zinc residues and catalytic residues. However, the biochemical function of the plant GPCIIs is currently unknown. The model species Arabidopsis has long been used to study dormancy at the genetic and physiological levels [1]. Received February 22, 2011; Accepted April 19, 2011; Published May 26, 2011 Received February 22, 2011; Accepted April 19, 2011; Published May 26, 2011 Copyright:  2011 Griffiths et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the CSIRO OCE postdoctoral fellowship program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: This work was supported by the CSIRO OCE postdoctoral fellowship program. T decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: frank.gubler@csiro.au Competing Interests: The authors have declared that no competing interests exist. * E-mail: frank.gubler@csiro.au Competing Interests: The authors have declared that no competing interests exist. * E-mail: frank.gubler@csiro.au ¤ Current address: Rothamsted Research, Harpenden, Hertfordshire, United Kingdom Jayne Griffiths¤, Jose M. Barrero, Jennifer Taylor, Chris A. Helliwell, Frank Gubler* Division of Plant Industry, CSIRO, Canberra, Australian Capital Territory, Australia Jayne Griffiths¤, Jose M. Barrero, Jennifer Taylor, Chris A. Helliwell, Frank Gubler* Division of Plant Industry, CSIRO, Canberra, Australian Capital Territory, Australia PLoS ONE | www.plosone.org Abstract Mutants in the rice PLASTOCHRON 3 and maize VIVIPAROUS 8 genes have been shown to have reduced dormancy and ABA levels. In this study we used several mutants in the orthologous gene ALTERED MERISTEM PROGRAM 1 (AMP1) to determine its role in seed dormancy in Arabidopsis. Here we report that there are accession-specific effects of mutations in AMP1. In one accession, amp1 mutants produce seeds with higher dormancy, while those in two other accessions produce seeds of lower dormancy. These accession-specific effects of mutating AMP1 were shown to extend to ABA levels. We assayed global gene transcription differences in seeds of wild-type and mutant from two accessions demonstrating opposing phenotypes. The transcript changes observed indicate that the amp1 mutation shifts the seed transcriptome from a dormant into an after-ripened state. Specific changes in gene expression in the mutants give insight into the direct and indirect effects that may be contributing to the opposing dormancy phenotypes observed, and reveal a role for AMP1 in the acquisition and/or maintenance of seed dormancy in Arabidopsis. JM, Taylor J, Helliwell CA, Gubler F (2011) ALTERED MERISTEM PROGRAM 1 Is involved in Development of Seed Dormancy in 20408. doi:10.1371/journal.pone.0020408 Editor: Edward Newbigin, University of Melbourne, Australia Role of AMP1 in Seed Dormancy All the mutations described here are recessive, and all mutant phenotypic traits are inherited in agreement with that. All the mutant plants displayed the same vegetative phenotype (Figure 2, Figure S1, S2 and S3), which has been described previously, such as an increased rate of leaf initiation (Figure 2B) and early flowering (Figure 2C). All the mutants studied showed complete penetrance but variable expressivity. Seeds produced by the mutant plants had a lighter pigmentation compared with their respective wild-types (Figure 2D). All the mutations described here are recessive, and all mutant phenotypic traits are inherited in agreement with that. All the mutant plants displayed the same vegetative phenotype (Figure 2, Figure S1, S2 and S3), which has been described previously, such as an increased rate of leaf initiation (Figure 2B) and early flowering (Figure 2C). All the mutants studied showed complete penetrance but variable expressivity. Seeds produced by the mutant plants had a lighter pigmentation compared with their respective wild-types (Figure 2D). The Arabidopsis orthologue of VP8 and PLA3 is ALTERED MERISTEM PROGRAM 1 (AMP1) [10–11]. Mutations in AMP1 also give rise to pleiotropic phenotypes including an altered number of cotyledons, de-etiolation in dark grown seedlings, increased leaf initiation, dwarfing, earlier flowering time and semi- sterility [10]. Whilst there are similarities in the phenotypes of amp1, vp8 and pla3 mutants, there are also some species specific effects. For example, Arabidopsis amp1 mutants have been shown to have increased cytokinin content [10]. A similar phenotype was also found in rice pla3 but at a lower extent [8]. No difference in cytokinin levels has been observed in maize vp8 [7]. Mutations in vp8 and pla3 in maize and rice have been shown to produce plants with a viviparous phenotype [7,8], similarly a mutant allele of AMP1 has been reported to eliminate dormancy [15]. The nature of this mutation is not known. In order to characterise the effect of amp1 mutations on dormancy, germina- tion was measured in the Col-0, C24 and Ler wild-types and in the amp1-1, amp1-21 and pt mutants (all recessive mutations showing the same vegetative phenotypes) at 0, 1 and 4 weeks after harvest. Under our experimental conditions Col-0 seeds displayed almost no dormancy, with 100% germination from week 0. In contrast, amp1-1 showed increased dormancy, with values of 5% germina- tion at week 0, 30% at week 1 and around 100% at week 4 after harvest (Figure 3A). Phenotypes of different amp1 mutant alleles in Col-0, Ler and C24 accessions The effects of AMP1 mutations on the vegetative and floral stages of the plant life cycle have already been reported [10,12]. However, little is known about the effect of these mutations on seed dormancy. To determine the effect on seed dormancy we have studied several mutant alleles in different accessions. In the Col-0 background we studied the amp1-1 mutation (encoding a premature stop codon) [11], three previously described knock-out T-DNA insertion mutants, (amp1-10, -11, and -13) [13] and one new knock-out insertion mutant (amp1-14) (Figure 1). The primordia timing allele (pt) [14] identified in Ler and a new C24 allele (amp1- 21, that carries a T-DNA insertion in the second exon at nucleotide 1268 from the translation start) were studied (Figure 1). Results In order to demonstrate that the increased dormancy phenotype of the amp1-1 mutant was not an allele-specific effect, we also analysed the dormancy of the other four Col-0 mutants amp1-10, - 11, -13 and -14. These four T-DNA insertion mutants share the same vegetative phenotype as amp1-1 (Figure S3) and also produced seeds that were more dormant (40–75% germination) than Col-0 (90% germination) after 7 days imbibition (Figure 3D). The germination rate of after-ripened seeds was also altered in the five amp1 Col-0 mutants, with a requirement for a slightly longer imbibition time to reach 100% germination (data not shown). Role of AMP1 in Seed Dormancy C24 seeds displayed strong dormancy, with 10% germination after 4 weeks of after-ripening. Surprisingly, amp1-21 showed a dramatic dormancy loss, with 80% germination in week 0 after harvest (Figure 3B). Ler showed an intermediate level of dormancy in our experimental conditions, having 50% germination at week 0 and around 90% at week 4 after harvesting. The pt mutant did not show a significant change in dormancy (Figure 3C). y p [ ] There have been a number of studies on the effect of amp1 alleles on whole plant development [10,11] and seedling [12], however, little research has been done into the seed phenotype of these mutants. In the present study we focus on the role of AMP1 on seed dormancy and germination in Arabidopsis by analysing amp1 mutant alleles in different accessions which display different levels of seed dormancy. We have analysed the ABA content and global gene expression changes in amp1 mutant seeds to gain insight into the possible mechanism(s) by which AMP1/VP8/PLA3 orthologues are influencing dormancy. We found that in Arabidopsis the amp1 mutation has different effects on dormancy and on ABA levels in different accessions. More importantly we show that AMP1 plays a conserved role during seed development in the acquisition or maintenance of seed dormancy, and that when mutated shifts the dormancy program into a germination- like program. Introduction These studies have revealed the importance of abscisic acid (ABA), gibberellins, light, temperature, nutrition and seed coat [2]. Genetic studies and more recent large scale studies investigating transcript changes, including transcription factor profiling [3–5], have highlighted the importance of ABA signalling in mediating loss of dormancy by after-ripening (a period of dry storage). Several studies in Arabidopsis and maize have also focused on the control of seed maturation and the acquisition of seed dormancy [6]. AFL (ABI3/ FUS3/LEC2) B3 domain factors have been found to be critical in the seed maturation program. Mutations in these genes compro- mise desiccation tolerance, embryo identity and dormancy, and May 2011 | Volume 6 | Issue 5 | e20408 1 PLoS ONE | www.plosone.org Role of AMP1 in Seed Dormancy Gene expression analysis in wild-type and amp1 mutant alleles The temporal expression pattern of AMP1 was studied during seed development in Col-0 seeds. AMP1 expression peaked at 8 Figure 1. Positions of the mutations in AMP1 used in this work. The structure of the AMP1 gene is presented. Exons are shown as boxes, T- DNA insertion locations are indicated by triangles and the amp1-1 and pt mutations by arrows. doi:10.1371/journal.pone.0020408.g001 Figure 1. Positions of the mutations in AMP1 used in this work. The structure of the AMP1 gene is presented. Exons are shown as boxes, T- DNA insertion locations are indicated by triangles and the amp1-1 and pt mutations by arrows. doi:10.1371/journal.pone.0020408.g001 May 2011 | Volume 6 | Issue 5 | e20408 PLoS ONE | www.plosone.org 2 Role of AMP1 in Seed Dormancy Figure 2. Phenotypes of amp1-1. Rosettes of Col-0 (A) and amp1-1 (B) at 21 days after sowing. (C) Images of Col-0 and amp1-1 27 days after sowing. (D) Dry seeds from Col-0 and from amp1-1. doi:10.1371/journal.pone.0020408.g002 PLoS ONE | www.plosone.org days after pollination (DAP) and then began to decline as maturation occurred, suggesting a role during early-to-mid seed development (Figure 4A). We studied the expression of genes with roles in seed development and ABA regulation in several amp1 mutants. Gene expression analysis was carried out in developing seeds removed from the silique at 10 DAP, just after AMP1 expression reached its maximum. We studied the expression of the AFL B3 factors (LEC1, ABI3 and FUS3), and several ABA metabolic and signalling genes, the two ABA biosynthetic genes NINE-CIS EPOXYCAR- OTENOID DIOXYGENASE 9 (NCED9) and NCED6 [16], the two ABA catabolic genes CYP707A1 and A2 [17,18] and the ABA signalling gene ABA-INSENSITIVE 5 (ABI5) [19]. All these genes were expressed predominantly in developing and mature seeds. In general these genes were similarly expressed between wild-type and mutant seed (demonstrating they are at a similar develop- mental stage), with some minor differences (Figure 4B). NCED6 was slightly up-regulated in amp1-1 in comparison with Col-0. ABI3 was slightly down-regulated in amp1-21 in comparison with C24. LEC1 was up-regulated in amp1-21 with respect to C24. Interestingly we found that ABI3, FUS3 and LEC1 were down- regulated in C24 in comparison with Col-0. Although we could only detect a few changes driven by the amp1 mutations common to both accessions, we identified several allele-specific and accession-specific changes. Gene expression analysis in wild-type and amp1 mutant alleles PLoS ONE | www.plosone.org ABA quantification in wild-type and amp1 mutant alleles In maize vp8 and rice pla3 mutants, a significant decrease in ABA levels in seeds and seedlings, respectively, was observed [7,8]. In order to determine whether ABA levels were also changed in the different Arabidopsis amp1 mutants, ABA levels were assayed. ABA levels were quantified in freshly harvested dry seeds from Col-0, C24, and Ler accessions and from amp1-1, amp1-21 and pt mutants. On a fresh weight basis amp1-1 mutants contained significantly more ABA than Col-0 (p,0.01), amp1-21 contained less ABA than C24, and pt contained more ABA than Ler (Figure 5). For amp1-1 and Col-0, ABA levels were also determined in developing seeds at 7, 10, 14 and 17 DAP, and an increase in ABA in amp1-1 was found from 14 DAP to the dry seed state (data not shown). The levels of ABA in imbibed seeds were not quantified because the different germination rates of mutants and wild-type could affect the ABA measurements. Microarray analysis of amp1-1, Col-0, amp1-21 and C24 developing seeds Microarray analysis of amp1-1, Col-0, amp1-21 and C24 developing seeds Of the selection of genes affected by amp1-1, At2g33830 is associated with bud dormancy and was up-regulated 20.4-fold; At2g16060 encodes the HEMO- GLOBIN1 protein and was up-regulated 5.6-fold; MYB118 was up-regulated 2.5-fold; and the gibberellin biosynthetic genes KAO1 and GA20OX2 were down-regulated 22.4 and 23.3-fold respec- tively. Among the genes affected by amp1-21, CLAVATA 1 (CLV1) [20] was up-regulated 2.6-fold; MYB51 and MYB67 were down- regulated 22.5 and 24.5-fold, respectively; and ABA3 was down- regulated 22.8-fold. There are several interesting genes that were up-regulated by both amp1-1 and amp1-21, such as the meristematic gene CLAVATA 3 (CLV3) [20], CYTOKININ RE- SPONSE FACTOR 2 (CRF2), and CYP78A5. There are also several genes that were down-regulated by both mutations, including SCARECROW-LIKE 1 (SCL1) [21], DELAY OF GERMINATION 1 (DOG1) [22], TRANSPARENT TESTA 10 (TT10) [23], GAST1 PROTEIN HOMOLOG 2 (GASA2) [24], GASA3, and ETHYLENE RESPONSE FACTOR 72 (ERF72). Given the opposite dormancy phenotypes of the amp1-1 and amp1-21 mutations, we have listed all 17 genes that were regulated in an opposing manner by the two mutations (Table 2), as these may contribute to the opposite phenotypes. Some of the genes from this list were expressed at different levels between the wild- types Col-0 and C24, as is the case of the ABA responsive gene RESPONSIVE TO ABA 18 (RAB18) [25]. This gene was expressed more highly in C24 than in Col-0, however the amp1-1 mutation increased the expression of this gene in Col-0, while the amp1-21 mutation decreased it in C24. Another gene from this group was EARLY LIGHT-INDUCED PROTEIN 1 (ELIP1) [26], which is implicated in light signalling, and was up-regulated in amp1-21 and down-regulated in amp1-1. Figure 3. Dormancy phenotypes of amp1 mutants. (A) % Germination seven days after sowing of bulk harvested Col-0 and amp1-1 (A), C24 and amp1-21 (B) and Ler and pt (C) after-ripened for the indicated time. (D) Germination of all amp1 mutant alleles in Col-0 background freshly-harvested seeds. Means of three biological repli- cates with their SE are shown. *Statistically significant difference in a Student’s t-test (P,0.05; n = 3). doi:10.1371/journal.pone.0020408.g003 We analysed further the 527 genes specifically de-regulated in amp1-1 (Table S3) and the 734 genes specifically de-regulated in amp1-21 (Table S5). In a study of Cvi seeds [3], mRNA expression was analysed in several dormancy and after-ripening states, using microarrays. Microarray analysis of amp1-1, Col-0, amp1-21 and C24 developing seeds To further analyse the effect of AMP1 on dormancy, we compared the global gene expression changes produced by amp1 mutations in two different accessions, Col-0 and C24. We chose these accessions because they displayed different dormancy levels: Col-0 produces seeds with very weak dormancy while C24 produced strongly dormant seeds. To identify gene expression differences that may confer the opposing dormancy phenotypes in Col-0 and C24 amp1 knock-out mutants, microarray analyses were performed on Col-0 and amp1-1 and on C24 and amp1-21 developing seeds at 10 DAP. Analysis of the data demonstrated that there were extensive changes in gene transcript levels in amp1 mutants in both accessions, the majority of them probably being indirect effects. PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org May 2011 | Volume 6 | Issue 5 | e20408 3 Role of AMP1 in Seed Dormancy Figure 3. Dormancy phenotypes of amp1 mutants. (A) % Germination seven days after sowing of bulk harvested Col-0 and amp1-1 (A), C24 and amp1-21 (B) and Ler and pt (C) after-ripened for the indicated time. (D) Germination of all amp1 mutant alleles in Col-0 background freshly-harvested seeds. Means of three biological repli- cates with their SE are shown. *Statistically significant difference in a Student’s t-test (P,0.05; n = 3). doi:10.1371/journal.pone.0020408.g003 amp1-21 but not in amp1-1 (Table S6). Only 337 genes were commonly de-regulated in both mutants (Table S4). These 337 genes can be further split into two groups, those that were similarly regulated in both amp1-1 and amp1-21 (320 genes) and those that were regulated in opposing ways (17 genes). The diversity of gene classes found was notable. GeneOntology (GO) annotation and TAGGIT re-annotation of the genes with germination/dormancy related terms [4] were used to determine whether there were any biological functions that were altered in opposing manners between the more dormant amp1-1 and less dormant amp1-21 and their wild-types, but we could not find any global change related with the phenotypes (Figures S4 and S5). However, it is notable that many genes found have functions in hormone synthesis and responsiveness. We did find several gene expression differences that could explain, at least in part, the mutant phenotypes. Table 1 shows selections of genes de-regulated in both mutants in the same manner, genes found only in amp1-1 and genes found only in amp1-21. Microarray analysis of amp1-1, Col-0, amp1-21 and C24 developing seeds In that work, a core list of 442 genes was found to be more highly expressed in all dormant states analysed and 779 genes was highly expressed in all the after-ripened states. In C24, 330 genes were up-regulated by amp1-21, 10% of them were classified into the ‘‘after-ripened’’ group by Cadman et al. 2006 (only 2.5% expected by random distribution); 404 were down- regulated by amp1-21 and again 11% (only 1.5% expected) of them were classified into the ‘‘dormant’’ group (Figure 6). However only 1% (more than 2.7% expected) of the down-regulated genes appear in the ‘‘after-ripened’’ set, and 0.3% (more than 1.5% expected) of the up-regulated genes appear in the ‘‘dormant’’ data set. In the case of Col-0, 278 genes were up-regulated by amp1-1, Genes that were differentially regulated in amp1-1 and/or amp1-21 compared to wild-type (.2-fold change in expression and an adjusted P-value,0.01) were selected (Table S1). In this group of 1597 genes, 863 were differentially regulated in amp1-1 in comparison with Col-0 (Table S2), and 527 of them were specific to amp1-1 and were not found in amp1-21 (Table S5). In amp1-21, 1071 genes were differentially expressed in comparison with C24 (Table S3), and 734 of them were differentially regulated only in Genes that were differentially regulated in amp1-1 and/or amp1-21 compared to wild-type (.2-fold change in expression and an adjusted P-value,0.01) were selected (Table S1). In this group of 1597 genes, 863 were differentially regulated in amp1-1 in comparison with Col-0 (Table S2), and 527 of them were specific to amp1-1 and were not found in amp1-21 (Table S5). In amp1-21, 1071 genes were differentially expressed in comparison with C24 (Table S3), and 734 of them were differentially regulated only in May 2011 | Volume 6 | Issue 5 | e20408 May 2011 | Volume 6 | Issue 5 | e20408 PLoS ONE | www.plosone.org 4 Role of AMP1 in Seed Dormancy Figure 4. mRNA expression of AMP1 and other genes involved in dormancy acquisition. (A) AMP1 mRNA expression during seed development in Col-0. Under our experimental conditions seeds were considered mature at 14–15 days after pollination (B) Expression of several ABA-related genes and of AFL group of genes in Col-0, amp1-1, C24 and amp1-21 developing seeds at 10 days after pollination. Means of three biological replicates with their SE are shown. Microarray analysis of amp1-1, Col-0, amp1-21 and C24 developing seeds Letters above bars identify statistically significant different values for each gene in a Student’s t-test (P,0.05; n = 3). doi:10.1371/journal.pone.0020408.g004 Figure 4. mRNA expression of AMP1 and other genes involved in dormancy acquisition. (A) AMP1 mRNA expression during seed development in Col-0. Under our experimental conditions seeds were considered mature at 14–15 days after pollination (B) Expression of several ABA-related genes and of AFL group of genes in Col-0, amp1-1, C24 and amp1-21 developing seeds at 10 days after pollination. Means of three biological replicates with their SE are shown. Letters above bars identify statistically significant different values for each gene in a Student’s t-test (P,0.05; n = 3). doi:10.1371/journal.pone.0020408.g004 Figure 4. mRNA expression of AMP1 and other genes involved in dormancy acquisition. (A) AMP1 mRNA expression during seed development in Col-0. Under our experimental conditions seeds were considered mature at 14–15 days after pollination (B) Expression of several ABA-related genes and of AFL group of genes in Col-0, amp1-1, C24 and amp1-21 developing seeds at 10 days after pollination. Means of three biological replicates with their SE are shown. Letters above bars identify statistically significant different values for each gene in a Student’s t-test (P,0.05; n = 3). doi:10.1371/journal.pone.0020408.g004 doi:10.1371/journal.pone.0020408.g004 and again around 10% (only 2.9% expected) of them were classified into the ‘‘after-ripened’’ group and 2.5% (1.4% expected) in the ‘‘dormant’’. Out of the 249 genes down-regulated by amp1- 1, only 2% (1.6% expected) belonged to the ‘‘dormant’’ group and 4% (2.8% expected) to the ‘‘after-ripened’’ (Figure 6). and the similar phenotypes of alleles with insertions in different positions in the gene suggest these are lost-of-function mutant, although we cannot rule out the possibility that truncated peptides with altered activity are produced. Previously, AMP1 was identified as a putative glutamate carboxypeptidase with similar- ities to the human N-acetylated alpha-linked acidic dipeptidase (NAALADase) proteins [11]. It has been suggested that there could be conservation of the biochemical function of this protein family across plants and animals, due to the conserved N-terminal membrane spanning domain, zinc binding and catalytic residues in both plant and mammalian GPCIIs [8,11]. However the biochemical function of AMP1 has not been demonstrated. All amp1, pla3 and vp8 mutants display pleiotropic phenotypes. Whilst there are some similarities between the mutants, there are also significant differences. Microarray analysis of amp1-1, Col-0, amp1-21 and C24 developing seeds It has been shown that pla3 and vp8 are viviparous and there has been a previous report of a viviparous allele of amp1 in Arabidopsis [15]. We show that whilst dormant C24 can be made non-dormant by the amp1-21 mutation, non- dormant Col-0 can be made more dormant by mutating the same gene. We have studied five different Col-0 mutant alleles that are affected in different gene regions (Figure 1), all are recessive and show the same vegetative phenotypes, and all of them showed an increase in seed dormancy (Figure 3). It is unlikely that the different dormancy effects in Col-0 and C24 are caused by the differences between mutant alleles, because for example amp1-11 and amp1-21 are very similar alleles both carrying T-DNA insertions in the second exon. All these together strongly supports that mutation of AMP1 produces accession-specific effects. This phenomenon has been reported before in regards to flowering time in Arabidopsis. The pt allele of AMP1 in the Ler accession has Accession-specific effects of amp1 mutations on seed dormancy In this study we have investigated the effect on dormancy of the AMP1 gene in Arabidopsis, using several recessive mutations in different accessions. The recessive nature of the amp1 mutations Figure 5. ABA content of dry wild-type and amp1 mutant seeds. ABA was extracted from seeds one week after harvest. Means of three biological replicates with their SE are shown. *Statistically significant difference in a Student’s t-test (P,0.05; n = 4). doi:10.1371/journal.pone.0020408.g005 Figure 5. ABA content of dry wild-type and amp1 mutant seeds. ABA was extracted from seeds one week after harvest. Means of three biological replicates with their SE are shown. *Statistically significant difference in a Student’s t-test (P,0.05; n = 4). doi:10.1371/journal.pone.0020408.g005 PLoS ONE | www.plosone.org May 2011 | Volume 6 | Issue 5 | e20408 5 Role of AMP1 in Seed Dormancy Table 1. Selected genes from the microarray analysis. Table 1. Selected genes from the microarray analysis. AGI Annotation amp1-1 vs Col-0 amp1-21 vs C24 adj.P.Val Genes de-regulated in both mutants (same regulation) Fold Change Fold Change At2g27250 CLV3 (CLAVATA3) 4.1 8.0 2.71E-05 At4g23750 CRF2 (CYTOKININ RESPONSE FACTOR 2) 2.7 3.8 1.13E-03 At1g13710 CYP78A5 2.4 3.0 7.95E-05 At1g21450 SCL1 (SCARECROW-LIKE 1) 22.1 22.4 1.24E-02 At5g45830 DOG1 (DELAY OF GERMINATION 1) 22.7 22.6 2.05E-02 At5g48100 TT10 (TRANSPARENT TESTA 10) 23.5 22.6 1.44E-04 At4g09610 GASA2 (GAST1 PROTEIN HOMOLOG 2) 24.8 29.0 4.11E-04 At4g09600 GASA3 (GAST1 PROTEIN HOMOLOG 3) 26.7 28.8 6.14E-03 At3g16770 ERF72 (ETHYLENE-RESPONSIVE) 220.1 227.8 3.45E-05 Genes only de-regulated in amp1-1 At2g33830 dormancy/auxin associated family protein 20.4 1.6 6.89E-03 At2g16060 GLB1 (ARABIDOPSIS HEMOGLOBIN 1) 5.6 21.5 3.91E-02 At3g27785 MYB118 2.5 1.1 1.19E-04 At1g05160 KAO1 (ent-kaurenoic acid hydroxylase) 22.3 21.0 2.54E-03 At5g51810 GA20OX2 (GIBBERELLIN 20 OXIDASE 2) 23.3 21.8 2.05E-03 Genes only de-regulated in amp1-21 At1g75820 CLV1 (CLAVATA 1) 1.5 2.6 1.08E-03 At1g17950 MYB52 21.4 22.5 6.23E-03 At1g16540 ABA3 (ABA DEFICIENT 3) 21.2 22.8 3.78E-02 At3g12720 MYB67 21.8 24.5 3.80E-02 Comparisons were carried out using developing seeds harvested at 10 days after pollination. Fold change was calculated dividing the expression in the mutant by the expression in the wild-type. doi:10.1371/journal.pone.0020408.t001 amp1-1 vs Col-0 amp1-21 vs C24 adj.P.Val Comparisons were carried out using developing seeds harvested at 10 days after pollination. Fold change was calculated dividing the expression in the mutant by the expression in the wild-type. doi:10.1371/journal.pone.0020408.t001 Table 2. Genes de-regulated in both mutants in opposing manners. Accession-specific effects of amp1 mutations on seed dormancy AGI Annotation amp1-1 vs Col-0 amp1-21 vs C24 adj.P.Val At5g38780 S-adenosyl-L-methionine:carboxyl methyltransferase 17.8 22.1 1.27E-04 At3g20950 CYP705A32 6.5 22.3 2.36E-04 At1g75030 ATLP-3 (THAUMATIN-LIKE PROTEIN 3) 3.3 21.9 3.70E-02 At5g09570 Unknown 2.9 23.3 3.41E-04 At2g23240 plant EC metallothionein-like family 15 protein 2.7 22.1 1.19E-02 At5g48850 SDI1 (SULPHUR DEFICIENCY-INDUCED 1) 2.2 211.0 5.21E-02 At5g49360 BXL1 (BETA-XYLOSIDASE 1) 2.1 22.0 2.36E-02 At5g66400 RAB18 (RESPONSIVE TO ABA 18) 2.1 22.8 7.58E-02 At5g57240 ORP4C (OSBP-RELATED PROTEIN 4C) 22.1 2.7 1.84E-03 At5g54470 zinc finger (B-box type) family protein 22.1 2.8 6.63E-04 At3g12955 auxin-responsive protein-related 22.3 3.1 5.63E-03 At2g28900 OEP16-1 (OUTER PLASTID ENVELOPE PROTEIN 16-1) 22.3 4.7 7.34E-06 At3g06360 AGP27 (ARABINOGALACTAN PROTEIN 27) 22.5 2.1 2.97E-02 At2g17850 Unknown 22.5 2.8 1.33E-02 At4g14980 Similar to DC1 domain containing protein 22.7 10.3 6.49E-06 At3g22840 ELIP1 (EARLY LIGHT-INDUCABLE PROTEIN) 22.8 2.0 1.45E-02 At1g43590 Transposable element gene 22.8 2.0 1.56E-02 Comparisons were carried out using developing seeds harvested at 10 days after pollination. Fold change was calculated dividing the expression in the mutant by the expression in the wild-type. doi:10.1371/journal.pone.0020408.t002 Comparisons were carried out using developing seeds harvested at 10 days after pollination. Fold change was calculated dividing the expression in the mutant by the expression in the wild-type. doi:10.1371/journal.pone.0020408.t002 May 2011 | Volume 6 | Issue 5 | e20408 PLoS ONE | www.plosone.org 6 Role of AMP1 in Seed Dormancy Figure 6. Analysis of the genes de-regulated in amp1-1 and amp1-21. The percentage of genes observed and expected (considering random distribution) for each category is shown. The number of genes observed and expected is also indicated above each column. D (Dormant) and AR (After-Ripened) groups were defined in Cadman et al. (2006), containing 442 and 779 genes, respectively. doi:10.1371/journal.pone.0020408.g006 Figure 6. Analysis of the genes de-regulated in amp1-1 and amp1-21. The percentage of genes observed and expected (considering random distribution) for each category is shown. The number of genes observed and expected is also indicated above each column. D (Dormant) and AR (After-Ripened) groups were defined in Cadman et al. (2006), containing 442 and 779 genes, respectively. doi:10.1371/journal.pone.0020408.g006 seed development, the most abundant being CYP707A1 during the mid maturation stage and CYP707A2 at late maturation [29]; we did not detect expression differences for these genes. Finally the expression of the ABA signalling-related genes ABI3 and ABI5 appears not to be effected in amp1-1, and only ABI3 expression is slightly reduced in amp1-21. ABA-related effects of amp1 mutations ABA concentration increases during later stages of seed development, inducing seed dormancy and desiccation tolerance [28]. In the maize vp8 mutant, the lack of dormancy is correlated with decreased ABA levels in the developing grain [7]. We have shown that in Arabidopsis, ABA levels in dry seeds are not clearly correlated with the level of dormancy observed in the seeds from three different mutants in three different wild-type accessions. However in the case of amp1-1 and Col-0 and of amp1-21 and C24, the level of ABA could be one of the reasons explaining the increase and the decrease in dormancy, respectively. Accession-specific effects of amp1 mutations on seed dormancy Therefore we could not find a consistent effect of the amp1 mutations on ABA metabolism or sensitivity in different accessions, suggesting the differences in the ABA level may be due to indirect regulation, for example by feedback signals coming from changes in other hormones (a change in cytokinins and in gibberellins and ethylene signalling has been already reported in amp1-1 [10,12]). been shown to have no effect on flowering time, whereas mutants in a Col-0 background require fewer days to bolt than wild-type [27]. These differential dormancy and flowering time phenotypes suggest that genetic background can have a strong impact on the effect of mutations in AMP1. Accession-specific effects of mutations in AMP1 orthologues have also been reported in maize, where the viviparous phenotype is observed in the SC background but not the W22 background, where it produces a defective embryo rather than vivipary [7]. Beside the accession-specific dormancy phenotype, we have found a common mutant trait in the seeds of amp1-1, amp1-21 and pt. The seed coats of the mutant seeds are lighter in colour than the wild-type seed coats. Another trait, out of the scope of this manuscript, common to the three mutants is the highly variable phenotype expressivity in the vegetative tissues (Figure 2, Figure S1, S2 and S3). We have also tested the putative role of AMP1 in regulating the AFL B3 domain factors (ABI3/FUS3/LEC2). In maize and rice, AMP1 orthologues have been shown to regulate the expression of this important set of genes during seed development [6]. We were able to validate that role in the accession C24, where amp1-21 affected the expression of ABI3 and LEC1. In contrast, in Col-0, amp1-1 did not appear to affect the expression of any of the AFL genes. PLoS ONE | www.plosone.org Specific gene expression changes in amp1-1 and amp1- 21 mutants We have grouped our microarray results into different categories: genes differentially regulated only in amp1-1 or only in amp1-21, genes differentially regulated by both mutations in both accessions in the same manner (Table 1), and genes differentially regulated by both mutations but in opposing manners (Table 2). Those that are similarly regulated in amp1-1 and amp1- 21 are likely to be specific to the amp1 mutation rather than due to an effect of the background accession. Those regulated in one or the other accession will most likely be due to a background specific effect of the amp1 mutation. And finally those differentially We analysed the expression level of some of the key genes involved in ABA synthesis, catabolism and signalling in the seed. We looked at the expression of NCED6 and NCED9 and saw a minor increase in the expression of NCED6 only in amp1-1, and no change in NCED9. ABA catabolic genes are expressed throughout May 2011 | Volume 6 | Issue 5 | e20408 PLoS ONE | www.plosone.org 7 Role of AMP1 in Seed Dormancy regulated in both accessions but in a different manner could be specific AMP1 targets also regulated by accession-specific effects. involved in bud dormancy in several species. The mechanism controlling bud and seed dormancy is very similar and as this gene is up-regulated 20-fold in amp1-1, it is a good candidate to explain the change in dormancy observed in the mutant. GLB1 is also very significantly up-regulated in amp1-1. This gene encodes the Arabidopsis HEMOGLOBIN 1 protein, which has been found to scavenge nitric oxide (NO) [37]. NO is a well known promoter of germination, and its depletion could lead to an increased dormancy [38]. A third gene up-regulated in amp1-1 is the transcription factor MYB118 that was found to be up-regulated in dormant seeds [5]. Interestingly two gibberellin biosynthetic genes, KAO1 and GA20OX2, are down-regulated in amp1-1. The effect of gibberellins as ABA antagonists have been widely studied and a reduction in gibberellin levels could explain an increase in seed dormancy. p g g y p Among the genes that are similarly regulated in both Col-0 and C24 mutants are genes that have been previously identified as being involved in meristem regulation, such as CLV3 [13]. Specific gene expression changes in amp1-1 and amp1- 21 mutants This gene is up-regulated in both mutants, and although the increased meristem size of amp1 mutants does not correlate well with the over-expression of CLV3, this could be compromising meristem development. Another gene that appears in this list is CYP78A5, which was previously identified in a microarray study of amp1 mutant rosettes [11], suggesting it is specifically regulated by AMP1 in at least two different tissues. We have highlighted a third gene up-regulated in both mutants, CRF2, that may be a consequence of the increased cytokinins in amp1 previously described [10]. A gene that is expressed specifically in the developing seed (Arabidopsis eFP Browser [30,31]) and that is down-regulated in both amp1 mutants is TT10. TT10 is involved in the oxidation of procyanidins, which are clear in colour, to the brown oxidised procyanidins [23,32]. Mutants in TT10 have pale yellow seeds that darken gradually similar to amp1 mutant seeds, suggesting that the down-regulation of TT10 causes the colour phenotype. We also found several other genes down-regulated in amp1-1 and amp1-21 that could be related to seed dormancy and germination. Several of these are related to gibberellins; the SCL1 transcription factor, a putative gibberellin regulator, and GASA2 and 3, two seed specific gibberellin-responsive genes [24]. Another interesting gene that is down-regulated more than 20-fold is ethylene-response factor ERF72; ethylene has been shown to promote germination and to be antagonistic to ABA [33–35] and it seems to act in the micropylar endosperm, a key seed tissue controlling germination [34–35]. A role of AMP1 in ethylene and GA signalling pathways during seedling development has already been described [12]. Finally we identified one of the better known dormancy-related genes, DOG1 [22]. This gene reduces dormancy when mutated and is down-regulated in both amp1-1 and amp1-21 compared with wild-type seeds. This gene was first discovered as a QTL involved in seed dormancy using a Cvi x Ler mapping population. The different DOG1 alleles from Cvi and Ler were correlated with high and low seed dormancy, respectively [22]. If C24 carries a DOG1 allele that confers the high dormancy observed in C24 (similarly to the Cvi allele), its down-regulation by amp1-21 would then explain the lack-of-dormancy phenotype. Conversely if Col-0 carries weak-dormancy DOG1 allele (func- tionally similar to the Ler allele), the down-regulation of this gene by amp1-1 would not have any affect. Specific gene expression changes in amp1-1 and amp1- 21 mutants Other explanations for the change in expression of DOG1, relate to changes in response to ABA, can be found. For example, the down-regulation of DOG1 in the amp1-21 mutant, which has reduced levels of ABA, could be related to the finding that the Cvi allele of DOG1 is up-regulated by ABA [36]. Genes de-regulated by both amp1-1 and amp1-21 in opposing manners could explain the opposite effect on seed dormancy caused by the two mutations. In Table 2 we have described all the genes belonging to this category, eight of them being up-regulated in amp1-1 and nine of them in amp1-21. Many of the genes from this group have not been reported before to be related with dormancy or germination, but some are more than 10-fold differentially expressed in one mutant or the other. A number of these genes are also differentially expressed between the wild-types so could explain the differences in dormancy level between Col-0 and C24. From this group RAB18, an ABA-responsive factor commonly used as an ABA-response marker gene [25], is of particular interest. This gene is up-regulated in amp1-1 and down- regulated in amp1-21, suggesting that the ABA pathway has been activated by the first mutation and repressed by the second, fitting with the phenotype of both mutants. Finally a second gene that may be important in the discussion of differences in dormancy is ELIP1, a light-induced gene described in many species [26,39] and related with grain dormancy in barley [40]. In Arabidopsis, light is needed to promote germination and break dormancy. Interest- ingly in amp1-1 the gene ELIP1 is down-regulated as if the light activated pathway were repressed. In contrast, in amp1-21, ELIP1 is up-regulated as would happen in a non-dormant seed. It is notable that the amp1 mutation affects, directly or indirectly, two key elements in the control of dormancy and germination, the ABA and the light signalling pathways, in opposing manners in Col-0 and C24. These suggest that AMP1 has a conserved role in dormancy acquisition during seed development, and could be used to investigate interactions between different dormancy regulatory pathways. PLoS ONE | www.plosone.org Plant lines and growth conditions To generate seeds for dormancy and germination rate experiments, 10 Arabidopsis plants were grown in an 11 cm pot in soil (Debco seed raising mix +2 g litre21 osmocote). Plants were grown in controlled environment cabinets set at a 16 hour photoperiod, with a light intensity of 180 mmol m22 s21 at a temperature of 20uC. Seeds were harvested in bulk 1 week after flowering had ceased when the plant was completely dry. For QRT-PCR 0.5 mg of RNA was used to synthesise cDNA in a 20 ml reaction using SuperScript II (Invitrogen Life Sciences; http://www.invitrogen.com) using the protocols supplied. cDNA was diluted 25- fold and 10 ml of this was used in a 20 ml PCR reactions with Platinum Taq and SYBR Green (Invitrogen). Specific primers were designed, and are listed in Table S7. Reactions were run on a RG-3000A real-time PCR machine (Corbett Research; http://www.corbettlifescience.com) and data were analysed with Rotor-Gene software using the comparative quantification tool. The expression of Cyclophillin (At2g29960) was used as a control gene [41]. amp1-1 and amp1-21 were donated by Dr. Chris Helliwell. T- DNA mutants were identified using the Salk T-DNA insertion database and seeds were obtained from the Arabidopsis Biological Resource Center (http://biosci.ohio-state.edu/pcmb/Facilities/ abrc/abrchome.htm). Homozygous mutant plants were then generated and dormancy and germination rate tested under our standard conditions. Microarray and Quantitative real time PCR g y y g y For transcriptome analysis, total RNA was isolated from 10 DAP developing seeds (for amp1-1 around 20 siliques were dissected per replicate, and for wild-type around 10 siliques were dissected per replicate) from three independent biological replicates. Resulting microarray data was analysed using the limma package from Bioconductor (http://www.bioconductor. org/) in R (R Development Core Team, 2008). Microarray data are available in the ArrayExpress database (www.ebi.ac.uk/ arrayexpress) under accession number E-MEXP-3181, following MIAME recommendations. For transcriptome analysis, total RNA was isolated from 10 DAP developing seeds (for amp1-1 around 20 siliques were dissected per replicate, and for wild-type around 10 siliques were dissected per replicate) from three independent biological replicates. Resulting microarray data was analysed using the limma package from Bioconductor (http://www.bioconductor. org/) in R (R Development Core Team, 2008). Microarray data are available in the ArrayExpress database (www.ebi.ac.uk/ arrayexpress) under accession number E-MEXP-3181, following MIAME recommendations. Supporting Information Developing seeds were obtained from plants grown under the same conditions. Figure S1 Phenotypes of amp1-21. Rosettes of C24 (A) and amp1-21 (B) at 21 days after sowing. (TIF) Figure S1 Phenotypes of amp1-21. Rosettes of C24 (A) and amp1-21 (B) at 21 days after sowing. (TIF) Global gene expression changes in amp1-1 or only by amp1-21 mutants All the seed used in an experiment were harvested from the same batch of plants, which were grown together at the same time and in the same environment. Plates were then sealed with Parafilm and incubated at 20uC under continuous fluorescent light (100 mmol m-2 s-1) for 7 days. Germination was scored as the emergence of the radicle outside the seed coat and the endosperm. For dormancy experiments involving the Col-0 accession we found that the filter paper reduced dormancy, and therefore experiments were performed on 3 ml of 0.6% agarose in 35 mm Petri dishes [35]. Experiments were performed in triplicate for each line examined. All the seed used in an experiment were harvested from the same batch of plants, which were grown together at the same time and in the same environment. regulated in amp1-21 were significantly enriched in genes from the ‘‘dormant’’ set and depleted in genes from the ‘‘after-ripened’’ set (Figure 6). The germination phenotype observed in amp1-21 correlates perfectly with these results. This effect of amp1-21 turning C24 seeds into an ‘‘after-ripened-like’’ state is only partially observed in amp1-1. Again, the group of genes up-regulated by amp1-1 is enriched in genes from the ‘‘after-ripened’’ set but is not depleted in genes from the ‘‘dormant’’ set. More importantly there is no significant over-representation of the ‘‘dormant’’ or ‘‘after- ripened’’ sets in the down-regulated genes (Figure 6). This correlation between the transcript changes found in C24 vs. amp1-21 and between the ‘‘dormant’’ and ‘‘after-ripened’’ states in Cvi, suggests that the amp1 mutation is affecting specific pathways regulating dormancy. This supports the hypothesis that AMP1 and orthologues in maize and rice are controlling a similar seed development mechanism involved in the acquisition of seed dormancy. However, mutation of AMP1 in Col-0 appears to be affecting genes in a non-phenotype specific manner. An increase in the ‘after-ripened’’ group of genes is seen in the up-regulated set, but not in the down-regulated set. A hypothesis for that could be a dormancy model where Col-0 would produce seeds with a very weak dormancy because the dormancy mechanism existing in Cvi and C24 is absent in Col-0. In fact, more than 50% of the ‘‘dormant’’ set of genes that are down-regulated by the amp1-21 mutation in comparison with C24 are also down-regulated in Col- 0 wild-type in comparison with C24. ABA quantification The ABA content was measured from developing seeds and matured seeds. Between 100 and 300 developing seeds were extracted from siliques and transferred to pre-weighed foil boats on dry ice, seeds were then weighed. For the mature seed 50 mg of dry seed was used for the extraction. ABA for both developing and mature seed was extracted from quadruplicate replicates as described previously [42]. The ABA measurements were carried out using a Phytodetek Competitive ELISA kit (Agdia) following the manufacturer’s protocol. Global gene expression changes in amp1-1 or only by amp1-21 mutants Mutations in AMP1 in C24 and Col-0 show some opposite phenotypes, suggesting that genetic factors in the background of the mutation are important. Microarray data generated from amp1 mutations in the Col-0 and C24 backgrounds demonstrated that the functional classification of the de-regulated transcripts is largely similar. Genes regulated .2 fold in amp1 mutants in Col-0 and C24 were compared to published data sets of dormant and after-ripened related genes in Cvi [3]. Very interestingly this analysis showed that the transcript signature of freshly harvested amp1-21 seeds is mimicking the one of Cvi after-ripened seeds. The number of genes de-regulated in amp1-21 found in the ‘‘dormant’’ or in the ‘‘after-ripened’’ sets was significantly different from the expected number of genes from a random distribution (Figure 6). Genes up-regulated in amp1-21 were significantly enriched in genes from the ‘‘after-ripened’’ set from Cadman et al. (2006), and depleted in genes from the ‘‘dormant’’ set. Contrarily, genes down- From the group of genes de-regulated in amp1-21 but not in amp1-1, we found a number that correlate well with the putative role of AMP1 in other species. We also found another well characterised meristematic gene, CLV1, to be up-regulated. The ABA biosynthesis gene ABA3 is down-regulated, consistent with the reduction in the levels of this hormone found in amp1-21 seeds. Finally the transcription factors MYB52 and MYB67, which were previously found to be up-regulated in dormant seeds [5], are significantly down-regulated in amp1-21 again consistent with the after-ripened-like behaviour of this mutant. In the Col-0 amp1-1 mutant, we found several genes only de- regulated in this genotype that could explain the increase in dormancy produced by this mutation. At2g33830 encodes a dormancy/auxin associated family protein, previously shown to be May 2011 | Volume 6 | Issue 5 | e20408 May 2011 | Volume 6 | Issue 5 | e20408 8 Role of AMP1 in Seed Dormancy Plates were then sealed with Parafilm and incubated at 20uC under continuous fluorescent light (100 mmol m-2 s-1) for 7 days. Germination was scored as the emergence of the radicle outside the seed coat and the endosperm. For dormancy experiments involving the Col-0 accession we found that the filter paper reduced dormancy, and therefore experiments were performed on 3 ml of 0.6% agarose in 35 mm Petri dishes [35]. Experiments were performed in triplicate for each line examined. PLoS ONE | www.plosone.org Microarray and Quantitative real time PCR Developing seeds for microarray and QRT-PCR experiments were removed from siliques at intervals after pollination and flash- frozen in liquid nitrogen. Total RNA was isolated from those developing seeds using the Qiagen RNA easy Plant mini kit (Qiagen), with Plant RNA isolation AID (Ambion) added to the extraction buffer. Isolated RNA was then treated with TurboD- NAse (Ambion). The quality of RNA intended for microarrays was assessed on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) before being sent to Australian Genome Research Facility Ltd. (www.agrf.org.au; Melbourne, Victoria, Australia) for labelling and hybridisation to the Affymetix ATH1 genome array. For transcriptome analysis, total RNA was isolated from 10 DAP developing seeds (for amp1-1 around 20 siliques were dissected per replicate, and for wild-type around 10 siliques were dissected per replicate) from three independent biological replicates. Resulting microarray data was analysed using the limma package from Bioconductor (http://www.bioconductor. org/) in R (R Development Core Team, 2008). Microarray data are available in the ArrayExpress database (www.ebi.ac.uk/ arrayexpress) under accession number E-MEXP-3181, following MIAME recommendations. Developing seeds for microarray and QRT-PCR experiments were removed from siliques at intervals after pollination and flash- frozen in liquid nitrogen. Total RNA was isolated from those developing seeds using the Qiagen RNA easy Plant mini kit (Qiagen), with Plant RNA isolation AID (Ambion) added to the extraction buffer. Isolated RNA was then treated with TurboD- NAse (Ambion). The quality of RNA intended for microarrays was assessed on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) before being sent to Australian Genome Research Facility Ltd. (www.agrf.org.au; Melbourne, Victoria, Australia) for labelling and hybridisation to the Affymetix ATH1 genome array. From these results we speculate that the some effects of the amp1- 21 mutation in eliminating dormancy in C24 may be conserved and direct, affecting the expression of dormancy-related genes like DOG1 and affecting ABA and light sensitivity. The effect of amp1-1 increasing dormancy in Col-0 could be a side effect resulting from the initial lack of deep dormancy in this accession and of fortuitous gene expression changes that partially restore dormancy levels. This is also in agreement with the effect of the pt mutation in Ler, another low-dormancy accession. The pt mutant has increased levels of ABA in seeds, but its dormancy remained very similar to wild-type. In conclusion we propose a role for AMP1 in the acquisition and/or maintenance of seed dormancy in Arabidopsis. References 16. Schwartz SH, Qin XQ, Zeevaart JAD (2003) Elucidation of the indirect pathway of abscisic acid biosynthesis by mutants, genes, and enzymes. Plant Physiol 131: 1591–1601. 1. 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Vidaurre DP, Ploense S, Krogan NT, Berleth T (2007) AMP1 and MP antagonistically regulate embryo and meristem development in Arabidopsis. Development 134: 2561–2567. 29. Okamoto M, Kuwahara A, Seo M, Kushiro T, Asami T, et al. (2006) CYP707A1 and CYP707A2, which encode abscisic acid 89-hydroxylases, are indispensable for proper control of seed dormancy and germination in Arabidopsis. Plant Physiol 141: 97–107. 14. Mordhorst AP, Voerman KJ, Hartog MV, Meijer EA, van Went J, Koornneef M, et al. Acknowledgments The authors wish to thank Trijntje Hughes and Kerrie Ramm for their expert technical assistance and Drs. Julien Curaba, Megan Hemming, Stephen Swain and Colleen MacMillan for comments on the manuscript. The authors wish to thank Trijntje Hughes and Kerrie Ramm for their expert technical assistance and Drs. Julien Curaba, Megan Hemming, Stephen Swain and Colleen MacMillan for comments on the manuscript. Table S1 Probes differentially expressed in amp1-1 and/or in amp1-21. (XLS) Author Contributions Conceived and designed the experiments: JG FG CAH. Performed the experiments: JG JMB. Analyzed the data: JG JMB JT. Contributed reagents/materials/analysis tools: JG JT. Wrote the paper: JMB JG. Conceived and designed the experiments: JG FG CAH. Performed the experiments: JG JMB. Analyzed the data: JG JMB JT. Contributed reagents/materials/analysis tools: JG JT. Wrote the paper: JMB JG. Conceived and designed the experiments: JG FG CAH. Performed the experiments: JG JMB. Analyzed the data: JG JMB JT. Contributed reagents/materials/analysis tools: JG JT. Wrote the paper: JMB JG. Table S2 Probes differentially expressed in amp1-1. (XLS) Germination assays For dormancy experiments involving C24, approximately 100 seeds were placed in 85 mm Petri dishes with 3 ml of water and three Whatman No. 1 70 mm diameter filter papers (Whatman International, Maidstone, England), as previously described [41]. Figure S2 Phenotypes of pt. Rosettes of Ler (A) and pt (B) at 21 days after sowing. (TIF) Figure S2 Phenotypes of pt. Rosettes of Ler (A) and pt (B) at 21 days after sowing. (TIF) PLoS ONE | www.plosone.org May 2011 | Volume 6 | Issue 5 | e20408 9 Role of AMP1 in Seed Dormancy Table S3 Probes differentially expressed in amp1-21. (XLS) Figure S3 Phenotypes of all Col-0 amp1 mutant alleles studied in this work. Rosettes of Col-0 (A), amp1-1 (B), amp1-10 (C), amp1-11 (D), amp1-13 (E) and amp1-14 (F) at 21 days after sowing. (TIF) Table S4 Probes differentially expressed in both amp1-1 and amp1-21. (XLS) Figure S4 GO classification of the genes differentially expressed in the microarray experiment. Classification of genes expressed highly in Col-0 than in amp1-1 (Col-0.amp1-1), amp1-1.Col-0, C24.amp1-21 or amp1-21.C24 using the Gene Ontology (GO) molecular function. The proportional representa- tion of the total gene set is shown. (TIF) Table S5 Probes differentially expressed only in amp1-1. (XLS) Table S6 Probes differentially expressed only in amp1-21. (XLS) Table S7 Primers used in this work. (XLS) Table S7 Primers used in this work. Figure S5 Classification of the genes differentially expressed from the microarray experiment using TAG- GIT annotation. Classification of genes expressed more highly in Col-0 than in amp1-1 (Col-0.amp1-1), amp1-1.Col-0, C24.amp1-21 or amp1-21.C24 using TAGGIT annotation [4]. (TIF) References (1998) Somatic embryogenesis in Arabidopsis thaliana is facilitated by mutations in genes repressing meristematic cell divisions. Genetics 149: 549–563. 30. Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, et al. (2007) An ‘‘Electronic Fluorescent Pictograph’’ browser for exploring and analyzing large- scale biological data sets. PLoS ONE 2: e718. 15. Evans MMS, Poethig RS (1997) The viviparous8 mutation delays vegetative phase change and accelerates the rate of seedling growth in maize. Plant J 12: 769–779. 31. Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, et al. (2005) A gene expression map of Arabidopsis thaliana development. Nat Genet 37: 501–506. PLoS ONE | www.plosone.org May 2011 | Volume 6 | Issue 5 | e20408 10 Role of AMP1 in Seed Dormancy 32. Liang MX, Davis E, Gardner D, Cai XN, Wu YJ (2006) Involvement of AtLAC15 in lignin synthesis in seeds and in root elongation of Arabidopsis. Planta 224: 1185–1196. 37. Perazzolli M, Dominici P, Romero-Puertas MC, Zago E, Zeier A, et al. (2004) Arabidopsis nonsymbiotic hemoglobin AHb1 modulates nitric oxide bioactivity. Plant Cell 16: 2785–2794. 38. Bethke PC, Libourel IGL, Jones RL (2007) Nitric oxide in seed dormancy and germination. In: Annual Plant Reviews Volume 27, Seed development, dormancy and germination. pp 153–175. 33. Cheng W, Chiang M, Hwang S, Lin P (2009) Antagonism between abscisic acid and ethylene in Arabidopsis acts in parallel with the reciprocal regulation of their metabolism and signaling pathways. Plant Mol Biol 71: 61–80. metabolism and signaling pathways. Plant Mol Biol 71: 61–80. 39. Grimm B, Kloppstech K (1987) The early light-inducible proteins of barley. Characterization of two families of 2-h-specific nuclear-coded chloroplast proteins. Eur J Biochem 67: 493–499. 34. Linkies A, Mu¨ller K, Morris K, Tureckova´ V, Wenk M, et al. (2009) Ethylene interacts with abscisic acid to regulate endosperm rupture during germination: a comparative approach using Lepidium sativum and Arabidopsis thaliana. Plant Cell 12: 3803–3822. 40. Barrero JM, Talbot MJ, White RG, Jacobsen JV, Gubler F (2009) Anatomical and transcriptomic studies of the coleorhiza reveal the importance of this tissue in regulating dormancy in barley. Plant Physiol 150: 1006–1021. 35. Morris K, Linkies A, Mu¨ller K, Oracz K, Wang X, et al. (2011) Regulation of Seed Germination in the Close Arabidopsis Relative Lepidium sativum: A Global Tissue-Specific Transcript Analysis. Plant Physiol 155: 1851–1870. 41. Millar AA, Jacobsen JV, Ross JJ, Helliwell CA, Poole AT, et al. 41. Millar AA, Jacobsen JV, Ross JJ, Helliwell CA, Poole AT, et al. (2006) Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 89- hydroxylase. Plant J 45: 942–954. 42. Gubler F, Hughes T, Waterhouse P, Jacobsen J (2008) Regulation of dormancy in barley by blue light and after-ripening: Effects on abscisic acid and gibberellin metabolism. Plant Physiol 147: 886–896. References (2006) Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 89- hydroxylase. Plant J 45: 942–954. 36. Teng S, Rognoni S, Bentsink L, Smeekens S (2008) The Arabidopsis GSQ5/ DOG1 Cvi allele is induced by the ABA-mediated sugar signalling pathway, and enhances sugar sensitivity by stimulating ABI4 expression. Plant J 3: 372–381. 42. Gubler F, Hughes T, Waterhouse P, Jacobsen J (2008) Regulation of dormancy in barley by blue light and after-ripening: Effects on abscisic acid and gibberellin metabolism. Plant Physiol 147: 886–896. PLoS ONE | www.plosone.org May 2011 | Volume 6 | Issue 5 | e20408 11
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Are we preparing for collaboration, advocacy and leadership? Targeted multi-site analysis of collaborative intrinsic roles implementation in medical undergraduate curricula
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Griewatz et al. BMC Medical Education (2020) 20:35 https://doi.org/10.1186/s12909-020-1940-0 Griewatz et al. BMC Medical Education (2020) 20:35 https://doi.org/10.1186/s12909-020-1940-0 Open Access Are we preparing for collaboration, advocacy and leadership? Targeted multi- site analysis of collaborative intrinsic roles implementation in medical undergraduate curricula Jan Griewatz* , Amir Yousef, Miriam Rothdiener, Maria Lammerding-Koeppel and on behalf of the Collaborators of the MERlin Mapping Group Abstract Background: The Collaborator, Health Advocate and Leader/Manager roles are highly relevant for safe patient management and optimization of healthcare system in rehabilitation and prevention. They are defined in competency-based frameworks and incorporate competencies empowering physicians to master typical daily tasks in interdisciplinary, interprofessional and institutional collaboration. However, appropriate implementation of roles remains difficult in undergraduate medical education (UME) and needs to be closely monitored. The aim of this cross-institutional mapping study was to examine for the roles of Collaborator, Health Advocate and Leader/ Manager: (1) To what extent do German UME programs explicitly meet the given standards after 5 years of study? (2) Which information may be obtained from multi-site mapping data for evidence-based reflection on curricula and framework? Methods: In a joint project of eight German UME programs, 80 to 100% of courses were mapped from teachers’ perspective against given national standards: (sub-)competency coverage, competency level attainment and assessment. All faculties used a common tool and consented procedures for data collection and processing. The roles’ representation was characterized by the curricular weighting of each role content expressed by the percentage of courses referring to it (citations). Data were visualized in a benchmarking approach related to a general mean of the intrinsic roles as reference line. Results: (Sub-)competencies of the Health Advocate are consistently well-integrated in curricula with a wide range of generally high curricular weightings. The Collaborator reveals average curricular representation, but also signs of ongoing curricular development in relevant parts and clear weaknesses regarding assessment and achieved outcomes. The Leader/Manager displays consistently lowest curricular weightings with several substantial deficiencies in curricular representation, constructive alignment and/or outcome level. Our data allow identifying challenges to be considered by local curriculum developers or framework reviewers (e.g. non-achievement of competency levels, potential underrepresentation, lacking constructive alignment). (Continued on next page) (Continued on next page) * Correspondence: jan.griewatz@med.uni-tuebingen.de Competence Centre for University Teaching in Medicine, Eberhard-Karls University of Tuebingen, Baden-Wuerttemberg, Elfriede-Aulhorn-Str. 10, D-72076 Tuebingen, Germany * Correspondence: jan.griewatz@med.uni-tuebingen.de Competence Centre for University Teaching in Medicine, Eberhard-Karls University of Tuebingen, Baden-Wuerttemberg, Elfriede-Aulhorn-Str. 10, D-72076 Tuebingen, Germany Background directly [12, 13]. Secondly, the Health Advocate addresses human health and well-being beyond clinical care: it em- phasizes the professional contribution to collectively developing practicable concepts for change in health care system [5, 14–16]. Thirdly, the Leader/Manager deals with complex situations and scarce resources in the ongoing evolving healthcare system, envisioning doctors as a “spearhead of change” [17, 18]. Reforming health care by changing health professionals’ education is still a highly topical and challenging issue worldwide: Today’s “health professionals are the service providers who link people to technology, information, and knowledge” [1]. Physicians are expected to be capable of promoting patient safety and providing efficient patient care together with other healthcare professionals. Add- itionally, medical doctors are responsible for prevention and policymaking as collaborative leaders, respecting the expectations and needs of patients, and to ensure the community is served in an ethical and resource-economic manner in a continuously evolving health care system [2]. In response to the profound shift in the healthcare envir- onment, competency-based medical education (CBME) has been gradually incorporated and integrated in a super- ordinate role framework of additional psychosocial com- petencies [3–5]. Role concepts, originally developed for postgraduate medical education (PME), have been defined to cover all relevant and typical facets of tasks in the daily practice of physicians. CanMEDS is only one of several frameworks, but it is the most widely adopted worldwide [5]. It specifies seven professional roles with the Medical Expert as central integrative role. All others are considered intrinsic roles [6, 7]. In view of the special significance of the Collaborator, Health Advocate and Leader/Manager for safe patient management and optimization of healthcare system, these roles are often requested to be introduced as an integral part into UME [17]. According to the practice-based learning theory, the interrelationship of defined compe- tencies and the way they are enacted in educational prac- tice are highly relevant for sustainable learning oriented towards professional demands [10]. For residency, stu- dents should be fundamentally prepared for the roles, in order to take over gradually growing responsibilities in interaction with persons and institutions quicker [10, 17]. However, there is an ongoing debate about equivalent im- portance of competencies, required competency levels and suitable curricular interventions across UME [14]. Yet it remains unclear in large part which design, frequency and intensity of teaching, learning and assessment is most ap- propriate to foster the acquisition of these competencies. © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 12 Griewatz et al. BMC Medical Education (2020) 20:35 Griewatz et al. BMC Medical Education Background Although CBME concepts exist since the late 1990s [3, 8], the appropriate implementation of the superordinate roles in undergraduate medical education (UME) still represents a major challenge for medical faculties inter- nationally [2, 4, 9–11]: This particularly matters for the Collaborator, Health Advocate and Leader/Manager. These professional roles are characterized by a high need for interaction with different people and groups within the healthcare system, thus resulting in relational complexities and intersecting sets of competencies. The roles encom- pass competencies empowering physicians to participate effectively and appropriately in the healthcare sector with health professional groups and institutions, with the over- all aim to ensure the well-being of patients and of the population. Despite these strong interrelations each role is setting key priorities: firstly, the Collaborator is focused on preventing, negotiating and resolving interpersonal and interprofessional conflicts, thus affecting patient safety Based on the above considerations, the process of roles implementation should be evaluated systematically from the beginning. Germany is at an early stage of trans- forming UME to CBME. Using Germany as an example, the overall aim of this study was to evaluate the status quo of developing the above three roles in UME curric- ula. The study was guided by the following questions: (1) To what extent do the German UME programs explicitly meet the given standards at present after 5 years of study? (2) Which information may be obtained from multi-site mapping data for evidence-based reflection on curricula and framework? (Continued from previous page) p p g Conclusion: Our non-normative, process-related benchmarking approach provides a differentiated crosscut snapshot to compare programs in the field of others, thus revealing shortcomings in role implementation, especially for Leader/Manager and Collaborator. The synopsis of multi-site data may serve as an external reference for program self-assessment and goal-oriented curriculum development. It may also provide practical data for framework review. Keywords: Undergraduate medical education, Competence orientation, CBME framework, NKLM, Intrinsic roles, Curriculum development, Curriculum mapping, Reference data, Benchmarking Mapping software, data collection and data control Mapping software, data collection and data control For the normalisation of the process and to ensure data quality, all faculties used the MERlin mapping database [22] as a common instrument and followed consented procedures, supported by hands-on instruction and indi- vidual counselling by the CCMD staff. Each faculty en- tered its curricular data into a separate protected data repository within the web-based MERlin-database appli- cation. Mapping tool, procedures as well as methods of data collection and processing have been described in detail earlier [21, 22]. A short summary is given below. Mapping of courses against given NKLM standards was conducted on sub-competency level by selecting pre-set menu options in the database: (1) the highest achieved competency level; (2) transparency in teaching (“explicit” standing for written in a study-guide, module manual or other material); (3) extent of sub-competency complete- ness as calculated automatically from underlying learn- ing objectives that were ticked off in case they are taught (“objective covered”); (4) formative and/or summative or no assessment. To ensure content validity, the mapping was carried out by 47–101 faculty members per site: individuals from each discipline, often preceptors with content-related expertise of courses or senior teachers with educational background, coordinating and/or supervising courses of the department. Plausibility con- trols were carried out by authorized local representatives and/or staff of the dean’s offices. The global administra- tor of CCMD carried out regular consistency checks. p g The multi-site project was led by the Competence Centre of University Teaching in Medicine Baden- Wuerttemberg in Tuebingen (CCMD). A pre-study focusing on multi-site depiction of intrinsic roles in German UME, revealed differentiated role patterns re- garding the varying realization of roles and programs’ agreement, thus providing a general diagnostic orienta- tion [20]. However, further methods are required sub- sequently for comprehensive insight. Benchmarking approaches have shown to provide detailed curricular reference data: firstly, by linking mapping data to the given NKLM standards, secondly, by comparing own results with data from other programs to determine one’s position in the field of others [21]. Multi-site ref- erence data at learning objectives level promises to facilitate targeted local curriculum development as well as to support the review process of the framework regarding e.g. role prioritization and topical foci. Mapping software, data collection and data control Eight faculties (Tuebingen, Freiburg, Ulm, Hannover, Bonn, Magdeburg, Frankfurt and Wuerzburg) contributed to the current analysis of mapping data focusing on the explicit curricular representation of three professional roles: Collaborator, Health Advocate, and Leader/Manager (corresponding to NKLM chapters 8–10 [19];). In 4 of the 8 programs, 95–100% of courses were mapped. The other participating programs documented at least 80% of courses, a percentage considered to be sufficient for inclusion. Data processing and statistics Relative weighting Mapping data were calculated in percentages to fa- cilitate comparison of the programs. The intrinsic roles representation was described by the curricular weighting of each objective expressed by the per- centage of citations, assuming the more courses present an objective (mapping citations) the higher its curricular emphasis. To compute the curricular weighting a two-step relativization was necessary to make up for (1) site-specific and (2) framework- specific differences. Site-specific differences are dis- played by the huge range of quantity of mandatory courses, respectively the amount of citations (Table 1). To achieve realistic comparability of the curricu- lar weighting of an objective (obj) at a medical fac- ulty (MF_x), the number of an objective’s citations (ncit) was put in the context of the total number of citations at that specific site for a defined reference size (Ncit). As framework-specific differences, the Setting and sample In 2015, the National Competency-based Learning Objectives Catalogue for Undergraduate Medical Education Page 3 of 12 Griewatz et al. BMC Medical Education (2020) 20:35 Griewatz et al. BMC Medical Education (2020) 20:35 Griewatz et al. BMC Medical Education shortened versions of the original wordings (Table 2; for long versions refer to Additional file 1). (NKLM) was adopted in its first version as guiding frame- work for Germany [19]. The catalogue will be reviewed, commented and modified by end of 2020 before becoming compulsory within a new medical licensure regulation. The comprehensive framework contains 21 chapters with the professional roles based on the CanMEDs concept being in- troduced in the beginning (chapters 5–11). Since 2016, medical faculties from all over Germany have participated in a joint project, mapping courses of five-year undergradu- ate programs against the NKLM in order to visualise cur- ricular implementation of roles and competencies, to follow-up their evolvement and to gain reference data. Terminology Local datasets of medical faculties (MF) were anon- ymized by consecutive numbers in random order (e.g. MF_1, MF_2). Programs and roles were characterized in an overview in Table 1. The basic organizational unit was defined as “course”, although it might be differenti- ated further by different lengths. A “citation” refers to any objective taught in a course and ticked off by the mapper in the mapping process. Teaching an objective on one or more occasions in a course corresponds to one citation. Competencies (C), sub-competencies (SC) and underlying objectives (O) of each intrinsic role were identified by their NKLM chapter code numbers and Page 4 of 12 Griewatz et al. BMC Medical Education (2020) 20:35 Griewatz et al. BMC Medical Education (2020) 20:35 NKLM framework for the milestone of 5 years of study, the value of the next milestone to come was filled in. In the cases that different objective levels are given in a sub-competency, the reference value was discussed and determined in the project group. Usually the highest level reached was assumed as reference, if (1) there were other objectives on that level, and/or (2) the respective objectives were of higher relevance for the superior com- petency. All reference levels for learning objectives are given in the Additional file 1 for completeness. Assess- ments of sub-competencies were outlined roughly di- vided in summative and/or formative formats. If no assessment was specified for a sub-competency, this in- formation was listed separately (Table 2). varying sizes of roles (number of learning objectives in resp. chapter, Nobj) were considered. Due to the differing numbers of objectives of roles, the resulting values were aligned with the defined reference size (∑roles) to get them equally levelled and enable role comparison. Because of the underlying concept of intrinsic roles, the reference size of relativization was theoretically set for the intrinsic roles (Note: The role of the Com- municator had to be excluded, because in the NKLM the Communicator is described only on competency level and its objectives have been relocated within the framework. Thus, the consented reference size is reduced to 5 roles). The relativized results were multiplied by a hundred, thus scaled on a percentage base: A value of hundred reliably showing average representation. The consider- ations led to the following formula: Statistical analyses l Data were analysed using the Statistical Package for the Social Sciences SPSS, version 25 and Excel of the Microsoft Office Package, version 2010. The descrip- tive statistics including frequencies, mean, minimum, maximum, first and third quartiles were performed for faculties mapping data (citations). For cross-role com- parisons the Kruskal H Test was performed. To get more precise markers for the position of an objective in relation to the general mean in the Additional file 1, a value for the tendency of representation was calcu- lated using the Wilcoxon Signed-Ranks Test (cp. Additional file 1). The degree of agreement on objec- tives between programs was measured with Average Pairwise Percent Agreement. relative weighting obj; MF x ð Þ ¼ ncit obj;MF x ð Þ Ncit Σ roles;MF x ð Þ ∙Nobj Σ roles ð Þ∙100 For greater clarity in presentation of the objectives, boxplot diagrams were created with data of the eight programs, depicting the detailed weighting profile for every role (Figs. 1.1–3), although its usage is seen critic- ally for smaller sample sizes. In the boxplot diagrams outliers were not excluded or especially emphasized. A “general mean” was implemented as a reference line to indicate high and low weightings and facilitate cross-role comparison: Assuming equal distribution of citations on objectives, multiplication of all average values by the total number of all roles’ objectives resulted in a com- mon mean of 100 for every reference size (here: concept of intrinsic roles). Results General role description in the UME programs Quantitative key data about the programs’ general curricular status reveals a huge range of quantity of mandatory courses (Table 1). This is due to the heterogenous granularity of organizational structure (teaching units) within the scope of governmental reg- ulations guaranteeing minimum standards of UME. The total number of role citations discloses that all programs explicitly represented the intrinsic roles examined; however, the Collaborator, Health Advocate and Leader/Manager clearly differed in their percent- age share. Degree of implementation For analysing the actual quality of performance in teachers’ perspective, the highest competency levels achieved after 5 years of studies according to the map- ping data were listed for each program in an overview in Table 1. Additionally, it was documented in how far competency parts are assessed. The site-specific specifi- cations for the levels of objectives were compared to the NKLM requirements as given reference. The framework defines level requirements on objective level and expli- citly refers to them as minimal standards. In Table 2 the number of programs that meet, exceed or fall below the standard are shown. In this study, analyses were carried out on sub-competency level to enable a greater degree of granularity without losing sight of the role perspective when being faced with comprehensive sets of objectives. For some objectives, competency level requirements were missing or heterogenous in a sub-competency: In the rare cases that level values were not given by the In inter-role comparison, the Health Advocate repre- sented the highest amount of explicit citations, ranking significantly higher than the other intrinsic roles ana- lysed (H(2) = 41,514; p < .001). In contrast, the Leader/ Manager role was mapped clearly at the lowest rate of all (mean ranks: Health Advocate 65,67; Collaborator 45, 25; and Leader/Manager 24,11). These data correspond to the general impression gained from the boxplot diagrams, indicating the relative importance of the Griewatz et al. BMC Medical Education (2020) 20:35 Griewatz et al. BMC Medical Education Page 5 of 12 Table 1 Curricular status quo of the selected roles Medical Faculty Total number of courses (n) Total number of citations (n) Percentage (%) Collaborator (Chap. 8) Health Advocate (Chap. 9) Leader/Manager (Chap. 10) courses citations courses citations courses citations MF_1 137 495 22 46 19 33 18 21 MF_2 121 310 10 22 19 50 17 28 MF_3 178 731 26 36 23 36 25 28 MF_4 146 293 14 22 21 62 14 16 MF_5 98 473 38 34 30 38 37 28 MF_6 133 650 26 22 43 56 29 22 MF_7 102 659 36 28 38 36 34 37 MF_8 112 531 29 32 39 52 21 16 The overview includes all curricular courses at the participating faculties in which objectives of the three intrinsic roles (Collaborator Chap. 8, 24 objectives; Health Advocate Chap. 9, 18 objectives; Leader/Manager Chap. 10, 37 objectives) are addressed (citations). Degree of implementation Data of medical faculties is presented in anonymized form (MF_1–8) and in random order Table 1 Curricular status quo of the selected roles The overview includes all curricular courses at the participating faculties in which objectives of the three intrinsic roles (Collaborator Chap. 8, 24 objectives; Health Advocate Chap. 9, 18 objectives; Leader/Manager Chap. 10, 37 objectives) are addressed (citations). Data of medical faculties is presented in anonymized form (MF 1–8) and in random order values. To give an idea, the lowest value of O-9.1.1.3 is still twice above the roles reference line. intrinsic roles and competencies (Fig. 1.1–3). The Health Advocate’s citations clearly exceed the general mean as a cross-role reference line indicating the highest degree of explicit curricular representation of the three roles. The amount citations for the Health Advocate shows a range between 33 and 62% of all citations. The mapping cita- tions of the Collaborator’s objectives are positioned quite balanced around the general mean, ranging from 22 to 46 of percentage shares. The Leader/Manager role with its citations lies clearly below the reference line and extends from 16 to 37% of citations. According to the mapping data (Table 2), most pro- grams achieve an above-standard competency level for the objectives of the Health Advocate in 5 years of study. All SCs are assessed in summative formats in all UME programs and in most programs also in formative for- mats. Thus, this role is considered to be strongly repre- sented and comprehensively integrated in all UME curricula. To examine the degree of programs’ matching the given NKLM role standards, the roles representation in teaching and assessment were described in detail (Table 2): (1) curricular weighting; (2) competency level achieved; and (3) presence of summative and/or forma- tive assessment of sub-competencies. The roles are ranked based on amount of citations in descending order, starting with the Health Advocate showing the highest explicit mapping citations. Collaborator Most objectives of the Collaborator are weighted around or above the reference line, indicating this role’s respect- able amount of curricular representation regarding most objectives (Fig. 1.1). Compared to the Health Advocate, the interquartile ranges appear less widely spread indi- cating the relatively high agreement of most programs (cp. Additional file 1). A growing number of programs appear to set a special focus on reflective collaboration in multiprofessional teams (SC-8.2.1, SC-8.2.2), but in a greater range of representation. However, SC-8.2.3 “Recognize interprofessional conflicts and actively con- tribute to solutions” as well as the complete competency C-8.4. “Collaboratively develop structures, processes and concepts contributing to solve relevant healthcare issues” display a special characteristic: they all show a noticeably low weighting, dropping even to zero. Regarding compe- tency level (Table 2), the majority of programs achieve the minimal NKLM-standards of Level 3a (competency in practice, supervised) for this role. In most cases even a higher competency level is reached for the sub- competencies, except for the two from the low-weighted C-8.4. Their underlying objectives are also assessed to a lesser extent, if at all. In contrast, the higher weighted objectives of the Collaborator are otherwise assessed The Table shows the competency levels reached after 5 years of study in comparison to the given NKLM standards for sub-competencies: Level 1: knowledge/ understanding/basic skills, Level 2: applied knowledge and skills in training, Level 3: competency in practice (3a: supervised, 3b: independent). Sub-competencies are specified in translations. For insight in content of superordinate competencies or sub-ordinate objectives and full-text wording see Additional file 1. Higher consent between the faculties (8–7 of eight faculties) in a column is indicated with darker colour (green = standard or above; yellow = sub-standard or none), consent above average (6–4) in lighter colour, allowing quicker diagnoses. Sum summative assessment; form formative assessment Leader/manager In comparison to the Health Advocate and the Collabor- ator, the Leader/Manager generally appears to be the role with the largest amount of low values in weightings, competency level and assessment. Few prominent com- ponents of this role can easily be identified in Table 2 and Fig. 1.3, because of superior or around average weighting. They reflect legal conditions and social values of the health care system and address key aspects of dealing with errors and patient safety (O-10.1.1.1 and O- 10.6.1.2 to O-10.6.1.4). The competency levels intended for most of the objectives, are achieved at minimal or above standard by the greater majority of programs (Table 2; SC-10.1.1 to SC-10.7.1). Additionally, these objectives are assessed in summative and formative formats at all sites. In contrast to the few well-integrated role compo- nents, most other objectives show consistently low cur- ricular weighting at the participating faculties or in some cases even none at all (Fig. 1.3). It is particularly striking that four to seven programs achieve minimal-standard or sub-standard competency level in 5 of 16 SC, but simultaneously do not to perform any assessment (Table 2; SC-10.8.1 to SC-10.10.2). This group include relevant issues of general competencies like time man- agement, career planning, and leadership (e.g. SC-10.8.1, SC 10.9.2, SC-10.10.2). In summary, the Leader/Manager role shows consented low representation in most objec- tives. However, a third of the role demonstrates obvious shortcomings of curricular integration (including assess- ment) – a finding that needs to be addressed particularly in sense of constructive alignment. The benchmarking approach with a common mapping database and consented procedures [22] applied in this study, appears an appropriate strategy to support moni- toring of CBME implementation [21]. Mapping data can be applied in that way at any time during curricular de- velopmental processes. The current data set documents a crosscut snapshot to indicate the program’s current positioning in relation to others in the field. In the context of UME, this approach allows to gain (external) reference data, to identify potential for optimization and to realize best practices. In any case, the data are consid- ered as non-normative but descriptive in benchmarking process. However, the anonymity of the program is ensured since individual data is accessible only by the respective faculties. Health advocate mean = general mean of reference set Griewatz et al. BMC Medical Education (2020) 20:35 Page 8 of 12 Page 8 of 12 Griewatz et al. BMC Medical Education stronger summatively and/or formatively. Thus, it can be concluded that most objectives of the Collaborator role currently appear to be integrated with average curricular weighting and assessed correspondingly, achieving at least given minimal standard. description and visualisation, a common reference, different perspectives and scalability in focus and permanent availability. y In a previous, preliminary mapping study, multi-site curricular weightings of role’s objectives and programs’ agreement were compared in a matrix map. By applying Roger’s theory of diffusions of innovations [23], the role-specific patterns gave an orienting overview to identify the roles in various stages of curricular devel- opment [20]. In a next step, the present multi-site study provides more detailed diagnostic data focussing on the curricular status quo of the (sub-)competencies and objectives of the Collaborator, Health Advocate and Leader/Manager. These roles are all highly relevant for safe patient management and optimization of the healthcare system in rehabilitation and prevention. In overview, the Health Advocate can be highlighted as a positive example of how sub-competencies are consist- ently well-integrated in curricula, though in wide ranges of generally high curricular weightings. In con- trast, the Collaborator role indicates average curricular representation, but reveals signs of ongoing curricular development in relevant parts, as well as obvious weak- nesses regarding assessment and achieved outcome. Finally, the Leader/Manager displays the consistently lowest curricular weightings of its objectives with sev- eral substantial deficiencies in curricular representa- tion, constructive alignment and/or outcome level [24]. Discussion International experiences show that implementing the superordinate role concept in UME is not an automatic process [2, 4, 10] but needs to be closely monitored with suitable tools. There are several ways to evaluate the im- plementation of intrinsic roles at certain stages, mostly relying on (focus group) interviews, surveys or observa- tion of practice of the different target groups involved. The strengths of these common approaches can be espe- cially seen in including individual viewpoints, identifica- tion of practical needs and fundamental freedom of positioning. Mapping approaches provide another re- source that can be combined with existing methods or form their basis. Mapping approaches, especially in web- based databases, enable e.g. comprehensive curricular Health advocate The boxplots give graphical information on the location, spread and skewness of the role’s curricular data. With very few exceptions, all learning objectives are taught intensively in all UME programs, although they show a relatively high variance in median curricular weighting and a wide spread of data regarding individual objectives (Fig. 1.2). The SC 9.1.1 “Recognize health status of individual persons as well as health imbalances, causes and consequences” is most strongly pronounced. Within this SC, the learning objective O-9.1.1.3 “Identify key factors, parameters and individual resources for changing overall health situation” is the most prominent: It shows the highest median weightings and broadly scattered Griewatz et al. BMC Medical Education (2020) 20:35 Griewatz et al. BMC Medical Education Page 6 of 12 Table 2 Competency level achievement and assessment of the selected roles ompetency level achievement and assessment of the selected roles ows the competency levels reached after 5 years of study in comparison to the given NKLM standards for sub-competencies: Level 1: know g/basic skills, Level 2: applied knowledge and skills in training, Level 3: competency in practice (3a: supervised, 3b: independent). Sub-com in translations. For insight in content of superordinate competencies or sub-ordinate objectives and full-text wording see Additional file 1. th f lti (8 7 f i ht f lti ) i l i i di t d ith d k l ( t d d b ll b t d d Griewatz et al. BMC Medical Education (2020) 20:35 Page 7 of 12 Griewatz et al. BMC Medical Education (2020) 20:35 Griewatz et al. BMC Medical Education Page 7 of 12 Fig. 1 Curricular profiles of the selected intrinsic roles. In the sub-diagrams the roles of the Collaborator (Fig. 1.1), Health Advocate (Fig. 1.2) and Leader/Manager (Fig. 1.3) are displayed in boxplots. Competencies are specified in abbreviated translations. Subordinated objectives are identified by NKLM code numbers: e.g. 8.1.1.1. For insight in content and full-text wording see Additional file 1. Gen. mean = general mean of reference set Fig. 1 Curricular profiles of the selected intrinsic roles. In the sub-diagrams the roles of the Collaborator (Fig. 1.1), Health Advocate (Fig. 1.2) and Leader/Manager (Fig. 1.3) are displayed in boxplots. Competencies are specified in abbreviated translations. Subordinated objectives are identified by NKLM code numbers: e.g. 8.1.1.1. For insight in content and full-text wording see Additional file 1. Gen. Taught but not assessed content Taught but not assessed content Several examples illustrate missing formal constructive alignment in a considerable number of programs (e.g. in the Collaborator SC-8.2.3 addressing interprofessional conflicts; or in the Leader/Manager: again SC-10.10.1 and SC-10.10.2). There is an ongoing debate regarding the testability as well as the necessity of obligatory as- sessment of every competency facet, especially consider- ing the rare opportunities to explicitly experience and practice certain role aspects in clinical context [27–29]. Faculties often perceive classical assessment methods as suboptimal for non-medical content. At the same time, unawareness and lack of familiarity with alternatives like qualitative methods lead to adherence to traditional habits. However, multifaceted assessment facilitating developmental progression of competence is crucial in CBME. More information and training are essential to “create a shared mental model of required learner’s behaviour and expected level of performance” [28] and build up “continuous, comprehensive and elaborate assessment and feedback systems” [4, 27]. Besides highlighting the role of feedback, research efforts are to be intensified for further development of additional for- mative assessment instruments and formats [25, 29, 30]. Thus, particularly faculty and curriculum development as well as quality management measures are required to Implications of role profiles Thus, basic competency levels achieved should be further de- veloped in the practical year and residency [17, 26]. However, inappropriate educational context and envir- onment as well as lacking students’ involvement could result in too rare opportunities to: (1) see role modelling (e.g. cooperative leadership), (2) reflect on the realisation and (3) practice it themselves [10]. The given representa- tion may indicate deep-rooted curricular patterns. In view of the low curricular representation in German UME programs and the development of societal needs, both curriculum developers and framework reviewers are recommended to rethink competency levels and weightings as well as to place greater emphasis on un- derrepresented qualities. priorities. Whilst, on the other hand, multi-site practice- based evidences support framework reviews by critically reflecting its content and currently valid standards for perspective adjustment. There are typical constellations of diagnostic findings down to detailed objective level, affecting both or one of the target groups. In the follow- ing, frequently occurring challenges are exemplified in increasing degree of difficulty; it is discussed how they may be interpreted and dealt with, from different perspectives. Implications of role profiles Mapping current UME curricula against consented stan- dards (here: the German CBME framework NKLM [19]) reveals detailed information on conformities and dis- crepancies between curricular reality and given stan- dards in teaching, assessment and competency level. On the one hand, these diagnostic data can help curriculum developers to identify curricular challenges in their local program. Based on that information they can decide whether and how to deal with these problems and set Page 9 of 12 Griewatz et al. BMC Medical Education (2020) 20:35 Griewatz et al. BMC Medical Education (2020) 20:35 Griewatz et al. BMC Medical Education Manager. They are mapped in only few or none courses but well-consented in this lowest amount of curricular representation: e.g. SC-10.10.1 and SC-10.10.2 focusing leadership personality and styles as well as management functions. Internationally, collaborative leadership skills are increasingly recognized as indispensable for every physician - a fact that has already been considered in the drafting (and revision) of various national frameworks [1, 18]. Because of the well-known leadership impact on patient care and safety, these objectives are recom- mended to be integrated stably into UME programs for advanced medical students in their clinical years. Thus, basic competency levels achieved should be further de- veloped in the practical year and residency [17, 26]. However, inappropriate educational context and envir- onment as well as lacking students’ involvement could result in too rare opportunities to: (1) see role modelling (e.g. cooperative leadership), (2) reflect on the realisation and (3) practice it themselves [10]. The given representa- tion may indicate deep-rooted curricular patterns. In view of the low curricular representation in German UME programs and the development of societal needs, both curriculum developers and framework reviewers are recommended to rethink competency levels and weightings as well as to place greater emphasis on un- derrepresented qualities. Manager. They are mapped in only few or none courses but well-consented in this lowest amount of curricular representation: e.g. SC-10.10.1 and SC-10.10.2 focusing leadership personality and styles as well as management functions. Internationally, collaborative leadership skills are increasingly recognized as indispensable for every physician - a fact that has already been considered in the drafting (and revision) of various national frameworks [1, 18]. Because of the well-known leadership impact on patient care and safety, these objectives are recom- mended to be integrated stably into UME programs for advanced medical students in their clinical years. Role parts exceeding given standards The Health Advocate offers characteristic examples for this data constellation. At a first glance it appears rather unproblematic: In line with international demands [1, 14–16], this role is an essential part of the UME curric- ula and well-integrated in many programs, though vary- ing in frequency and intensity. It is assessed in all programs. A closer inspection shows that many sites clearly surpass the given minimal competency level in most sub-competencies, except in SC-9.1.3 and SC-9.2.3 addressing interprofessional health promotion in popula- tion groups and systems. Here, few programs fall short of the desired competence level and thus give the re- sponsible local curriculum developers cause to act. In the overall evaluation, the Health Advocate shows itself as a positive role that does not currently require imme- diate urgent attention except in some local curricula. Non-achievement of competency level Despite the wide range of curricular weightings, heteroge- neous attainments of competency levels were mapped, in some UME programs below the minimum requirements (Table 2). Typical examples are some sub-competencies of the Collaborator role, esp. SC-8.2.1 to SC-8.2.3, encompass- ing interpersonal skills for interdisciplinary and interprofes- sional collaboration. These topics are evidenced as a key aspect of successful inter-professional teams being closely related to patient safety [1, 13]. In case of sub-standard rep- resentation, sub-competencies appear to be taught rather in theory (Level 2: applied knowledge and skills in training) than in practice as specified in the NKLM (Level 3a: com- petency in practice, supervised). Presumably this is because of missing learning opportunities, inadequate context or impeding cultural environment [25]. After review of the NKLM framework and its re-acceptance, German curricu- lum developers are most likely challenged to revise and in- tensify UME interventions ensuring that graduates are appropriately prepared for mastering collaborative practice on day 1 of residency [10]. Low curricular weighting but (potentially) underrepresented Typical examples of this characteristic feature are (sub-)competencies and objectives of the Leader/ Page 10 of 12 Page 10 of 12 Griewatz et al. BMC Medical Education Griewatz et al. BMC Medical Education (2020) 20:35 facilitate institutional and programmatic change regard- ing Collaborator and Leader/Manager. visualization in boxplots and following definite inter- pretation about underlying structures. Nevertheless, the display in boxplots instead of dot columns pro- vides more clarity and enough orientation to catch an informative tendency of roles development on object- ive level at a glance. General accumulation of curricular weaknesses Some framework content is characterized by clear defi- ciencies in the majority of programs regarding the criteria included: none or very low curricular weightings, sub-standard level attainment and missing assessment (e.g. non-specific sub-competencies from Leader/Man- ager field addressing time management, career planning and personal qualification needs; or Collaborator role features focusing on advanced aspects of interprofes- sional work). This may be caused by lack of conceptual clarity in terms of definitions, role characteristics, personal-specific and context-specific features, as sys- tematically reviewed for the Leader/Manager [31]. Not- withstanding, some roles (esp. Collaborator, Leader/ Manager) appear to be less affected by external pressure like e.g. legal regulation or politics than others (e.g. Health Advocate) during the last decades. Instead, mod- erate signs of increasing curricular emphasis like e.g. in the Collaborator role, seem to be based rather on internal efforts of individual programs. More than in any other case mentioned above, reviewers of the framework may seek clarification on whether, and if so to what extent, a (sub-)competency should be integrated into UME (e.g. personal-specific features of planning, system- related interprofessional collaboration). If re-affirmed, every effort for institutional change must be strength- ened. If identified as inappropriate, it should be removed from UME and potentially transferred to PME. Conclusions d h Faced with disillusioning international experiences, faculties are well-advised to carefully monitor the pro- gress of the implementation of the Collaborator, Health Advocate and Leader/Manager in detail. The results must be made transparent, if the intrinsic role concept is to be beneficial. The type of data evalu- ation applied in this study focuses on targeted in- depth analyses of defined competencies down to ob- jectives level, to unfold a differentiated picture of the roles’ status: Conformities, deficiencies and typical constellations within the role profiles can be identified and categorized for constructive data interpretation and informed discussions. This more detailed look is a second step in a systematic concept with continuing refinement of mapping analyses. It builds on data from a preliminary mapping approach, giving orient- ing insight in the stages of roles curricular diffusion and enabling the identification problematic fields by using Matrix Map analysis. In this non-normative, process-related benchmarking, multi-site reference data is generated with evidences for informed self- assessment of a curriculum and decision-making on goal-oriented, tailored development at faculties. In the ensuing discourse, local culture and contexts, such as educational resources, traditions, faculty attitudes and institutional readiness for change can be considered and related to (inter-)national requirements. Thus, in quality assurance these data from practice supports local curriculum developers and framework reviewers alike. This systematic approach can be transferred and adapted to any other framework content. The key elements for its replication in other contexts are (1) a flexible web-based database depicting the framework, (2) defined mapping procedures to create meaningful data sets and (3) commitment and integration of mul- tiple sites referring to the same (or comparable) framework. The mapping approach promises the chance to handle the enormous amount of curricular data in a productive and resource-efficient way with- out putting too much pressure on participating facul- ties and thereby hindering the change process. In perspective, regular, transparent and graduated hand- ling of mapping data (site-specific as well as cross- site) may foster a shared mental concept regarding given standards, expected student outcomes and edu- i l i i Consent for publication C f bli i i Consent for publication is not applicable for this study. The usage of data was approved in all participating medical faculties by the deans’ office and/ or local authorized persons. Competing interests Th h d l h PME: Postgraduate medical education; UME: Undergraduate medical education The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 10 February 2019 Accepted: 17 January 2020 Received: 10 February 2019 Accepted: 17 January 2020 References k 1. Frenk J, Chen L, Bhutta ZA, Cohen J, Crisp N, Evans T, et al. Health professionals for a new century: transforming education to strengthen health systems in an interdependent world. Lancet. 2010;376:1923–58. https://doi.org/10.1016/S0140-6736(10)61854-5. 1. Frenk J, Chen L, Bhutta ZA, Cohen J, Crisp N, Evans T, et al. Health professionals for a new century: transforming education to strengthen health systems in an interdependent world. Lancet. 2010;376:1923–58. https://doi.org/10.1016/S0140-6736(10)61854-5. 2. Caverzagie KJ, Nousiainen MT, Ferguson PC, Ten Cate O, Ross S, Harris KA, Busari J, Bould MD, Bouchard J, Iobst WF, Carraccio C. Frank JR; ICBME collaborators. Overarching challenges to the implementation of competency-based medical education. Med Teach. 2017;39(6):588–93. https://doi.org/10.1080/0142159X.2017.1315075. On behalf of the Collaborators of the MERlin Mapping Group: Olaf Fritze (Tuebingen, olaf.fritze@med.uni-tuebingen.de), Alessandro Dall’Acqua (Freiburg, alessandro.dallacqua@uniklinik-freiburg.de), Mara Geißinger (Ulm, mara.geissinger@uni-ulm.de), Sandra Steffens (Hannover, steffens.sandra@mh-hannover.de), Bernhard Steinweg (Bonn, bernhard.steinweg@ukb.uni-bonn.de), Katrin Borucki (Magdeburg, katrin.borucki@med.ogvu.de), Aleksandra Germanyuk (Frankfurt, aleksandra.germanyuk@kgu.de), Sarah Koenig (Wuerzburg, koenig_sarah@ukw.de). 3. Frank JR, Snell L, Englander R, Holmboe E. On the behalf of the ICBME collaborators. Implementing competency-based medical education: moving forward. Med Teach. 2017;39(6):568–73. https://doi.org/10.1080/ 0142159X.2017.1315069. 4. Holmboe ES, Sherbino J, Englander R, Snell L. Frank JR; ICBME collaborators. A call to action: the controversy of and rationale for competency-based medical education. Med Teach. 2017;39(6):574–81. https://doi.org/10.1080/ 0142159X.2017.1315067. 5. Frank JR, Snell L, Sherbino J, editors. The draft CanMEDS 2015 physician competency framework – series IV. Ottawa: The Royal College of Physicians and Surgeons of Canada; 2015. Ethics approval and consent to participate This research was performed in accordance with the Declaration of Helsinki. The study does not involve any human subjects, including research on identifiable human material and data. The study was approved by the deans of studies of all participating medical faculties. The ethics committee of the Medical Faculty of Tuebingen University confirmed that no formal ethics approval was required for this study (Project-No: 622/2018BO2). Acknowledgements The authors would like to thank the entire MERlin Project Group for stimulating discussions, the deans of study and teaching as well as the staff of all the deans’ offices of student affairs for their eminent support of the mapping project. Particularly, we wish to thank all teaching coordinators and those responsible for modules for their energetic participation in the mapping process and the discussions. They contributed important information and gave impulses for optimizing the instruments and the process. We further thank Dr. M Salker, University of Oxford for her time proof-reading this manuscript. Availability of data and materials pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12909-020-1940-0. The datasets analysed during the current study are not publicly available due to contract agreements. Data are available from the corresponding author in anonymized version upon reasonable request and with permission of the participating medical faculties as third party and owners of their local curricular data. Supplementary information accompanies this paper at https://doi.org/10. 1186/s12909-020-1940-0. Additional file 1. NKLM Objectives of Collaborator, Health Advocate and Leader/Manager in detail. This supplement includes translated full- text wordings and weightings of the objectives of three professional roles given in the NKLM framework: Collaborator (chapter 8), Health Advocate (chapter 9) and Leader/Manager (chapter 10). Competencies are dis- played in dark grey, sub-competencies in light grey. The weighting of fac- ulties is differentiated in a) tendency (high weighting, above the general median = ➚; low weighting, below the general median = ➘; medium weighting, on the general median = ➔) and b) agreement between fac- ulties (100–75 - full/good agreement =; < 75–50 - major agreement =; < 50–25 - minor agreement =; < 25 - no agreement =). Authors’ contributions JG and MLK developed the concept and study design. MR and JG were concerned with literature review as well as data acquisition and control. Statistical analyses were conducted by JG and AY. JG, AY, MR and MLK discussed and interpreted the data. JG and MLK drafted the manuscript and critically revised it with AY and MR. The other collaborators of the MERlin Mapping Group (OF, AD, MG, SS, BS, KB, AG and SK) were integrated in data acquisition, consulted during the entire process (regarding actual procedures and data control) and involved in critical revision of the manuscript. All authors read and approved the final manuscript. 6. Sherbino J, Frank JR, Flynn L, Snell L. “Intrinsic roles” rather than “Armour”: renaming the “non-medical expert roles” of the CanMEDS framework to match their intent. Adv Health Sci Educ Theory Pract. 2011;16:695–7. https:// doi.org/10.1007/s10459-011-9318-z. 7. Whitehead CR, Austin Z, Hodges BD. Flower power: the armoured expert in the CanMEDS competency framework? Adv Health Sci Educ Theory Pract. 2011;16:681–94. https://doi.org/10.1007/s10459-011-9277-4. p g 8. CanMEDS: http://www.royalcollege.ca/rcsite/canmeds/canmeds-framework-e. Accessed: 30 Sept. 2018. 8. CanMEDS: http://www.roy Accessed: 30 Sept. 2018. Abbreviations CBME: Competency-based medical education; CCMD: Competence Centre Medical Didactics (short form of: Competence Centre for University Teaching in Medicine, Baden-Wuerttemberg); NKLM: National Competency-based Learning Objectives for Undergraduate Medical Education; PME: Postgraduate medical education; UME: Undergraduate medical education CBME: Competency-based medical education; CCMD: Competence Centre Medical Didactics (short form of: Competence Centre for University Teaching in Medicine, Baden-Wuerttemberg); NKLM: National Competency-based Learning Objectives for Undergraduate Medical Education; Limitations Some restrictions of our approach have to be consid- ered. Over−/underestimations of competency repre- sentation cannot be excluded despite the instruction of mappers and data quality control. Mapping data may be positively or negatively biased by certain fac- tors: e.g. knowledge, framework terminology, percep- tion of intrinsic roles, CBME acceptance, institutional culture and enthusiasm for teaching. Therefore, map- ping data cannot be taken as hard and accurate values for curriculum depiction. It is rather an actual snap- shot and crosscut of curricula in the continuously changing field of UME, which must be regularly up- dated to show its merit. The teachers view is an im- portant but single-sided perspective on the explicit curriculum (taught curriculum), even though results were controlled in probability checks by Dean’s offices and senior teachers. However, the students view, ad- dressed in another project, is relevant for a multi- perspective curriculum evaluation (learned curricu- lum). Regarding the graphical representation of data, the sample size is considered to be rather small for Page 11 of 12 Page 11 of 12 Page 11 of 12 (2020) 20:35 Griewatz et al. BMC Medical Education Funding Th A systematic review of medical leadership in hospital settings. PLoS One. 2017;12(9):e0184522. https://doi.org/10.1371/journal. pone.0184522. Funding Th Ottawa: The Royal College of Physicians and Surgeons of Canada; 2015. 19. Nationaler Kompetenzbasierter Lernzielkatalog Medizin [National Competency-based Learning Objective Catalogue] (NKLM). 2015. Berlin: MFT Medizinischer Fakultaetentag der Bundesrepublik Deutschland. [accessed 30 Sept. 2018]. http://www.nklm.de. 20. Griewatz J, Lammerding-Koeppel M. Intrinsic roles in the crosshair – strategic analysis of multi-site role implementation with an adapted matrix map approach. BMC Med Educ. 2019;19:237. https://doi.org/10. 1186/s12909-019-1628-5. 21. Lammerding-Koeppel M, Fritze O, Giesler M, Narciss E, Steffens S, Wosnik A, Griewatz J. Benchmarking for research-related competencies – a curricular mapping approach at medical faculties in Germany. Med Teach. 2018;40(2): 164–73. https://doi.org/10.1080/0142159X.2017.1395403. 22. Fritze O, Lammerding-Koeppel M, Boeker M, Narciss E, Wosnik A, Zipfel S, Griewatz J. Boosting competence-orientation in undergraduate medical education - a web-based tool linking curricular mapping and visual analytics. Med Teach. 2019;41(4):422–32. https://doi.org/10.1080/ 0142159X.2018.1487047. 23. Rogers EM. Diffusion of innovations. 4th ed. New York: Free Press; 1995. 24. Biggs J. Enhancing teaching through constructive alignment. High Educ. 1996;32:347–64. https://doi.org/10.1007/BF00138871. 25. Gruppen L, Frank JR, Lockyer J, Ross S, Bould MD, Harris P, et al. Toward a research agenda for competency-based medical education. Med Teach. 2017;39(6):623–30. https://doi.org/10.1080/0142159X.2017.1315065. 26. Fürstenberg S, Schick K, Deppermann J, Prediger S, Berberat PO, Kadmon M, Harendza S. Competencies for first year residents - physicians' views from medical schools with different undergraduate curricula. BMC Med Educ. 2017;17(1):154. https://doi.org/10.1186/s12909-017-0998-9. 27. Holmboe ES, Sherbino J, Long DM, Swing SR, Frank JR. The role of assessment in competency-based medical education. Med Teach. 2010; 32(8):676–82. https://doi.org/10.3109/0142159X.2010.500704. 27. Holmboe ES, Sherbino J, Long DM, Swing SR, Frank JR. The role of assessment in competency-based medical education. Med Teach. 2010; 32(8):676–82. https://doi.org/10.3109/0142159X.2010.500704. 28. Lockyer J, Carraccio C, Chan MK, Hart D, Smee S, Touchie C, Holmboe ES. Frank JR; ICBME Collaborators. Core principles of assessment in competency-based medical education. Med Teach. 2017;39(6):609–16. https://doi.org/10.1080/0142159X.2017.1315082. 28. Lockyer J, Carraccio C, Chan MK, Hart D, Smee S, Touchie C, Holmboe ES. Frank JR; ICBME Collaborators. Core principles of assessment in competency-based medical education. Med Teach. 2017;39(6):609–16. https://doi.org/10.1080/0142159X.2017.1315082. 29. Gruppen LD, Ten Cate O, Lingard LA, Teunissen PW, Kogan JR. Enhanced requirements for assessment in a competency-based, time-variable medical education system. Acad Med. 2018:17–21. https://doi.org/10.1097/ACM. 0000000000002066. 30. Harris P, Bhanji F, Topps M, Ross S, Lieberman S, Frank JR, et al. Evolving concepts of assessment in a competency-based world. Med Teach. 2017; 39(6):603–8. https://doi.org/10.1080/0142159X.2017.1315071. 31. Berghout MA, Fabbricotti IN, Buljac-Samardzlic M, Hilders CGJM. Medical leaders or masters? Funding Th 9. Griewatz J, Wiechers S, Ben-Karacobanim H, Lammerding-Koeppel M. Medical teachers’ perception of professional roles in the framework of the German National Competence-Based Learning Objectives for undergraduate medical education (NKLM) – a multicenter study. Med Teach. 2016;38:1157– 65. https://doi.org/10.3109/0142159X.2016.1170777. The joint MERlin-Project (Phase I: 2012–2016, Phase II: 2017–2020) is funded by the Federal Ministry of Education and Research of Germany (BMBF; reference numbers: 01PL12011A, 01PL17011A). Since 2011, the Federal Ministry has been supporting the improvement of study conditions and teaching quality at German universities via the Quality Pact for Teaching (QPL) program. This program serves public educational goals and not economic interests. The joint project of the Medical Faculties of Baden- Wuerttemberg includes Freiburg, Heidelberg, Mannheim, Tuebingen, Ulm. It is focused on medical education research within a cooperative, state-wide teaching network regarding competency-based education and assessment. The Tuebingen mapping project is one of the subprojects in which external institutions may participate at their own charge. 10. Renting N, Raat JAN, Dornan T, Wenger-Trayner E, van der Wal MA, Borleffs JCC, et al. Integrated and implicit: how residents learn CanMEDS roles by participating in practice. Med Educ. 2017;51:942–52. https://doi.org/10.1111/medu.13335. 11. Nousiainen MT, Caverzagie KJ, Ferguson PC. Frank JR; ICBME collaborators. Implementing competency-based medical education: what changes in curricular structure and processes are needed? Med Teach. 2017;39(6):594–8. https://doi.org/10.1080/0142159X.2017.1315077. Page 12 of 12 Griewatz et al. BMC Medical Education (2020) 20:35 Griewatz et al. BMC Medical Education (2020) 20:35 12. Berger E, Chan MK, Kuper A, Albert M, Jenkins D, Harrison M, Harris I. The CanMEDS role of collaborator: how is it taught and assessed according to faculty and residents? Paediatr Child Health. 2012;17(10):557–60. y 13. Thistlethwaite J. Interprofessional education: 50 years and counting. Med Educ. 2016;50(11):1082–6. https://doi.org/10.1111/medu.12959. Thistlethwaite J. Interprofessional education: 50 years and counting. M Educ. 2016;50(11):1082–6. https://doi.org/10.1111/medu.12959 14. Luft LAM. The essential role of physician as advocate: how and why we pass it on. CMEJ. 2017;8(3):e109–16. 15. Hubinette M, Dobson S, Voyer S, Regehr G. 'We' not 'I': health advocacy is a team sport. Med Educ. 2014 Sep;48(9):895–901. 16. Hubinette MM, Regehr G, Cristancho S. Lessons from rocket science: reframing the concept of the physician health advocate. Acad Med. 2016;91(10):1344–7. 17. Sonsale A, Bharamgoudar R. Equipping future doctors: incorporating management and leadership into medical curriculums in the United Kingdom. Perspect Med Educ. 2017;6:71–5. https://doi.org/10.1007/ s40037-017-0327-3. 18. Dath D, Chan M-K, Abbott C. CanMEDS 2015: from manager to leader. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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PENGARUH PENGETAHUAN MASYARAKAT TENTANG AKAD MUDHARABAH TERHADAP MINAT MENABUNG DI BANK SYARIAH
Jurnal Al-Fatih Global Mulia
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77 Jurnal Al-Fatih Global Mulia Volume 4 Issue 2 (2022), Pages 77-88 Jurnal Al-Fatih Global Mulia pISSN 2580-8036, eISSN 2746-7058 https://jurnalglobalmulia.or.id/index.php/alfatih PENGARUH PENGETAHUAN MASYARAKAT TENTANG AKAD MUDHARABAH TERHADAP MINAT MENABUNG DI BANK SYARIAH Volume 4 Issue 2 (2022), Pages 77-88 Jurnal Al-Fatih Global Mulia pISSN 2580-8036, eISSN 2746-7058 https://jurnalglobalmulia.or.id/index.php/alfatih Volume 4 Issue 2 (2022), Pages 77-88 Jurnal Al-Fatih Global Mulia pISSN 2580-8036, eISSN 2746-7058 https://jurnalglobalmulia.or.id/index.php/alfatih Riska Restapia1, Nurajizah2 Sekolah Tinggi Ilmu Ekonomi Dan Bisnis Islam (STEBI) Global Mulia Cikarang riskarestapia@gmail.com1, nurajizah@globalmulia.ac.id2 Abstract Abstrak Pengetahuan masyarakat tentang perbankan syariah masih sedikit dibandingkan dengan perbankan konvensional. Hal ini tidak sebanding dengan masyarakat Indonesia yang sebagian besar memeluk agama Islam. Dengan kondisi tersebut, perbankan syariah harus lebih diminati oleh masyarakat Indonesia, khususnya masyarakat muslim. Dan masih banyak masyarakat yang belum memahami perbankan syariah sehingga belum banyak masyarakat yang menggunakan layanan perbankan syariah. Tujuan penelitian ini adalah untuk mengetahui pengaruh pengetahuan masyarakat tentang akad Mudharabah terhadap minat menabung di bank syariah pada masyarakat desa Malangnengah. Penelitian ini menggunakan pendekatan kuantitatif dengan metode pengumpulan data atau angket, wawancara dan dokumentasi. Populasi dalam penelitian ini adalah masyarakat Desa Malangnengah dan sampel sebagai responden penelitian akan diambil dengan menggunakan teknik Purposive Sampling dengan rumus slovin yaitu sebanyak 94 orang. Data yang diperoleh dari penelitian ini dianalisis dengan menggunakan program IBM SPSS 25. Dalam penelitian ini penulis menggunakan teori pengetahuan menurut Gazalba (1992) pengetahuan adalah apa yang diketahui atau pekerjaan mengetahui. Pekerjaan mengetahui ini adalah hasil dari mengetahui, menyadari, menyadari, memahami, dan menjadi pandai. Pengetahuan adalah semua yang dimiliki atau isi pikiran. Teori mudharabah menurut Fatwa Dewan Syari'ah Nasional NO: 07/DSN-MUI/IV/2000 tentang Pembiayaan Mudharabah (Qiradh) menyatakan bahwa Mudharabah adalah akad kerjasama usaha antara dua pihak dimana pihak pertama (shaibul mal, LKS ) menyediakan seluruh modal, sedangkan pihak kedua (mudharib, nasabah) bertindak sebagai pengelola, dan keuntungan usaha dibagi di antara mereka sesuai dengan kesepakatan yang dituangkan dalam akad. Teori minat Menurut Muhibbin Syah (2001) minat adalah kecenderungan dan kegairahan yang tinggi atau keinginan yang besar terhadap sesuatu. Hasil penelitian ini menunjukkan bahwa variabel pengetahuan masyarakat tentang akad mudharabah (X) berpengaruh terhadap minat menabung di bank syariah pada masyarakat Desa Malangnengah (Y), hal ini terlihat dari hasil uji t terhadap variabel pengetahuan masyarakat tentang akad mudharabah diketahui nilai t_hitung sebesar 7,744 > dari nilai t_tabel sebesar 1,661. Kata kunci: Pengetahuan, Akad Mudharabah, Minat Menabung Abstract Public knowledge about Islamic banking is still small compared to conventional banking. This is not comparable to the people of Indonesia who mostly embrace Islam. Under these conditions, Islamic banking should be more attractive to the Indonesian people, especially the Muslim community. And there are still many people who do not understand Islamic banking, so not many people use Islamic banking services. The purpose of this study was to determine the effect of community knowledge about the Mudharabah contract on the interest in saving at Islamic banks in the Malangnengah village community. This study uses a quantitative approach to data collection methods or questionnaires, interviews and documentation. The population in this study is the community of Malangnengah Village and the sample as research respondents will be taken using the Purposive Sampling technique with the slovin formula, namely 94 people. The data obtained from this study were analyzed using the IBM SPSS 25 program. In this study the authors used the theory of knowledge according to Gazalba (1992) knowledge is what is known or the work of knowing. This knowing work is the result of knowing, realizing, realizing, understanding, and being clever. Knowledge is all owned or the contents of the mind. Mudharabah theory according to Fatwa of the National Syari'ah Council NO: 07/DSN- MUI/IV/2000 concerning Mudharabah Financing (Qiradh) states that Mudharabah is a business cooperation contract between two parties in which the first party (shaibul mal, LKS) provides all the capital , while the second party (mudharib, customer) acts as manager, and business profits are shared between them according to the agreement set forth in the contract. Interest theory According to Muhibbin Syah (2001) interest is a tendency and high excitement or a great desire for something. The results of this study indicate that the community's knowledge variable about mudharabah contracts (X) influences the interest in saving in Islamic banks in the Malangnengah Village community (Y), this can be seen from the results of the t test on the variable community knowledge about mudharabah contracts that it is known that the t_value is 7.744 > from the t_table value of 1.661. Keywords: Knowledge Mudharabah Contract Interest in Saving f f Keywords: Knowledge, Mudharabah Contract, Interest in Saving. 77 Jurnal Al-Fatih Global Mulia Pengaruh Pengetahuan Masyarakat... 78 Jurnal Al-Fatih Global Mulia PENDAHULUAN Keberadaan bank syariah di Indonesia semakin kokoh dengan Undang-Undang Nomor 10 Tahun 1998 tentang Perubahan Atas Undang-Undang Nomor 7 Tahun 1992 tentang Perbankan. Undang-Undang ini cakupanya lebih luas, bahwa bank syariah tidak semata-mata adalah bank yang melakukan kegiatan usahanya berdasarkan bagi hasil melainkan bank yang memberikan produk-produknya berdasarkan prinsip syariah. Menurut Sudarsono (2008) “Bank Syariah adalah lembaga keuangan negara yang memberikan kredit dan jasa-jasa lainnya di dalam lalu lintas pembayaran dan juga peredaran uang yang beroperasi dengan menggunakan prinsip-prinsip syariah atau islam (Indi, 2019) Fungsi bank syariah secara garis besar tidak berbeda dengan bank konvensional, yakni sebagai lembaga internediasi (Intermediary Institution) yang mengerahkan dana dari masyarakat dan menyalurkan kembali dana-dana tersebut kepada masyarakat yang membutuhkannya dalam bentuk fasilitas pembiayaan. Perbedaannya, bila bank konvensional mendasarkan keuntungannya dari pengambilan bunga, maka bank syariah dari apa yang disebut imbalan, baik berupa jasa (fee-base income) maupun mark-up atau profit margin, serta bagi hasil (loss and profit sharing) (Sari, Fadilla, & Aravik, 2021). Mudharabah saat ini merupakan wahana utama bagi lembaga keuangan syari’ah untuk memobilisasi dana masyarakat dan untuk menyediakan berbagai fasilitas, seperti fasilitas pembiayaan bagi para pengusaha. Menurut Quardhawi (2000) Mudharabah dengan dasar profit and loss sharing principle merupakan salah satu alternatif yang tepat bagi lembaga keuangan Riska Restapia1, Nurajizah2 syari’ah yang menghindari sistem bunga (interest free) yang oleh sebagian ulama dianggap sama dengan riba yang diharamkan (Andiyansari C. N., 2020). Akad Mudharabah adalah akad kerjasama usaha antara 2 (dua) pihak, di mana pihak pertama bertindak sebagai pemilik dana (shaibul mal) yang menyediakan seluruh modal (100%), sedangkan pihak lainnya sebagai pengelola usaha atau mudharib. Secara teknis, mudharabah adalah kemitraan laba, dimana satu pihak (rabbul mal) menyediakan modal dan pihak yang lain (mudharib) menyediakan tenaga kerja. Beberapa ahli fiqih, seperti para ulama Hanafi dan Hanbali, menggunakan istilah mudharabah, sedangkan para ulama Maliki dan Syafi’i menggunakan istilah qiradh. Akad mudharabah pernah dilakukan oleh Nabi Muhammad SAW ketika bekerjasama dengan Siti Khadijah sebelum Muhammad SAW diangkat menjadi seorang Nabi dan Rasul. Kala itu Siti Khadijah berperan sebagai pemilik modal (shahibul maal) sedangkan Nabi Muhammad SAW berperan sebagai pelaksana usaha (mudharib). Disana Siti Khadijah mempercayakan barang dengannya untuk dijual Nabi Muhammad SAW. ke luar negeri (Sari, Fadilla, & Aravik, 2021). Akad Mudharabah adalah akad kerjasama usaha antara 2 (dua) pihak, di mana pihak pertama bertindak sebagai pemilik dana (shaibul mal) yang menyediakan seluruh modal (100%), sedangkan pihak lainnya sebagai pengelola usaha atau mudharib. Pengetahuan g Secara etimologi pengetahuan berasal dari kata dalam bahasa Inggris yaitu knowledge. Dalam Encyclopedia of Philosophy menurut (Edwards, 1972) dijelaskan bahwa “definisi pengetahuan adalah kepercayaan yang benar knowledge is justified true belief”. Sedangkan secara terminology definisi pengetahuan dikemukakan dalam beberapa definisi. Menurut (Gazalba, 1992) definisi pengetahuan adalah apa yang diketahui atau hasil pekerjaan tahu. Pekerjaan tahu tersebut adalah hasil dari kenal, sadar, insaf, mengerti, dan pandai. Pengetahuan itu adalah semua milik atau isi pikiran. Menurut Notoadmodjo pengetahuan merupakan hasil dari tahu dan ini terjadi setelah orang melakukan pengindraan terhadap suatu objek tertentu. Pengindraan terjadi melalui pancaindra manusia, yakni indra penglihatan, pendengaran, penciuman, rasa, dan raba dengan sendiri. Sebagian besar pengetahuan manusia diperoleh melalui mata dan telinga (Wawan & Dewi , 2010). Dalam kamus filsafat dijelaskan bahwa pengetahuan (knowledge) adalah proses kehidupan yang diketahui manusia secara langsung dari kesadarannya sendiri. Dalam peristiwa ini yang mengetahui (subjek) memiliki yang diketahui (objek) di dalam dirinya sendiri sedemikian aktif sehingga yang mengetahui itu menyusun yang mengetahui itu menyusun yang diketahui pada dirinya sendiri dalam kesatuan aktif (Bagus, 1996). Dari berbagai definisi pengetahuan yang telah dikemukakan oleh beberapa ahli dapat disimpulkan bahwa pengetahuan adalah proses dari beberapa usaha manusia untuk tahu, sehingga dengan pengetahuan manusia mempunyai kepercayaan untuk dapat memberi putusan yang benar dan pasti. PENDAHULUAN Secara teknis, mudharabah adalah kemitraan laba, dimana satu pihak (rabbul mal) menyediakan modal dan pihak yang lain (mudharib) menyediakan tenaga kerja. Beberapa ahli fiqih, seperti para ulama Hanafi dan Hanbali, menggunakan istilah mudharabah, sedangkan para ulama Maliki dan Syafi’i menggunakan istilah qiradh. Akad mudharabah pernah dilakukan oleh Nabi Muhammad SAW ketika bekerjasama dengan Siti Khadijah sebelum Muhammad SAW diangkat menjadi seorang Nabi dan Rasul. Kala itu Siti Khadijah berperan sebagai pemilik modal (shahibul maal) sedangkan Nabi Muhammad SAW berperan sebagai pelaksana usaha (mudharib). Disana Siti Khadijah mempercayakan barang dengannya untuk dijual Nabi Muhammad SAW. ke luar negeri (Sari, Fadilla, & Aravik, 2021). Prinsip mudharabah adalah bagian dari produk perbankan syariah yang unik, karena memiliki perbedaan filosofis antara sistem perbankan konvensional dengan perbankan syariah yang menganut prinsip bagi keuntungan atau kerugian. Prinsip bagi hasil dikenal sebagai profit and loss sharing, dimana ketika mudharib mendapatkan hasil dari pengembangan modal usaha dari shaibul mal maka keuntungan yang didapat dibagi sesuai dengan perjanjian. Begitu pula dengan kerugian, maka antara mudharib dengan shaibul mal sama-sama menanggung. Konsep inilah yang diusung oleh syariah bawasannya skim mudharabah ini menerapkan sistem kerjasama berbasis keadilan (Andiyansari C. N., 2020). Minat merupakan salah satu faktor yang cukup penting dalam mempengaruhi preferensi masyarakat/nasabah dalam menabung. Pengetahuan adalah yang mendasar dalam mengembangkan produk- produk perbankan syariah di kalangan masyarakat. Pengetahuan sendiri informasi yang bisa diperoleh melalui berbagai macam media, contohnya iklan pada majalah, televisi, koran, radio, bahkan bisa juga diperoleh dari pengalaman seseorang. Malang Nengah adalah salah satu desa dengan jumlah penduduk 5.284 jiwa dengan 1.620 kepala keluarga. Desa ini terletak di Kecamtan Sukatani, Kabupaten Purwakarta, Jawa Barat, Indonesia. Kenapa penulis memilih penelitian ini, karena masyarakat desa Malang Nengah sendiri tidak banyak menggunakan jasa perbankan syariah baik dalam bentuk transaksi penyimpanan dana maupun transaksi pendanaan. 79 Jurnal Al-Fatih Global Mulia Pengaruh Pengetahuan Masyarakat... 80 Jurnal Al-Fatih Global Mulia Jenis Pengetahuan Ada beberapa jenis pengetahuan menurut (Salam, 1997) yang mengemukakan bahwa pengetahuan yang dimiliki oleh manusia ada empat, yaitu: Pengetahuan Biasa (Common Sense),. ada pengetahuan ini diartikan bahwa seseorang memiliki sesuatu dimana seorang tersebut dapat menerimanya secara baik. Dengan common sense, semua orang sampai pada keyakinan secara umum tentang sesuatu, dimana meraka akan berpendapat sama semuanya dari pengalaman sehari-hari yang diperolehnya. Contohnya air dapat dipakai untuk menyiram bunga, makanan dapat memuaskan rasa lapar. Pengetahuan Ilmu (Science), Ilmu pada prinsipnya merupakan usaha untuk mengorganisasikan dan mensistematisasikan common sense, suatu pengetahuan yang berasal dari pengalaman dan pengamatan dalam kehidupan sehari-hari. Namun, dilanjutkan dengan suatu pemikiran secara cermat dan teliti dengan menggunakan berbagai metode, diperoleh melalui observasi, eksperimen, dan klasifikasi. Pengetahuan Filsafat Pengetahuan yang diperoleh dari pemikiran yang bersifat kontemplatif dan spekulatif. Pengetahua Filsafat lebih menekan pada universalitas dan kajian kedalam tentang sesuatu. Misalnya, kalau ilmu hanya pada satu bidang pengetahuan yang sempit dan rigid, filsafat membahas hal yang lebih luas dan mendalam. Filsafat biasanya memberikan yang reflektif dan kritis, sehingga ilmu yang tadinya kaku dan cenderung tertutup menjadi longgar sekali. Pengetahuan Agama. Pengetahuan yang hanya diperoleh dari Tuhan lewat para utusan- Nya yang bersifat mutlak dan wajib diyakini oleh para pemeluk agama. Pengetahuan ini mengandung beberapa hal pokok, yaitu ajaran tentang cara berhubungan dengan Tuhan atau hubungan vertikal dan cara berhubungan dengan sesama manusia atau hubungan horizontal. Sumber Pengetahuan g Pengetahuan yang telah diperoleh dengan menggunakan berbagai alat yang merupakan sumber pengetahuan tersebut. Ada beberapa pendapat tentang sumber pengetahuan antara lain: 80 Jurnal Al-Fatih Global Mulia Riska Restapia1, Nurajizah2 Empirisme. Berdasarkan aliran ini manusia memperoleh pengetahuan melalui pengalamannya. John Locke (1632-1704), Bapak empiris Britania mengemukakan teori tabula rasa (sejenis buku catatan kosong). Maksudnya adalah bahwa manusia itu pada mulanya kosong dari pengetahuan, lantas pengalamannya mengisi jiwa yang kosong itu, lantas ia memiliki pengetahuan. Mula-mula tangkapan indera yang masuk itu sederhana, lama-kelamaan menjadi kompleks, lalu tersusunlah pengetahuan yang berarti. Jadi, bagaimanapun kompleks pengetahuan manusia, selalu dapat dicari ujungnya pada pengalaman indera. Sesuatu yang tidak dapat diamati oleh indera bukanlah pengetahuan yang benar. Jadi pengalaman indera itulah sumber pengetahuan yang benar. Namun aliran ini memiliki banyak kelemahan misalnya seperti indera terbatas, indera menipu, objek yang menipu, dan berasal dari indera dan objek sekaligus. Rasionalisme. Dalam aliran ini menyatakan bahwa akal adalah dasar kepastian pengetahuan. Pengetahuan yang benar dapat di ukur dengan akal. Manusia mampu memperoleh pengetahuan melalui kegiatan menangkap objek. Jenis Pengetahuan Konsep tersebut mempunyai wujud dalam alam nyata dan bersifat universal, dengan prinsip ini yang dimaksud adalah abstraksi dari benda-benda konkret. Para penganut rasionalsme yakin bahwa kebenaran dan kesesatan terletak dalam ide dan bukunya di dalam diri barang sesuatu. Jika kebenaran mengandung makna dan mempunyai ide yang sesuai atau dengan menunjuk pada kenyataan,kebenaran hanya dapat ada di dalam pikiran kita dan hanya dapat diperoleh dari akal budi saja (Kattsoff, 1996). y p p j Intuisi. Menurut Henry Bergason intuisi adalah hasil dari evolusi pemahaman yang tertinggi. Kemampuan ini mirip dengan insting, tetapi berbeda dengan kesadaran dan kebebasannya. Pengembangan kemampuan ini atau intuisi memerlukan suatu usaha. Intuisi mengatasi sifat lahiriah pengetahuan simbolis, yang pada dasarnya bersifat analisis, menyeluruh, mutlak, dan tanpa dibantu oleh penggambaran secara simbolis. Oleh sebab itu, intuisi adalah sarana untuk mengetahui secara langsung dan seketika. Analitis atau pengetahuan yang diperoleh lewat pelukisan tidak menggantikan hasil pengenalan intuisi. Karena intuisi bersifat personal dan tidak bisa diramalkan, Wahyu. Wahyu adalah pengetahuan yang disampaikan oleh Allah SWT kepada manusia lewat perantara para nabi. Para nabi memperoleh pengetahuan dari Tuhan tanpa upya, Tuhan mensucikan jiwa para nabi dan diterangkan-Nya jiwanya untuk memperoleh kebenaran dengan jalan wahyu. Berdasarkan teori ini kebenaran pengetahuan berasal dari Tuhan. Kepercayaan inilah yang membuat titik tolak dalam agama dan lewat pengkajian selanjutnya yang dapat meningkatkan atau menurunkan kepercayaan itu. Sedangkan ilmu pengetahuan berbanding terbalik, yaitu dimulai mengkaji dengan riset, pengalaman, dan percobaan untuk sampai pada kebenaran yang faktual (afidburhanuddin, 2014). Rukun Dan Syarat Mudharabah Rukun mudharabah menurut jumhur ulama adalah: Pihak-pihak yang melakukan akad, shahib al-mal dan mudharib. Ma’qud modal (ra’s al-mal), usaha (al-‘amal/al-a’mal), dan keuntungan (al-ribh). Pernyataan mudharabah/shigat akad, yaitu pernyataan yang berupa ijab/penawaran dan qabul/penerimaan (al-Zuhaili, al-Fiqh al-Islami wa Adillatuh, 1997). Syarat-syarat mengenai akad mudharabah, antara lain: Pihak-pihak yang melakukan akad mudharabah dipersyaratkan memiliki kemampuan: cakap hukum untuk memberikan kuasa (bagi shahib almal) dan menerima perwakilan/kuasa karena dalam akad mudharabah terkandung akad wakalah/kuasa, yaitu mudharib melakukan usaha (bisnis) atas dasar kuasa dari shahib al-mal, mudharabah boleh dilakukan antara muslim dan non-muslim (dzimmi dan musta’min) di negara muslim (al-Zuhaili, al-Fiqh al-Islami wa Adillatuh, 1997). Dalam bisnis, syarat yang utama bagi mudharib (pelaku usaha) harus memiliki kemampuan, keahlian, dan/atau keterampilan usaha sehingga mampu mengembangkan modal usaha. Ra’s al-mal (modal usaha) dalam akad mudharabah harus memenuhi syarat-syarat berikut: Modal harus berupa alat tukar (nuqud/uang), bukan berupa barang. Modal harus dapat diketahui dan terukur. Modal harus tunai (bukan dalam bentuk piutang). Modal harus dapat diserahkan dari shahib al-mal kepada mudharib (al-Zuhaili, al-Fiqh al-Islami wa Adillatuh, 1997). ) Qiyas Mudharabah dapat diqiyaskan sebagai bentuk interaksi makhluk sosial. Sebagai makhluk sosial, kebutuhan akan kerjasama antara satu pihak dengan pihak lain guna meningkatkan taraf perekonomian dan kebutuhan hidup, atau keperluan-keperluan lain, tidak bisa diabaikan 82 Jurnal Al-Fatih Global Mulia Akad Mudharabah Secara kata bahasa, Mudharabah diambil dari kalimat dharaba fil ardh, artinya melakukan perjalanan dalam rangka berdagang (Rozalinda, 2016) Dalam Fatwa Dewan Syari’ah Nasional NO:07/DSN-MUI/IV/2000 tentang Pembiayaan Mudharabah (Qiradh) menyatakan bahwa Mudharabah yaitu akad kerjasama suatu usaha antara dua pihak di mana pihak pertama (shaibul mal, LKS) menyediakan seluruh modal, sedang pihak kedua (mudharib, nasabah) bertindak selaku pengelola, dan keuntungan usaha dibagi di antara mereka sesuai kesepakatan yang dituangkan dalam kontrak. Sedangkan definisi menurut Wahbah Az-Zuhaili mudharabah adalah “akad didalamnya pemilik modal memberikan modal (harta) pada ‘amil (pengelola) untuk mengelolanya, dan keuntungan menjadi milik bersama sesuai dengan apa yang mereka sepakati. Sedangkan kerugiannya hanya menjadi tanggungan pemilik modal saja, ‘amil tidak menanggung kerugian apapun kecuali usaha dan kerjanya saja (Az-Zuhaili, 2011). 81 Jurnal Al-Fatih Global Mulia Pengaruh Pengetahuan Masyarakat... Dari beberapa definisi diatas dapat disimpulkan bahwa akad mudharabah adalah kerjasama antara dua pihak yaitu pemilik modal dan pengelola usaha dengan keuntungan yang telah disepakati sebelumnya. Kerugian hanya menjadi tanggungan pemilik modal, kecuali pengelola melakukan kelalaian dalam tugasnya, maka pengelola ikut bertanggung jawab atas kerugian tersebut. Dasar Hukum Mudharabah Al-Qur’an Ada beberapa ayat Al-Qur’an yang terkait menjadi landasan hukum mudharabah salah satunya firman Allah QS. Al-Jumu’ah, (62):10)“...Apabila salat telah dilaksanakan, maka bertebaranlah kamu di bumi; carilah karunia Allah” Diriwayatkan oleh Ibn Majah bahwa Nabi Saw. Bersabda, “Terdapat berkat pada tiga transaksi: penjualan kredit, mudharabah dan pencampuran gandum dengan jelsi untuk konsumsi rumah tangga, bukan untuk perdagangan”. Bahkan keabsahan mudharabah adalah perbuatan Nabi Saw sendiri yang tadinya bekerja sebagai mudharib bagi Khadijah. Jadi praktek kerjasama mudharabah di perbolehkan dalam Islam dan terkandung keberkahan atau kemanfaatan di dalamnya. Imam Zailai dalam Syafii menyebutkan “Diriwayatkan oleh sejumlah sahabat menyerahkan (kepada orang, mudharib) harta anak yatim sebagai mudharabah dan tidak seorangpun mengingkari mereka. Karenannya, hal itu dipandang sebagai ijma” (Antonio M. S., 2017). Minat Menabung Minat muncul apabila individu tertarik kepada sesuatu karena sesuai dengan kebutuhannya atau merasakan bahwa sesuatu yang akan dipelajari dirasakan berarti bagi dirinya. Kebutuhan disini yaitu seperti kebutuhan akan aktualisasi diri, kebutuhan estetis, kebutuhan kognitif, kebutuhan akan penghargaan, kebutuhan cinta dan rasa memiliki, kebutuhan akan keamanan dan kebutuhan fisiologi (Nigel & Grove, 2000). Minat adalah "Perhatian yang mengandung unsur-unsur perasaan, minat adalah suatu sikap yang menyebabkan seseorang berbuat aktif dalam suatu pekerjaan. Dengan kata lain minat dapat menjadi sebab dari suatu kegiatan” (Shalahuddin, 1990) Unsur-unsur minat Sebagaimana yang dikemukakan oleh Abdurrahman Abror dalam bukunya Psikologi Pendidikan bahwa minat itu mengandung tiga unsur, yaitu :Unsur kognisi (mengenal) dalam pengertian bahwa minat itu didahului oleh pengetahuan dan informasi mengenai obyek yang dituju oleh minat tersebut. Unsur emosi (perasaan) karena dalam partisipasi atau pengalaman itu disertai dengan perasaan tertentu (biasanya perasaan senang), dan unsur konasi (kehendak) merupakan kelanjutan dari dua unsur diatasnyaitu diwujudkan dalam bentuk kemauan dan hasrat untuk melakukan suatunkegiatan (Abror, 1993). Pengakhiran Kontrak Mudharabah Akad mudharabah dinyatakan batal dalam hal-hal: Pertama, masing-masing pihak menyatakan akad batal, pekerja dilarang untuk bertindak hukum terhadap modal yang diberikan, atau pemilik modal menarik modalnya. Kedua, salah seorang yang berakad meninggal dunia. Jika pemilik modal yang wafat, menurut jumhur ulama, akad tersebut batal, karena akad mudharabah sama dengan akad wakalah (perwakilan yang gugur disebabkan wafatnya orang yang mewakilkan. Disamping itu, jumhur ulama berpendapat bahwa akad mudharabah tidak bisa diwariskan. Akan tetapi ulama madzhab maliki berpendapat bahwa jika salah seorang yang berakad itu meninggal dunia, akadnya tidak batal, tetapi tidak dilanjutkan oleh ahli warisnya karena, menurut mereka akad mudhharabah bisa diwariskan. Ketiga, salah seorang yang berakad gila, karena orang yang gila tidak cakap lagi bertindak hukum. Keempat pemilik modal murtad (keluar dari agama Islam), menurut Imam Abu Hanifah, akad mudharabah batal. Kelima, modal habis ditangan pemilik modal sebelum dikelola oleh pekerja. Demikian juga halnya, mudharabah batal apabila modal tersebut dibelanjakan oleh pemilik modal sehingga tidak ada lagi yang bisa dikelola oleh pemilik modal sehingga tidak ada lagi yang bisa dikelola oleh pekerja (Arifin & Sa’diyah, 2013). Jenis Mudharabah Mudharabah mutlaqah (investasi tidak terikat) yaitu pihak pengusaha diberi kuasa penuh untuk menjalankan proyek tanpa larangan atau gangguan apapun urusan yang berkaitan dengan proyek itu dan tidak terkait dengan waktu, tempat, jenis, perusahaan dan pelanggan. Investasi tidak terkait ini pada bank syari’ah diaplikasikan pada produk tabungan dan deposito. Riska Restapia1, Nurajizah2 Dari penerapan mudharabah muthlaqah ini dikembangkan produk tabungan dan deposito, sehingga terdapat dua jenis produk penghimpunan dana, yaitu tabungan mudharabah dan deposito mudharabah. Mudharabah Muqayyadah off Balance Sheet, merupakan jenis mudharabah yang penyaluran dananya langsung kepada pelaksana usahanya, bank bertindak sebagai perantara (arranger) yang mempertemukan antara pemilik dana dengan pelaksana usaha. Pemilik dana dapat menetapkan syarat-syarat tertentu yang harus dipatuhi oleh bank dalam mencari kegiatan usaha yang akan dibiayai dan pelaksanaan usahanya. (Mauludia, 2021). Faktor-Faktor Yang Memengaruhi Minat Masyarakat Menabung Faktor-Faktor Yang Memengaruhi Minat Masyarakat Menabung Faktor-faktor yang memengaruhi kurangnya minat masyarakat menabung yaitu: Pengaruh pengetahuan terhadap minat menabung. Pengetahuan merupakan hasil dari tahu dan ini terjadi setelah orang melakukan pengindraan terhadap suatu objek tertentu. Pengindraan terjadi melalui pancaindra manusia, yakni indra penglihatan, pendengaran, penciuman, rasa, dan raba dengan sendiri. Sebagian besar pengetahuan manusia diperoleh melalui mata dan telinga (Wawan & Dewi , 2010).Pengaruh pelayanan tehadap minat menabung adalah setiap tindakan atau kegiatan yang dapat ditawarkan oleh suatu pihak kepada pihak lain, yang pada dasarnya tidak berwujud dan tidak mengakibatkan kepemilikan apapun (Kotler, 2002). Pengaruh Pelayanan tehadap minat menabung. Menurut Kotler pelayanan adalah setiap tindakan atau kegiatan yang dapat ditawarkan oleh suatu pihak kepada pihak lain, yang pada dasarnya tidak berwujud dan tidak mengakibatkan kepemilikan apapun (Kotler, 2002). Pengaruh lokasi terhadap minat menabung Lokasi adalah letak pada daerah yang strategis sehingga dapat memaksimumkan laba (Basu Swasta dan Irawan dalam Rusdiana, 2014:248). Masyarakat Menurut paul, pengertian masyarakat ialah sekumpulan manusia yang secara relatif mandiri, yang hidup bersama-sama yang cukup lama, yang menepati suatu wilayah tertentu, mempunyai kebudayaan yang sama serta melakukan sebagian besar kegiatan dalam kelompok itu. Ciri-Ciri Masyarakat Manusia yang hidup berkelompok, secara bersama-sama dan kemudian membentuk kelompok yang dibentuk tersebut kemudian menjadi masyarakat. Melahirkan kebudayaan, tidak ada suatu masyarakat yang tidak memiliki budaya. Masyarakat yang melahirkan kebudayaan serta budata kemudian diwariskan dalam generasi ke generasi dengan adanya proses-proses penyesuaian. Melahirkan kebudayaan. Mengalami perubahan, bisa terjadi sebab beberapa faktor yang berasal dari amsyarakat itu sendiri, seperti terdapat penemuan baru yang mungkin saja bisa menimbulkan perubahan kepada suatu masyarakat (Guru, 2021). B k S i h Bank Syariah Menurut Sudarsono “Bank Syariah adalah lembaga keuangan negara yang memberikan kredit dan jasa-jasa lainnya di dalam lalu lintas pembayaran dan juga peredaran uang yang beroperasi dengan menggunakan prinsip-prinsip syariah atau islam. Sedangkan menurut undang-undang Nomor 21 Tahun 2008 tentang Perbankan Syariah segala sesuatu yang menyangkut bank syariah dan unit usaha syariah, mencakup kelembagaan, mencakup kegiatan usaha, serta tata cara dan proses di dalam melaksanakan kegiatan usahanya. Menabung Menurut Kamus Besar Bahasa Indonesia (2005), menabung merupakan sebuah kata kerja yang memiliki arti yaitu menyimpan uang (di celengan, pos, bank, dan sebagainya). Secara luas menabung dapat diartikan sebagai suatu kegiatan menyisihkan sebagian pendapatannya untuk dikumpulkan sebagai cadangan pada masa yang akan datang. Terkait dengan anjuran untuk menabung, maka diperlukan suatu lembaga keuangan yang tidak hanya sebagai tempat menyimpan uang masyarakat tetapi juga sebagai tempat yang berfungsi untuk mencegah masyarakat khususnya masyarakat muslim agar tidak terjebak dengan kegiatan ekonomi yang bertentangan dengan syariat islam. Oleh karena itu salah satu solusi yang 83 Jurnal Al-Fatih Global Mulia Pengaruh Pengetahuan Masyarakat... ditawarkan sebagai tempat menabung yang sesuai dengan syariat islam adalah menabung di bank syariah (Indi, 2019). Prinsip-prinsip Dasar Perbankan Syariah Prinsip dasar dalam penjalankan perbankan syariah menurut Antonio, Syafi’i.M: Prinsip titipan atau simpanan (Al-wadi’ah), Al-wadi’ah dapat diartikan sebagai titipan murni dari suatu pihak ke pihak lain. Prinsip Bagi Hasil, Secara umum, prinsip bagi hasil salam perbankan syariah dapat dilakukan dalam empat akad utama, yaitu mudharabah, al-musyarakah al- muzara’ah dan al-musaqah. Akan tetapi prinsip yang paling banyak digunakan adalah al- musyarakah dan al-mudharabah. Sedangkan al-muzara’ah dan al-musaqah dipergunakan khusus pembiayaan pertanian oleh bank Islam. Prinsip Jual Beli, Ada tiga jenis jual beli yang telah dikembangkan sebagai dasar dalam pembiayaan kerja dan investasi dalam perbankan syaraih, yaitu: bai’al-murabahah, bai’as-salam, bai’al-istisna. Prinsip Sewa, Dalam perbankan syariah, prinsip sewa ada dua jenis, yaitu al-ijarah, al-muntahia bit-tamlik (Saputro & Dzulkirom, 2015). 84 Jurnal Al-Fatih Global Mulia Riska Restapia1, Nurajizah2 Keistimewaan Bank Syariah y Bank Syariah sebagai alternatif perekonomian Indonesia dan bagi bank-bank konvensional yang dianggap kurang berhasil di dalam mengemban misi utamanya memiliki keistemewaan-keistimewaan yang juga merupakan perbedaan jika dibandingkan dengan Bank Konvensional. Keistimewaan-keistimewaan Bank Syariah tersebut adalah: Adanya kesamaan ikatan emosional yang kuat antara pemegang saham, pengelola bank dan nasabahnya.Diterapkannya sistem bagi hasil sebagai pengganti bunga akan menimbulkan akibat-akibat yang positif. Di dalam Perbankan Syariah, tersedia fasilitas kredit kebaikan (al- Qardhul Hasan) yang diberikan secara cuma-Cuma Keistimewan yang paling menonjol dari Perbankan Syariah adalah yang melekat pada konsep (build in concept) dengan berorientasib pada kebersamaan (Basir, 2009). y y g Teknik Pengumpulan Data Agar memperoleh hasil yang valid dalam suatu penelitian, dibutuhkan data-data yang dapat dipertanggung jawabkan kebenarannya. Teknik pengumpulan data dalam penelitian ini menggunakan cara mengumpulkan data dan informasi yang ada dilapangan melalui kuesioner/angket, observasi dan dokumentasi. METODE PENELITIAN Metode penelitian pada dasarnya merupakan cara ilmiah untuk mendapatkan data dengan tujuan dan kegunaan tertentu. Berdasarkan hal tersebut terdapat empat kata kunci yang perlu diperhatikan yaitu, cara ilmiah, data, tujuan, dan kegunaan. Cara ilmiah kegiatan penelitian itu didasarkan pada ciri-ciri keilmuan, yaitu rasional, empiris dan sistematis. Rasional berarti kegiatan penelitian itu dilakukan dengan cara-cara yang masuk akal, sehingga terjangkau oleh penalaran manusia. Empiris berarti cara-cara yang dilakukan itu dapat diamati oleh indera manusia, sehingga orang lain dapat mengamati dan mengetahui cara-cara yang digunakan. Sistematis artinya, proses yng dilakukan dalam peneliti itu menggunakan langkah- langkah tertentu yang bersifat logis (Supardi, 2017). Pendekatan dan metode penelitian yang digunakan dalam penelitian ini adalah penelitian kuantitatif. Kuantitatif adalah penelitian yang menggunakan format matematika dan statistik. Menurut (Bahruddin & Hamdi, 2014) penelitian kuantitatif menekankan pada fenomena-fenomena objektif dan dikaji secara kuantitatif. Jenis dan Sumber Data Sumber data adalah salah satu yang paling vital dalam penelitian. Adaun jenis sumber data yang penulis gunakan dalam penelitian ini adalah data primer dan data sekunder. Data primer yaitu data informasi yang diperoleh secara langsung oleh peneliti dari narasumber atau responden yang ada dilapangan (Bawono, 2006). Data primer secara khusus dikumpulkan oleh peneliti dari responden yang menjawab pertanyaan. Data Sekunder, merupakan data yang didapat secara tidak langsung yang diperoleh dari instansi-instansi yang mendokumentasikan dan mempublikasikan data tersebut seperti artikel, jurnal, skripsi ataupun lainnya yang masih berhubungan dengan materi penelitian (Supardi, 2017). Peneliti juga menggali informasi dari beberapa sumber buku, jurnal, skripsi dan dokumentasi laiinya yang berkaitan dengan materi penelitian. HASIL DAN PEMBAHASAN HASIL DAN PEMBAHASAN Pengaruh Pengetahuan Masyarakat Tentang Akad Mudharabah Terhadap Minat Menabung Di Bank Syariah Pengaruh Pengetahuan Masyarakat Tentang Akad Mudharabah Terhadap Minat Menabung Di Bank Syariah Salah satu faktor yang mendasar dalam mengembangkan produk- produk perbankan syariah di kalangan masyarakat adalah pengetahuan. Begitupun untuk pengetahuan masyarakat tentang akad mudharabah. Pengetahuan sendiri informasi yang bisa diperoleh melalui berbagai macam media, contohnya iklan pada majalah, televisi, koran, radio, bahkan bisa juga diperoleh dari pengalaman seseorang. Menurut (Gazalba, 1992) definisi pengetahuan adalah apa yang diketahui atau hasil pekerjaan tahu. Pekerjaan tahu tersebut adalah hasil dari kenal, sadar, insaf, mengerti, dan pandai. Pengetahuan itu adalah semua milik atau isi pikiran. Berdasarkan penelitian diatas dapat disimpulkan bahwa variabel (X) pengetahuan masyarakat tentang akad mudharabah berpengaruh positif terhadap variabel (Y) minat menabung di Bank Syariah. Hal ini mengindikasikan bahwa semakin bertambahnya pengetahuan masyarakat tentang akad mudharabah maka semakin tinggi minat menabung masyarakat di Bank Syariah. Hal ini dapat dibuktikan dengan hasil analisis data yang di uji menggunakan uji t yang menunjukkan nilai variabel pengetahuan tentang akad mudharabah (X) pada output sebesar 7,744 > 1,661. Penelitian ini juga memiliki data yang signifikan valid dan reliabel. Penelitian ini juga memilik uji normalitas signifikan, data ini juga terbebas dari gejala heterokedastisitas dan multikolinearitas. Dan diperkuat oleh penelitian terdahulu oleh Faisal Umardani Hasibuan, Rahma Wahyuni (2020) dengan judul yang sama tetapi variabel yang berbeda menunjukan bahwa, Variabel pengetahuan masyarakat berpengaruh signifikan secara parsial terhadap keputusan masyarakat menggunakan produk tabungan Perbankan Syariah. Dan variabel minat penerapan nilai-nilai Islam berpengaruh signifikan secara parsial terhadap keputusan masyarakat menggunakan produk Tabungan perbankan Syariah. Jadi dapat disimpulkan bahwa variabel pengetahuan tentang akad mudharabah berpengaruh positif terhadap variabel minat. Tabel 4. 11 Hipotesis Hipotesis Kesimpulan H Pengaruh Pengetahuan Masyarakat Tentang Akad Mudharabah Terhadap Minat Menabung Di Bank Syariah Diterima Tabel 4. 11 Hipotesis Hipotesis Kesimpulan H Pengaruh Pengetahuan Masyarakat Tentang Akad Mudharabah Terhadap Minat Menabung Di Bank Syariah Diterima Populasi dan Sampel Populasi adalah wilayah yang terdiri atas objek dan subjek yang mempunyai kualitas dan karakteristik tertentu yang ditetapkan oleh peneliti untuk mempelajari dan kemudian ditarik kesimpulannya (Supardi, 2017). Populasi dalam penelitian ini adalah masyarakat Desa Malang Nengah Kecamatan Sukatani Kabupaten Purwakarta yang berjumlah 1.620 Kepala keluarga yang diwakili 1 orang per kartu keluarga, populasi yang digunakan 1.620 orang. Sampel adalah bagian dari jumlah dan karakteristik yang dimiliki oleh populasi tersebut. Peneliti dapat menggunakan sampel yang diambil dari populasi tersebut (Supardi, 2017). 85 Jurnal Al-Fatih Global Mulia Pengaruh Pengetahuan Masyarakat... Sampel pada penelitian ini yaitu sebagian masyarakat Desa Malang Nengah Kecamatan Sukatani Kabupaten Purwakarta. KESIMPULAN Berdasarkan penelitian diatas dapat disimpulkan hasil analisa data, pengujian hipotesis dan pembahasannya, maka disimpulkan bahwa variabel X (pengetahuan) masyarakat tentang akad mudharabah berpengaruh positif dan signifikan sebesar 39,5% R Square terhadap variabel Y (minat) menabung di Bank Syariah. Hal ini mengindikasikan bahwa semakin positif bertambahnya pengetahuan masyarakat terhadap produk bank syariah maka semakin tinggi minat menabung masyarakat di Bank Syariah. Hal ini dapat dibuktikan dengan hasil analisis data yang di uji menggunakan uji t yang menunjukkan nilai variabel pengetahuan tentang akad mudharabah (X) pada output sebesar 7,744 > 1,661. 86 Jurnal Al-Fatih Global Mulia Riska Restapia1, Nurajizah2 DAFTAR PUSTAKA Abror, A. (1993). Psikologi Pendidikan (Vol. 4). Yogyakarta: PT. Tiara Wacana. afidburhanuddin. (2014). https://afidburhanuddin.wordpress.com/2014/05/13/jenis- pengetahuan-dan-ukuran-kebenarannya/. Diambil kembali dari jenis-pengetahuan- dan-ukuran-kebenarannya. al-Zuhaili, W. (1997). al-Fiqh al-Islami wa Adillatuh (Vol. 5). Beirut: Dar al-Fikr al-Mu'ashir. al-Zuhaili, W. (1997). al-Fiqh al-Islami wa Adillatuh (Vol. 5). Beirut: Dar al-Fikr al-Mu'ashir. Andiyansari, C. N. (2020). Akad Mudharabah dalam Perspektif Fikih dan Perbankan Syariah. al-Zuhaili, W. (1997). al-Fiqh al-Islami wa Adillatuh (Vol. 5). Beirut: Dar al-Fikr al-Mu'ashir. Andiyansari, C. N. (2020). Akad Mudharabah dalam Perspektif Fikih dan Perbankan Syariah. J l P didik d A I l 3(2) 43 Andiyansari, C. N. (2020). Akad Mudharabah dalam Perspektif Fikih dan Perbankan Syariah. Jurnal Pendidikan dan Agama Islam, 3(2), 43. Antonio, M. S. (2017). Bank Syariah Dari Teori Ke Praktik. Jakarta: Gema Insani. Arifin , & Sa’diyah. (2013, Desember). Mudharabah Dalam Fiqh Dan Perbankan Syariah. JURNAL Equilibrium, 1(2), 12. Az-Zuhaili, W. (2011). Fiqih Islam Wa Adillatuhu (Vol. 5). Jakarta: Gema Insani. Az-Zuhaili, W. (2011). Fiqih Islam Wa Adillatuhu (Vol. 5). Jakarta: Gema Insani. Bagus, L. (1996). Kamus Filsafat. Jakarta: Gramedia. aili, W. (2011). Fiqih Islam Wa Adillatuhu (Vol. 5). Jakarta: Gema Insani. L. (1996). Kamus Filsafat. Jakarta: Gramedia. Bagus, L. (1996). Kamus Filsafat. Jakarta: Gramedia. Bagus, L. (1996). Kamus Filsafat. Jakarta: Gramedia. Bahruddin, E., & Hamdi, A. S. (2014). Metode Penelitian Kuantitatif Aplikasi Dalam Pendidikan. Yogyakarta: Deeppublish. Basir, C. (2009). Penyelesaian Sengketa Perbankan Syariah di Pengadilan Agama dan Mahkamah Syariah. Jakarta: Kencana. Bawono, A. (2006). Multivariate Analysis dengan SPSS. Salatiga: STAIN Salatiga Press. Crow, L., & Crow, A. (1989). Psikologi Pendidikan Terjemahan dari Educational Psycologi (Vol. 1). Yogyakarta: s, P. (1972). The Encyclopedia of Philosophy. New York: Macmillan Publish Gazalba, S. (1992). Sistematika Filsafat. Jakarta: Bulan Bintang. a, S. (1992). Sistematika Filsafat. Jakarta: Bulan Bintang. Ghadah, A. a.-S. (2003). Buhut fi al-Mu'amalat wa al-Asalib al-Masharifiyah al-Islamiyyah (Vol. 1). t.t: Majmu'ah Dilah al-Barkah. Ghozali, I. (2006). Aplikasi analisis multivariate dengan program SPSS. , ( ) f f y yy (Vol. 1). t.t: Majmu'ah Dilah al-Barkah. Ghozali, I. (2006). Aplikasi analisis multivariate dengan program SPSS. (Vol. 1). t.t: Majmu ah Dilah al-Barkah. Ghozali, I. (2006). Aplikasi analisis multivariate dengan program SPSS. zali, I. (2006). Aplikasi analisis multivariate dengan program SPSS. Guru, P. (2021). Diambil kembali dari https://pendidikan.co.id/pengertian-masyarakat Indi, I. (2019). Faktor-Faktor Yang Memengaruhi Minat Masyarakat Menabung Di Bank Syariah (Studi Kasus Pada Masyarakat Di Kecamatan Rappocini Kota Makassar) (Doctoral dissertation, Universitas Negeri Makassar). Iskandarwasid , & Sunendar, D. (2011). 88 Jurnal Al-Fatih Global Mulia DAFTAR PUSTAKA Strategi Pembelajaran Bahasa (Vol. 3). Bandung: Rosda. Isma'il, U. M. (2006). Sanadat al-Muqaradhah wa Ahkamuha fi al-Fiqh al-Islami: Dirasah Muqaranah Tathbiqiyyah. Amman: Dar al-Nafa'is. ISRA. (2015). Sistem Keuangan Islam Prinsip & Oprasional. Jakarta: Rajawali Pers. Karim, H. (1993). Fiqh Muamalah. Jakarta: PT Raja Grafindo Persada. Kattsoff, L. O. (1996). Pengantar Filsafat. Yogyakarta: Tiara Wicana. Kattsoff, L. O. (1996). Pengantar Filsafat. Yogyakarta: Tiara Wicana. Kotler, P. (2002). Manajemen pemasaran. Jakarta: Prenhallindo. Mauludia, Z. ( 2021, Desember). IMPLEMENTASI AKAD MUDHARABAH DALAM PERBANKAN SYARIAH. Jurnal Kajian, Penelitian Ekonomi dan Bisnis Islam, 1(1), 40-42. Nasehudin, T. S., & Gozali , N. (2012). Metode Penelitian Kuantitatif. Nasehudin, T. S., & Gozali , N. (2012). Metode Penelitian Kuantitatif. Nigel , C. B., & Grove, S. (2000). Mengenal Psikologi For Beginners (Vol. 1). Bandun Nugroho, B. A. (2005). Strategi jitu memilih metode statistik penelitian dengan SPSS. Yogyakarta: Andi. 87 Jurnal Al-Fatih Global Mulia Pengaruh Pengetahuan Masyarakat... Priyastama, R. (2017). Buku Sakti Kuasai SPSS. Yogyakarta: Start Up. Rozalinda. (2016). Fikih Ekonomi Syariah. Jakarta: Rajawali Pers. Saputro , & Dzulkirom. (2015, April). Sistem Perhitungan Bagi Hasil Pembiayaan Mudharabah Pada PT. Bank Syariah Mandiri Cabang Malang. Jurnal Administrasi Bisnis (JAB), 21(2), 2. Sari, N. P., Fadilla, & Aravik, H. (2021). PENERAPAN AKAD MUDHARABAH PADA PRODUK TABUNGAN DI PT. BANK SYARIAH MANDIRI KC PRABUMULIH. Ekonomica Sharia: Jurnal Pemikiran dan Pengembangan Ekonomi Syariah, 6 (2), 212- 215. yono. (2017). Metode Penelitian Kuantitatif, Kualitatif, dan R&D. Bandung: ALFABETA Sugiyono. (2017). Metode Penelitian Kuantitatif, Kualitatif, dan R&D. Bandung: ALFABETA. Supardi, D. (2017). Statistik penelitian pendidikan. Depok: PT Raja Grafindo Persada. Sugiyono. (2017). 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Publisher Correction: Pushing the thinness limit of silver films for flexible optoelectronic devices via ion-beam thinning-back process
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Publisher Correction: Pushing the thinness limit of silver films for flexible optoelectronic devices via ion-beam thinning-back process Publisher Correction: Pushing the thinness limit of silver films for flexible optoelectronic devices via ion-beam thinning-back process Dongxu Ma, Ming Ji, Hongbo Yi, Qingyu Wang, Fu Fan, Bo Feng, Mengjie Zheng, Yiqin Chen & Huigao Duan In this article the wrong figure appeared as Fig. 1; the figure should have appeared as Dongxu Ma, Ming Ji, Hongbo Yi, Qingyu Wang, Fu Fan, Bo Feng, Mengjie Zheng, Yiqin Chen & Huigao Duan Correction to: Nature Communications https://doi.org/10.1038/s41467-024-46467-6, published online 13 March 2024 https://doi.org/10.1038/s41467-024-47014-z Check for updates Correction to: Nature Communications https://doi.org/10.1038/s41467-024-46467-6, published online 13 March 2024 https://doi.org/10.1038/s41467-024-47014-z Check for updates In this article the wrong figure appeared as Fig. 1; the figure should have appeared as shown below. In this article the wrong figure appeared as Fig. 1; the figure should have appeared as shown below. The original article has been corrected. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The original article has been corrected. The original article has been corrected. The original article has been corrected. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. Publisher Correction: Pushing the thinness limit of silver films for flexible optoelectronic devices via ion-beam thinning-back process If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2024 © The Author(s) 2024 Corrections&amendments Corrections&amendments nature communications (2024) 15:2587 | 1
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<i>In vivo</i> CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy
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In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy Gaia Colasante,1,* Yichen Qiu,2,* Luca Massimino,1 Claudia Di Berardino,1 Jonathan H. Cornford,2 Albert Snowball,2 Mikail Weston,2 Steffan P. Jones,2 Serena Giannelli,1 Andreas Lieb,2,# Stephanie Schorge,2,3 Dimitri M. Kullmann,2 Vania Broccoli1,4 and Gabriele Lignani2 Received August 23, 2019. Revised December 31, 2019. Accepted January 14, 2020 V C The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy Gaia Colasante,1,* Yichen Qiu,2,* Luca Massimino,1 Claudia Di Berardino,1 Jonathan H. Cornford,2 Albert Snowball,2 Mikail Weston,2 Steffan P. Jones,2 Serena Giannelli,1 Andreas Lieb,2,# Stephanie Schorge,2,3 Dimitri M. Kullmann,2 Vania Broccoli1,4 and Gabriele Lignani2 *These authors contributed equally to this work. doi:10.1093/brain/awaa045 BRAIN 2020: Page 1 of 15 | 1 Downloaded from https://academic.oup.com/brain/adv In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy Gaia Colasante,1,* Yichen Qiu,2,* Luca Massimino,1 Claudia Di Berardino,1 Jonathan H. Cornford,2 Albert Snowball,2 Mikail Weston,2 Steffan P. Jones,2 Serena Giannelli,1 Andreas Lieb,2,# Stephanie Schorge,2,3 Dimitri M. Kullmann,2 Vania Broccoli1,4 and Gabriele Lignani2 *These authors contributed equally to this work. doi:10.1093/brain/awaa045 BRAIN 2020: Page 1 of 15 | 1 Downloaded from https://academic.oup.com/brain/adv doi:10.1093/brain/awaa045 BRAIN 2020: Page 1 of 15 | 1 doi:10.1093/brain/awaa045 In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy *These authors contributed equally to this work. Epilepsy is a major health burden, calling for new mechanistic insights and therapies. CRISPR-mediated gene editing shows promise to cure genetic pathologies, although hitherto it has mostly been applied ex vivo. Its translational potential for treating non-genetic pathologies is still unexplored. Furthermore, neurological diseases represent an important challenge for the application of CRISPR, be- cause of the need in many cases to manipulate gene function of neurons in situ. A variant of CRISPR, CRISPRa, offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters. We asked if this strategy can effectively treat acquired focal epilepsy, focusing on ion channels because their manipulation is known be effective in changing network hyperactivity and hypersynchronziation. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding Kv1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated Kv1.1 upregulation led to a sub- stantial decrease in neuronal excitability. Continuous video-EEG telemetry showed that AAV9-mediated delivery of CRISPRa, upon doxycycline administration, decreased spontaneous generalized tonic-clonic seizures in a model of temporal lobe epilepsy, and rescued cognitive impairment and transcriptomic alterations associated with chronic epilepsy. The focal treatment minimizes concerns about off-target effects in other organs and brain areas. This study provides the proof-of-principle for a translational CRISPR-based ap- proach to treat neurological diseases characterized by abnormal circuit excitability. Introduction be switched simply by changing the sgRNA. Second, CRISPRa preserves the full range of native splice variants and protein biogenesis mechanisms (Liao et al., 2017). Third, CRISPRa is, in principle, safe because it only alters the promoter activity of genes that are already transcribed in targeted neurons. Finally, CRISPRa can be targeted to spe- cific neurons in the epileptic focus using established viral vectors (La Russa and Qi, 2015). Epilepsy affects up to 1% of the population, and 30% of patients continue to experience seizures despite the use of current medication (Kwan et al., 2011; Tang et al., 2017). Although the majority of drug-resistant epilepsies are focal, targeting drugs to a restricted brain region presents major challenges, and potentially curative surgery is limited to a minority of cases where the seizure focus is remote from elo- quent cortex (Kullmann et al., 2014). Gene therapy holds promise as a rational replacement for surgery for intractable pharmaco-resistant epilepsy, and could in principle improve the prospect for seizure freedom in many people (Kullmann et al., 2014; Lieb et al., 2018). Several approaches have been proposed to interfere with epileptogenesis or to decrease seizure frequency in chronic epilepsy (Simonato, 2014). Current experimental gene therapies mainly rely on viral vector-mediated expression of genes encoding normal CNS proteins or exogenous non-mammalian proteins (Richichi et al., 2004; Noe et al., 2008; Wykes et al., 2012, 2016; Krook-Magnuson et al., 2013; Ka¨tzel et al., 2014; Lieb et al., 2018; Agostinho et al., 2019). This approach has sev- eral potential limitations, including a finite packaging cap- acity of viral vectors, difficulty in ensuring normal splicing and post-transcriptional processing, and, in the case of non- mammalian membrane proteins, concerns about immuno- genicity. Modulating the expression of endogenous genes, in contrast, would represent an important step toward safe and rational treatment of intractable epilepsy and other neuro- logical diseases. Here, we report the use of CRISPRa to treat a mouse model of temporal lobe epilepsy, from in vitro validation to demonstration of efficacy in reducing seizure frequency and rescuing cognitive impairment in vivo. Animals and ethics All experimental procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986. Male and female C57BL/6J and Camk2a-Cre mice (2–3 months old, 20–30 g, Envigo and Jackson Laboratory, respectively) were used for the experiments. Animals were housed in an enriched environment, in groups before surgery and singly after surgery in individually ventilated cages in a specific pathogen-free facility. The DNA editor/regulator CRISPR/Cas9 (Konermann et al., 2015; Dominguez et al., 2016; Adli, 2018) represents a powerful tool to modify endogenous genes, not only in somatic cells but also in mammalian neurons (Heidenreich and Zhang, 2016; Suzuki et al., 2016). In addition to per- manently altering endogenous gene sequences, CRISPR/Cas9 can regulate the activity of genes through promoter modula- tion, an approach known as CRISPR activation (CRISPRa) (Dominguez et al., 2016; Liao et al., 2017; Matharu et al., 2019 ). CRISPRa is therefore a promising tuneable tool to increase the expression of genes encoding, for instance, ion channels, in chronic epilepsy in order to restore physiologic- al levels of network activity (Wykes et al., 2012; Wykes and Lignani, 2018; Colasante et al., 2020). The CRISPRa system consists of a nuclease-defective Cas9 (dCas9) fused to a tran- scription activator and a small guide RNA (sgRNA) that tar- gets dCas9 to the promoter of the gene of interest (Dominguez et al., 2016). There are multiple advantages of this system. First, it is versatile because the targeted gene can Study design This study aimed to test the hypothesis that upregulating en- dogenous genes (e.g. Kcna1) with CRISPRa can treat chemocon- vulsant-induced temporal lobe epilepsy. The experiments were designed to achieve a power 40.8 with an a = 0.05. For in vivo experiments the 3Rs guidelines for animal welfare were fol- lowed. Outliers were not excluded and at least three independ- ent repetitions were performed. Exclusion criteria were applied for all the recordings (see below). All the experiments were randomized and researchers were blinded during recordings and analysis. Keywords: CRISPR; gene therapy; epilepsy; gene promoter; potassium channels Abbreviations: AAV = adeno-associated virus; CRISPRa = CRISPR activation; sgRNA = small guide RNA Keywords: CRISPR; gene therapy; epilepsy; gene promoter; potassium channels Abbreviations: AAV = adeno-associated virus; CRISPRa = CRISPR activation; sgRNA = small guide RNA Stephanie Schorge E-mail: s.schorge@ucl.ac.uk Stephanie Schorge E-mail: s.schorge@ucl.ac.uk *These authors contributed equally to this work. ract/doi/10.1093/brain/awaa045/5780426 by UCL, London user on 11 March 2020 1 San Raffaele Scientific Institute, Via Olgettina 58, 20132, Milan, Italy 1 San Raffaele Scientific Institute, Via Olgettina 58, 20132, Milan, Italy 2 Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, University College Lo 2 Department of Clinical and Experimental Epilepsy, UCL Institute of Neuro p p p y, gy, y g , , of Pharmacology, UCL School of Pharmacy, University College London, London, UK te of Neuroscience, Via Vanvitelli 32, 20129, Milan, Italy 3 Department of Pharmacology, UCL School of Pharmacy, University College London, London, UK 4 CNR Institute of Neuroscience, Via Vanvitelli 32, 20129, Milan, Italy p gy, y, y g , , 4 CNR Institute of Neuroscience, Via Vanvitelli 32, 20129, Milan, Italy 4 CNR Institute of Neuroscience, Via Vanvitelli 32, 20129, Milan, Italy #Present address: Department of Pharmacology, Medical University of Innsbruck, Peter Mayr Strasse 1A, 6020 Innsbruck, Austria dress: Department of Pharmacology, Medical University of Innsbruck, Peter Mayr Strasse 1A, 6020 A i #Present address: Department of Pharmacology, Medical University of Innsbruck, Peter Mayr Strasse 1A, 6020 Innsbruck, Austria Correspondence to: Gabriele Lignani Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology Department of Clinical and Experimental Epilepsy, U University College London, London, UK University College London, London, UK E-mail: g.lignani@ucl.ac.uk Correspondence may also be addressed to: Vania Broccoli San Raffaele Scientific Institute, Via Olgettina 58, 20132, Milan, Italy E-mail: v.broccoli@hsr.it E-mail: v.broccoli@hsr.it 2 | BRAIN 2020: Page 2 of 15 2 | BRAIN 2020: Page 2 of 15 G. Colasante et al. P19 cell line Using the Galaxy web-tool (https://usegalaxy.org/) we generated two datasets: one containing sg19 off-target sequences predicted by the CRISPOR web tool (http://crispor.tefor.net) and one con- taining all the 500-bp genomic regions (NCBI37/mm9) up- stream to transcription start sites of annotated transcripts. Intersecting the two datasets, all sg19 off-target sequences in pu- tative gene promoters were derived. To identify genes regulated by putative promoters, the sequence of the predicted off-targets was aligned by IGV to the reference genome and to transcripts annotated in ENSEMBL database. Validation of expression lev- els of putative off-target genes was performed by RT-qPCR. P19 cells were cultured in alpha-MEM (Sigma-Aldrich) supple- mented foetal bovine serum non-essential amino acids, sodium pyruvate, glutamine and penicillin/streptomycin and split every 2–3 days using 0.25% trypsin. For transfection, LipofectamineTM 3000 (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. Plasmids Small guide RNAs (sgRNAs) were cloned into a lentiviral vector with a U6 promoter (pU6). Defective Cas9 fused to the VP160 activator domain was cloned into T2A with the Puromucin resistance cassette (PuroR) and under the control of the Eef1a1 (Ef1alpha) promoter (Ef1alpha-dCas9VP160-T2A- PuroR). The dCas9VP160-2A-PuroR cassette was obtained from pAC94-pmax-dCas9VP160-2A-PuroR (a gift from R. Jaenisch) (Addgene plasmid #48226), and subcloned in a the TetO-FUW vector followed by restriction digestion with HpaI/AfeI, then blunt cloned into an Ef1alpha-GFP vector after GFP removal by BRAIN 2020: Page 3 of 15 | 3 CRISPRa treats temporal lobe epilepsy SmaI/EcoRV digestion. Ef1alpha-dCas9VP160-T2A-GFP was obtained by restriction digestion of Ef1alpha-dCas9VP160-T2A- PuroR with AscI/XbaI, which removed VP160-T2A-PuroR; the VP160-T2A fragment was then obtained by AscI/XhoI digestion from Ef1alpha-dCas9VP160-T2A-PuroR while the GFP frag- ment was PCR amplified using primers containing XhoI/XbaI restriction sites; finally, the two fragments were ligated together into the vector. To obtain a single vector containing both dCas9A and sgRNA, the pU6-sgRNA cassette was HpaI digested and cloned into Ef1alpha-dCas9VP160. To generate an adeno-associated virus (AAV) with activating dCas9 (dCas9- VP64) under a doxycycline-inducible promoter, and tetracycline transactivator responsive element (TRE), we used AAV-SpCas9 (a gift from F. Zhang, Addgene #PX551) as the starting mater- ial: the Mecp2 promoter was removed by XbaI/AgeI digestion and the TRE promoter was amplified using the primers: FWXbaI: 50-GCTCTAGACCAGTTTGGTTAGATCTC-30; and RV AgeI: 50-GCACCGGTGCGATCTGACGGTTCACT-30. SpCas9 was removed using AgeI/EcoRI and Cas9m4-VP64 (a gift from G. Church, Addgene #47319) was digested with AgeI/EcoRI. The VP64 fragment was PCR-amplified using the following primers with EcoRI sites: F: 50-GATCATCGAGC AAATAAGCGAATTCTC-30 and R: 50-gctaaGAATTCTTA- TCTAGAGTTAATCAGCATG-30. The AAV vector containing the sgRNA cassette was derived from pAAV-U6sgRNA (SapI)_hSyn-GFP-KASH-bGH (PX552 was a gift from F. Zhang, Addgene #60958): sg19 or lacZ were cloned under the U6 promoter and the GFP was removed by KpnI/ClaI digestion and replaced by a DIO-rtTA-T2A-Tomato cassette. This vector was used for the work in Camk2a-Cre mice. For the work in C57/Bl6 mice, this vector was XbaI/ClaI-digested to remove the human SYN1 (hSyn) promoter, and the DIO-rtTA-T2A-Tomato cassette was replaced by a Camk2a promoter amplified with NheI-KpnI and a tTA T2a tomato cassette amplified with KpnI/ ClaI, ligated together in the vector. electrophysiology recordings were performed 14–16 days after transduction. electrophysiology recordings were performed 14–16 days after transduction. Western blot Total neuronal protein extracts were obtained from the lysis of primary neurons by RIPA lysis buffer (150 mM NaCl, 1% Triton, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sul- phate, Tris pH 8.0 50 mM, protease inhibitor cocktail) 2 weeks after infection with the CRISPRa-Kcna1 system. Lysates were kept on ice for 30 min by vortexing every 10 min and then cen- trifuged at 4C for 5 min at 5000 rpm. Supernatants with solu- bilized proteins were collected in new tubes and stored at –80C until use. Western blot analysis was performed using primary antibodies against the following proteins: anti-Kv1.1 (1:1000, NeuroMab) anti-a/bActin (1:10000, Sigma). tract/doi/10.1093/brain/awaa045/5780426 by UCL, London user on 11 March 2020 Bioinformatic analysis Encyclopedia of DNA Elements (ENCODE) and the Functional ANnoTation Of the Mammalian genome (FANTOM) (Carninci et al., 2006) databases were used to download transcriptomics and epigenetics NGS data. Tracks were visualized along the mm10 mouse reference genome with the Integrative Genome Viewer (IGV) (Thorvaldsdottir et al., 2013). RNA isolation and quantitative RT- PCR RNA was extracted from primary neurons and cells using TRI ReagentV R (Sigma) according to the manufacturer’s instructions. For quantitative RT-PCR (RT-qPCR), cDNA synthesis was obtained using the ImProm-IITM Reverse Transcription System (Promega) and RT-qPCR was carried out with custom designed oligonucleotides (Supplementary Table 1) using the Titan HotTaq EvaGreenV R qPCR Mix (BIOATLAS). Analysis of rela- tive expression was performed using the DDCT method, relative to the Ctrl-dCas9A condition. To determine Kcna1 expression in vivo and for RNA-Seq, RNA was extracted from frozen tis- sues. For qPCR, DDCT was determined in Ctrl-dCas9A or in Kcna1-dCas9A injected hippocampi relative to contralateral hip- pocampi in epileptic animals at the end of the recordings. Virus preparation Lentiviruses were produced as previously described with a titre of 107–108 IU/ml (Colasante et al., 2015). AAVs were produced as previously described with a titre 41012 vg/ml (Morabito et al., 2017). The TRE-dCas9-VP64 AAV was produced by VectorBuilder with a titre of 8  1012 vg/ml. Activity clamp The template simulating the barrage of synaptic conductances during epileptiform bursts was previously described (Morris et al., 2017). Dynamic clamp software (Signal 6.0, Cambridge Electronic Design, Cambridge, UK) and a Power3 1401 (CED) were used to inject both excitatory and inhibitory conductance templates simultaneously in a neuron recorded in current clamp configuration (iteration frequency 15 kHz). Erev was set to 0 mV and –75 mV for excitatory and inhibitory conductances, respectively, and corrected for a liquid junction potential of 14.9 mV. Incrementing synaptic conductances were injected in recorded neurons to establish the conductance threshold for ac- tion potential generation. Current clamp recordings for activity clamp were performed with the same external and internal solu- tions as given above. In vitro and ex vivo electrophysiology analysis In vitro and ex vivo electrophysiology analysis All the electrophysiology analysis was performed with an auto- mated Python script. Passive properties were calculated from the hyperpolarizing steps of the current clamp steps protocol. Input resistance was averaged from three current steps (two negative and one positive). Capacitance was calculated from the hyper- polarizing current step as follows. First, the input resistance was determined as the steady state DV/DI (voltage/current), then the cell time constant (tau) was obtained by fitting the voltage relax- ation between the baseline and the hyperpolarizing plateau. Capacitance was then calculated as tau/resistance. Single action potential parameters were calculated as previously described (Pozzi et al., 2013). An event was detected as an action potential if it crossed 0 mV and if the rising slope was 420 mV/ms in a range of injected currents from 0 pA to 500 pA. All the experi- ments were performed at room temperature (22–24C). All recordings and analyses were carried out blind to vector transduced. Differential gene expression and functional enrichment analy- ses were performed with DESeq2 (Love et al., 2014) and GSEA, respectively. Statistical and downstream bioinformatics analyses were performed within the R environment. Gene expression heat maps were produced with GENE-E (Broad Institute). Data of in vitro and in vivo experiments were deposited in the NCBI Gene Expression Omnibus repository with the GSE133930 GEO ID. Primary neuronal culture and lentivirus transduction Cortical neurons were isolated from postnatal Day 0 C57Bl/6J mouse pups as previously described (Beaudoin et al., 2012) and were transduced with lentiviruses at 1 day in vitro (DIV). Quantitative RT-PCR, RNA seq, western blot analysis and RNA-Seq RNA libraries for both in vitro and in vivo experiments were generated starting from 1 lg of total RNA extracted from 4 | BRAIN 2020: Page 4 of 15 G. Colasante et al. sglacz- and sg19-dCas9A neurons at 10 DIV. RNA quality was assessed using a TapeStation instrument (Agilent) and only RNA samples with integrity number (RIN) 58 were analysed. For in vitro experiments, RNA was processed according to the Lexogen QuantSeq 3’ mRNA-Seq Library Prep Kit protocol and the libraries were sequenced on an Illumina NextSeq 500 with 75-bp stranded reads at CTGB, Ospedale San Raffaele. Fastq files were aligned to the mouse genome (NCBI37/mm9) with Bowtie2. interface combined with Signal software (CED), filtered at 10 kHz and digitized at 50 kHz. sglacz- and sg19-dCas9A neurons at 10 DIV. RNA quality was assessed using a TapeStation instrument (Agilent) and only RNA samples with integrity number (RIN) 58 were analysed. For in vitro experiments, RNA was processed according to the Lexogen QuantSeq 3’ mRNA-Seq Library Prep Kit protocol and the libraries were sequenced on an Illumina NextSeq 500 with 75-bp stranded reads at CTGB, Ospedale San Raffaele. Fastq files were aligned to the mouse genome (NCBI37/mm9) with Bowtie2. In vitro For current-clamp recordings, the internal solution contained (in mM): 126 K-gluconate, 4 NaCl, 1 MgSO4, 0.02 CaCl2, 0.1 BAPTA, 15 glucose, 5 HEPES, 3 ATP-Na2, 0.1 GTP-Na, pH 7.3. The extracellular (bath) solution contained (in mM): 2 CaCl2, 140 NaCl, 1 MgCl2, 10 HEPES, 4 KCl, 10 glucose, pH 7.3. D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5; 50lM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10lM) and picrotoxin (PTX; 30lM) were added to block synaptic trans- mission. Transduced excitatory neurons were identified with EGFP fluorescence and from a pyramidal somatic shape. Neurons with unstable resting potential (or 4–50 mV), access resistance (Ra) 415 MX and/or holding current 4200 pA at –70 mV were discarded. Bridge balance compensation was applied and the resting membrane potential was held at –70 mV. A current step protocol was used to evoke action potentials by injecting 250-ms long depolarizing current steps of increasing amplitude from –20 pA (D10 pA). Recordings were acquired using a MultiClampTM 700A amplifier (Axon Instruments, Molecular Devices) and a Power3 1401 (CED) Slice preparation Camk2a-Cre mice of either sex (2–3 months old) were sacrificed by cervical dislocation under isoflurane. Brains were quickly dis- sected into ice-cold oxygenated slicing solution (in mM: 75 su- crose, 2.5 KCl, 25 NaHCO3, 25 glucose, 7 MgCl2, 0.5 CaCl2) and cut into 300 mm coronal slices using a Leica VT1200S vibratome (Leica). Slices were stored submerged in oxygenated recording artificial CSF (in mM: 25 glucose, 125 NaCl, 2.5 KCl, 25 NaHCO3, 1 MgCl2, 1.25 NaH2PO4.H2O and 2 CaCl2) at 32C for 30 min and at room temperature for a fur- ther 30 min before recording. Surgical procedures All surgical procedures were performed in anaesthetized adult mice (2–3 months) placed in a stereotaxic frame (Kopf). Ex vivo current clamp recordings Current clamp recordings were performed in standard external solution in the presence of DL-AP5 (50 lM), CNQX (10 lM) and PTX (30 lM) to block NMDA, AMPA/kainate, and GABAA receptors, respectively. The internal solution was the same as for in vitro patch clamp recordings. Neurons with hold- ing current 4100 pA and Ra 420 MX upon whole-cell break- in in voltage clamp mode and membrane potential less negative than –60 mV in current clamp were not considered for analysis. A 1440 DigidataV R (Molecular Devices) or Power3 1401 (CED) interface and MultiClampTM 700A (Molecular Devices) ampli- fier were used. For in vivo experiments, RNA was processed according to the TruSeq Stranded mRNA Library Prep Kit protocol. The libra- ries were sequenced on an Illumina HiSeq 3000 with 76 bp stranded reads using Illumina TruSeq technology at Genewiz. Image processing and base calling were performed using the Illumina Real Time Analysis Software. Fastq files were mapped to the mm10 mouse reference genome with the STAR aligner v2.7 (Dobin et al., 2013). EEG analysis Animal food was changed to doxycycline ad libitum pellet (TD.120769-BLUE 625 mg/kg) after baseline recordings for the following 2 weeks. Spontaneous seizures were detected from chronic recordings using a semi-automated supervised learning approach (Supplementary Fig. 7). First, a library containing examples of epileptiform activity was built using seizures identified from vis- ual inspection of ECoG data. The recordings were saved in hour-long files, and for each seizure this full hour was included in the library. Recordings were chunked into 5-s blocks that were labelled as either ‘ictal’ or ‘interictal’ if they contained epi- leptiform-labelled activity or not, respectively. For each 5-s chunk of recording, 15 features were extracted (Supplementary Fig. 7 and see online resource below). A random forest discrim- inative classifier was trained on the features and labels of each of the 5-s examples in the library (Breiman, 2001). In addition, cross-validation generated classifier predictions were used to parameterize a Hidden Markov Model in which the hidden states were the human annotations and the emissions the classi- fier predictions. For automated detection of epileptiform activity from unlabelled recordings, the discriminative classifier was first used to predict the class of consecutive 5-s chunks. We then applied the forward-backward algorithm to obtain the marginal probability of being in seizure state for each recording chunk given the surrounding classifier predictions. The smoothed pre- dictions were then manually verified, false positives removed from the analysis and start and end locations adjusted. To quan- tify the performance of our approach, we randomly selected four 2-week sets of recordings and visually examined the traces for seizures and compared them to classifier predictions (blinded). During the 8 weeks, we did not visually detect any seizures that were not marked by the classifier—as such, for this model of epilepsy, our false negative rate was 51/300. False positives were less of a concern, but in general we observed  n seizures for a given period of time. For further information and code, see: https://github.com/jcornford/pyecog. Transmitter implantation An electrocorticogram (ECoG) transmitter (A3028C-CC Open Source Instruments, Inc.) was subcutaneously implanted and the recording electrode was placed in the cortex above the viral in- jection site (antero-posterior: –2/3 bregma/lamda distance; medio-lateral: –3). The ground electrode was placed in the contralateral frontal hemisphere. The ECoG was acquired wirelessly using hardware and software from Open Source Instruments, Inc. The ECoG was sampled at a frequency of 256 Hz, band-pass filtered between 1 and 160 Hz, and recorded continuously for the duration of the experi- ments. The animals were housed independently in a Faraday cage. Epilepsy model Kainic acid (0.3 mg of 10 mg/ml, Tocris) was injected in a vol- ume of 200 nl (7.14 mM effective concentration) in the right amygdala (antero-posterior: –0.94; medio-lateral: 2.85; dorso- ventral: 3.75) at 200 nl/min under isoflurane anaesthesia BRAIN 2020: Page 5 of 15 | 5 CRISPRa treats temporal lobe epilepsy 5 (surgery time 10–15 min). The mice were allowed to recover from anaesthesia at 32C for 5 min and then moved back to their cage where they were monitored closely during status epi- lepticus. Status epilepticus (characterized by stage 5 seizures on the Racine scale) usually began 10–15 min after complete recov- ery and always ended 40 min after kainic acid injection with 10 mg/kg intraperitoneal diazepam. Only animals that exhibited at least one seizure per week were included in the subsequent study. contralateral skull. A cannula (Bilaney Consultants Ltd.) was implanted in the same location as the recording electrode for se- quential pilocarpine injections. Animals were allowed to recover for 2 weeks before induction of acute seizures by pilocarpine (3.5 M in saline) (Magloire et al., 2019) injected 0.5 mm below the cannula using a microinjection pump (WPI Ltd.), a 5-ml Hamilton syringe (Esslab Ltd.), and a 33-Gauge needle (Esslab Ltd.). The injection volume was incremented on consecutive days (180 nl, 300 nl and 500 nl) until spike-wave discharges were observed, and recorded as the threshold dose. If seizures failed to terminate spontaneously, the animal was excluded from the study. To assess the treatment, the animals were placed on a doxycycline diet for 7 days and only the threshold dose for the animal was repeated. ECoG monitoring was used to assess seizure severity for an hour after the pilocarpine injection. The researcher who acquired and analysed the data was blinded to the virus injected. Stereotaxic viral injection AAV9 viruses (300 nl, 1:1 ratio) were injected with a 5-ml Hamilton syringe (33-Gauge) at 100 nl/min in three different coordinates of the right ventral hippocampus (Paxinos Mouse Brain Atlas; antero-posterior: –2/3 bregma/lambda distance; medio-lateral: –3; dorso-ventral: 3.5/3/2.5). The needle was kept in place for 10 min after each injection. EEG (or ECoG) recordings Exclusion criteria Only animals recorded for the entire period of the experiment (6 weeks after kainic acid) were used in the analysis. At the end of the experiments some animal tissues were analysed with qRT-PCR and others were verified with immunofluorescence. Of the 42 mice injected with kainic acid, 34 (80%) were implanted and injected, 20% either did not reach stage 5 of the Racine scale or were excluded because of infections or unexpect- ed death in the first few days before planned implantation. Twenty-four mice were recorded for the entire duration of the experiment. Ten animals were not included in the analysis be- cause they either died before the end of the recordings (n = 2) or were culled because of technical issues (detachment of the head- piece) or transmitter failure. Two did not express dCas9 and were therefore excluded from the analysis. Twenty-two mice were used for the analysis (13 Ctrl-dCas9A and nine Kcna1- dCas9A). To avoid possible bias, all exclusions were made while researchers were blinded to treatment. Pilocarpine acute seizure model Data availability All the Python and Bonsai scripts are freely available. Plasmids will be deposited with Addgene, and transcriptomic data are deposited in the NCBI Gene Expression Omnibus repository with the GSE133930 GEO ID. Video recordings IP cameras from Microseven (https://www.microseven.com/ index.html) were used and synchronized via the Windows time server to the same machine as used to acquire the ECoG. Continuous video recordings produced six videos per hour. Data are plotted as box and whiskers, representing interquartile range (box), median (horizontal line), and maximum and min- imum (whiskers), together with all the points. The mean is fur- ther shown as a plus symbol. The statistical analysis performed is shown in each figure legend. Deviation from normal distribu- tions was assessed using D’Agostino-Pearson’s test, and the F- test was used to compare variances between two sample groups. Student’s two-tailed t-test (parametric) or the Mann-Whitney test (non-parametric) were used as appropriate to compare means and medians. Fisher’s exact test was used to analyse the contingency table. To compare two groups at different time points we used two-way repeated measure ANOVA, followed by Bonferroni post hoc test for functional analysis. Statistical analysis was carried out using Prism (GraphPad Software, Inc., CA, USA) and SPSS (IBM SPSS statistics, NY, USA). Immunohistochemistry Immunostaining was performed on 50-mm mouse brain PFA- fixed sections with the following antibodies: mouse anti- GAD67 (MAB5406, Merck), rabbit anti-RFP (600-401-379, Rockland), Alexa FluorV R 555 goat anti-rabbit (A32732, Invitrogen) and Alexa FluorV R 488 goat anti-mouse (A32723, Invitrogen). Images were acquired with ZEN software (Zeiss) on a LSM710 confocal microscope (Zeiss) and co-localization analysis of tdTomato and GAD67 were performed with ImageJ 1.51n (Wayne Rasband, National Institute of Health) plugin ‘JACoP’. Behaviour tests Trials started 2 weeks post-virus injection, all were carried out between 7 am and 7 pm, during the light phase. Animals (3 months old) were habituated in the designated behaviour room for at least 15 min in home cages prior to the test. Object Location Test For the familiarization phase, mice were placed individually in the arena (50 cm  50 cm  40 cm) and for 8 min, were allowed to explore two identical objects placed in the arena at least 5 cm away from the border. After a 6-h retention delay, the animals were returned to the same arena with one of the objects randomly relocated to a new location. The animal was allowed to explore for 8 min with video recordings. The arena and objects were thoroughly cleaned with ethanol between each session. We first asked if CRISPRa can be exploited to increase en- dogenous gene expression in glutamatergic neurons and de- crease their excitability. As a proof-of-principle, we chose the Kcna1 gene encoding the Kv1.1 channel, which is important for the regulation of neuronal action potential firing and syn- aptic transmission (Pinatel et al., 2017; Vivekananda et al., 2017). Lentivirus- or AAV-mediated overexpression of Kcna1 reduces neuronal excitability and, when targeted to principal cells, suppresses seizures in rodent models of epilepsy (Wykes et al., 2012; Snowball et al., 2019). We first conducted a bio- informatic analysis to identify its promoter region. Alignment of datasets of gene expression and epigenetic markers of ac- tively transcribed genes in perinatal and adult mouse brain identified peaks of enrichment for RNA PolII, mono- and tri- methylation of Lys4 and acetylation of Lys27 of H3 histone along the gene (Supplementary Fig. 1A). One of these regions was located immediately upstream to the annotated Kcna1 transcription start site and identified as a suitable target for CRISPRa. We submitted 200 bp from this region to the CRISPOR web tool for sgRNA design, and selected four can- didate guides (sg4, sg14, sg19 and sg30) for validation, ini- tially in the mouse P19 cell line that expresses many neuronal genes. SgRNAs were lipofected individually or in combin- ation, together with a construct carrying dCas9 fused to the transcriptional activator VP160 (dCas9-VP160) and a puro- mycin resistance cassette. dCas9-VP160 with sg4, sg14 or 6 | BRAIN 2020: Page 6 of 15 6 | BRAIN 2020: Page 6 of 15 G. Colasante et al. Results CRISPRa targeting the Kcna1 promoter region increases Kv1.1 and decreases neuronal excitability Pilocarpine acute seizure model Male wild-type C57BLC/6J mice (3 months old) were anaesthe- tized with isoflurane and placed in a stereotaxic frame (David Kopf Instruments Ltd.). The animals were injected with 1.5 ml AAV CamKII-CRISPR-Kcna1/CamKII-CRISPR-LacZ at 100 nl/min in layer 2/3–5 primary visual cortex (coordinates: antero-posterior –2.8 mm, medio-lateral 2.4 from the bregma, and dorso-ventral 0.7/0.5/0.3 from pia). For ECoG monitoring, the recording electrode of 256 Hz single-channel ECoG trans- mitter (A3028C-CC, Open Source Instruments Inc.) was placed at the same coordinates. A reference electrode was placed in the Novel Object Recognition Test The separation of the CRISPRa tool into two constructs, and the choice of dCas9-VP64 over dCas9VP160, was dictated by the reduced cargo capacity of AAV vectors. Nevertheless, the dual AAV9 system has been shown to give a good level of neuron co-transduction (Sun et al., 2019). This experimental design allowed the Kcna1- dCas9A system to be activated in forebrain principal neu- rons of Camk2a-cre mice transduced with both AAVs, but only after doxycycline administration (Fig. 2A and B). We co-injected both AAVs in the hippocampus of 2–3-month- old Camk2a-Cre mice, which were subsequently fed a doxy- cycline diet for 3 weeks before collecting acute brain slices for analysis. Pyramidal neurons in the CA1-subiculum of the ventral hippocampus were recorded with whole-cell patch clamp to measure their excitability (Fig. 2B, C and Supplementary Figs 3 and 4). Consistent with data from pri- mary cultures, neurons transduced with Kcna1-dCas9A showed a decreased firing rate and increased current thresh- old when compared with Ctrl-dCas9A expressing neurons (Fig. 2D and E). A minor difference from primary cortical cultures was that the half-width of the first spike was not significantly different between Kcna1-dCas9A- and Ctrl- dCas9A-expressing neurons (Fig. 1I). However, a significant decrease in half-width was seen when all the action poten- tials during the current step protocol were pooled (Fig. 2F). Finally, we applied activity clamp, a method to assess neur- onal excitability during epileptiform barrages of excitation (Morris et al., 2017). Neurons expressing Kcna1-dCas9A fired less than neurons expressing Ctrl-dCas9A when exposed to the same simulated synaptic input. Taken to- gether, these results support using Kcna1-dCas9A as a can- didate antiseizure gene therapy (Fig. 2G). The CRISPOR tool predicted 250 putative off-target genes for sg19, mostly with a very low likelihood score. To evalu- ate the specificity of CRISPRa we performed a gene expres- sion profile analysis in primary neurons treated with Kcna1- dCas9A and compared this with Ctrl-dCas9A transduced neurons. No consistent alteration in the transcriptome of sg19 treated neurons was observed, except for a significant increase in Kcna1 (Fig. 1E, red dot). Six of 250 predicted off- target genes for sg19 were located close to promoters of Mylpf, Efcab4a, Nudcd2, Pde4b, Gc and Vps16 genes. However, none of these genes showed a significant change in expression in either the transcriptome analysis (Fig. 1E, green dot) or in quantitative RT-PCR assays (Fig. 1F). Novel Object Recognition Test Twenty-four hours after the Object Location Test, the same ani- mals were subjected to the Novel Object Recognition Test. The familiarization session was the same as for the Object Location Test. After a 6-h retention delay, one of the objects was ran- domly replaced by a novel object with a different shape and sur- face texture. The animals were allowed to explore freely for 8 min (Leger et al., 2013). All trials were recorded with a Raspberry Pi 3B + equipped with a V1 camera module (https://www.raspberrypi.org/docu mentation/hardware/camera/) and using Raspivid version 1.3.12 as 1296  972 pixel, 30 frame/s MP4 video files. Automated analysis was carried out with custom scripts written in Bonsai version 2.4-preview (Lopes et al., 2015). A researcher blinded to the treatment assessed and scored the exploration time manually after automated analysis. Discrimination index (DI) was calcu- lated using the following formula: (time spent with altered ob- ject – time spent with unchanged object) / (total time spent exploring objects). BRAIN 2020: Page 7 of 15 | 7 CRISPRa treats temporal lobe epilepsy sg19, but not with sg30, significantly upregulated the expres- sion of the Kcna1 gene. We focused on sg4 and sg19, which induced the highest levels of Kcna1 expression (Supplementary Fig. 1B). When tested in combination, sg4 and sg19 together were also efficacious, but not sg4 and sg30 (Supplementary Fig. 1B). We confirmed that the highest effi- ciency of upregulation of Kcna1 in primary neurons was achieved with sg19 (Fig. 1A and B). Consequently, we gener- ated a construct carrying dCas9-VP160 driven by the Ef1a promoter and either the sg19 targeting the Kcna1 promoter (Kcna1-dCas9A) or a control sgRNA targeting LacZ (Ctrl- dCas9A). Western blot analysis confirmed increased Kv1.1 protein levels in sg19-treated neurons when compared to the sgLacZ control. Importantly, we detected increased levels of glycosylated Kv1.1, corresponding to mature protein, imply- ing normal processing of the upregulated potassium channel (Fig. 1C and D) (Watanabe et al., 2003). spread more efficiently than lentiviruses in the brain paren- chyma. One AAV vector carried the dCas9-VP64 under the control of a TRE, while the other vector included the sg19 (or sgLacZ as a control) element and, to confer specific neur- onal expression, a human synapsin promoter (hSyn) up- stream to an inverted rtTA-t2a-tdTomato cassette flanked by loxP and lox511 sites. Novel Object Recognition Test stract/doi/10.1093/brain/awaa045/5780426 by UCL, London user on 11 March 2020 Exogenous Kcna1 overexpression results in a decrease in neuronal excitability and neurotransmitter release (Heeroma et al., 2009; Wykes et al., 2012). To test the functional effi- cacy of the CRISPRa system, primary neurons were trans- duced at 1 DIV with a lentivirus expressing Kcna1-dCas9A or Ctrl-dCas9A. After 14–16 DIV we used whole-cell patch clamp recordings to analyse neuronal excitability of both ex- perimental groups (Fig. 1G). The maximal firing frequency was significantly decreased in neurons transduced with Kcna1-dCas9A when compared to Ctrl-dCas9A (Fig. 1H). Other excitability parameters sensitive to Kv1.1 were also changed in neurons transduced with Kcna1-dCas9A in com- parison with Ctrl-dCas9A: the current threshold was increased, and action potential width was decreased (Fig. 1I). However, passive membrane properties and other action potential properties were unchanged apart from a de- crease in the input resistance (Supplementary Fig. 2). This change suggests that the effect of expression of the endogen- ous promoter using CRISPRa is different than overexpres- sion using virally delivered cDNA (Heeroma et al., 2009). Kcna1-dCas9A decreases seizure frequency in a mouse model of temporal lobe epilepsy We administered Kcna1-dCas9A in a mouse model of acquired epilepsy. C57BL/6J wild-type animals were injected with kainic acid in the right amygdala (Jimenez-Mateos et al., 2012). This induced a period of status epilepticus, which was quantified by video recording to monitor seizure severity (Supplementary Fig. 5 and Supplementary Video 1). One week later, we injected either Kcna1-dCas9A or Ctrl- dCas9A AAVs in the right ventral hippocampus, and at the same time we implanted wireless EEG transmitters (band- width 1–256 Hz). For these experiments an AAV9 carrying a rtTA-t2a-tdTomato cassette without flanking recombin- ation sites but driven by a Camk2a promoter was delivered to bias expression towards excitatory neurons. After a week Kcna1-dCas9A decreases CA1 pyramidal cell excitability (C and D) Western blots were used to determine the increase in Kv1.1 and gly- cosylated Kv1.1 (glyc) in neurons transduced either with dCas9A and sg19 (red), or sgLacZ (blue). Student’s t-test. (E) MA plots showing log2 fold-change as a function of log2 base mean expression of Kcna1-dCas9A treated neurons with respect to Ctrl-dCas9A. Kcna1 = pink; off-targets = green; all other genes = grey. (F) Messenger RNA expression relative to sgLacZ for each off-target. The expression level of sgLacZ is repre- sented as the dashed line at 1. Multiple Student’s t-tests, each compared to control and corrected for multiple comparison (a = 0.0083). (G) Representative traces of recordings from neurons transduced either with Ctrl-dCas9A (sgLacZ, blue) or Kcna1-dCas9A (sg19, red) and injected with 300 pA steps in current clamp. (H) Average firing rates in response to different current injections for neurons transduced with ctrl- dCas9A or Kcna1-dCas9A. Two-way RM ANOVA. (I) Neuronal and action potential (AP) properties in neurons transduced with ctrl-dCas9A or Kcna1-dCas9A. Student’s t-test. Figure 1 CRISPRa increases endogenous Kcna1 expression reducing neuronal excitability in vitro. (A) Schematic representation of the CRISPRa approach to increasing Kcna1. Primary neurons were transduced with lentivirus 1 day after plating and experiments were performed after 10 days for qPCR and western blotting, and 14 days for electrophysiology. (B) Kcna1 mRNA expression normalized to the control LacZ sgRNA (blue) for the most effective sgRNAs and combinations of different sgRNAs tested in P19 cells (Supplementary Fig. 1). One-way ANOVA followed by Bonferroni multiple comparison test versus sgLacZ. (C and D) Western blots were used to determine the increase in Kv1.1 and gly- cosylated Kv1.1 (glyc) in neurons transduced either with dCas9A and sg19 (red), or sgLacZ (blue). Student’s t-test. (E) MA plots showing log2 fold-change as a function of log2 base mean expression of Kcna1-dCas9A treated neurons with respect to Ctrl-dCas9A. Kcna1 = pink; off-targets = green; all other genes = grey. (F) Messenger RNA expression relative to sgLacZ for each off-target. The expression level of sgLacZ is repre- sented as the dashed line at 1. Multiple Student’s t-tests, each compared to control and corrected for multiple comparison (a = 0.0083). (G) Representative traces of recordings from neurons transduced either with Ctrl-dCas9A (sgLacZ, blue) or Kcna1-dCas9A (sg19, red) and injected with 300 pA steps in current clamp. Kcna1-dCas9A decreases CA1 pyramidal cell excitability Kcna1-dCas9A decreases CA1 pyramidal cell excitability To test the efficacy of CRISPRa in vivo, we subcloned the CRISPRa elements in two separate AAV9 viral vectors that 8 | BRAIN 2020: Page 8 of 15 G. Colasante et al. 8 Figure 1 CRISPRa increases endogenous Kcna1 expression reducing neuronal excitability in vitro. (A) Schematic representation of the CRISPRa approach to increasing Kcna1. Primary neurons were transduced with lentivirus 1 day after plating and experiments were performed after 10 days for qPCR and western blotting, and 14 days for electrophysiology. (B) Kcna1 mRNA expression normalized to the control LacZ sgRNA (blue) for the most effective sgRNAs and combinations of different sgRNAs tested in P19 cells (Supplementary Fig. 1). One-way ANOVA followed by Bonferroni multiple comparison test versus sgLacZ. (C and D) Western blots were used to determine the increase in Kv1.1 and gly- cosylated Kv1.1 (glyc) in neurons transduced either with dCas9A and sg19 (red), or sgLacZ (blue). Student’s t-test. (E) MA plots showing log2 fold-change as a function of log2 base mean expression of Kcna1-dCas9A treated neurons with respect to Ctrl-dCas9A. Kcna1 = pink; off-targets = green; all other genes = grey. (F) Messenger RNA expression relative to sgLacZ for each off-target. The expression level of sgLacZ is repre- sented as the dashed line at 1. Multiple Student’s t-tests, each compared to control and corrected for multiple comparison (a = 0.0083). (G) Representative traces of recordings from neurons transduced either with Ctrl-dCas9A (sgLacZ, blue) or Kcna1-dCas9A (sg19, red) and injected with 300 pA steps in current clamp. (H) Average firing rates in response to different current injections for neurons transduced with ctrl- dCas9A or Kcna1-dCas9A. Two-way RM ANOVA. (I) Neuronal and action potential (AP) properties in neurons transduced with ctrl-dCas9A or Downloaded from https://academic.oup.com/brain/advance-article-abstract/doi/10.1093/brain/awaa045/5780426 by UCL, London user on 11 March 2020 Downloaded from https://academic.oup.com/brain/advance-article-abstract/doi/10.109 Figure 1 CRISPRa increases endogenous Kcna1 expression reducing neuronal excitability in vitro. (A) Schematic representation of the CRISPRa approach to increasing Kcna1. Primary neurons were transduced with lentivirus 1 day after plating and experiments were performed after 10 days for qPCR and western blotting, and 14 days for electrophysiology. (B) Kcna1 mRNA expression normalized to the control LacZ sgRNA (blue) for the most effective sgRNAs and combinations of different sgRNAs tested in P19 cells (Supplementary Fig. 1). One-way ANOVA followed by Bonferroni multiple comparison test versus sgLacZ. Kcna1-dCas9A decreases CA1 pyramidal cell excitability Number of action potentials for each burst showed as mean 6 standard error of the mean (SEM) (middle). Two-way ANOVA. The histogram represents the average number of action potentials for each neuron in the 24 bursts (right). Student’s t-test. CRISPRa treats temporal lobe epilepsy BRAIN 2020: Page 9 of 15 | BRAIN 2020: Page 9 of 15 | 9 CRISPRa treats temporal lobe epilepsy Figure 2 CRISPRa delivered with AAV9 increases endogenous Kcna1 expression and reduces CA1 pyramidal neuron excitabil- Downloaded from https://academic.oup.com/brain/advance-article-abstract/doi/10.109 Downloaded from https://academic.oup.com/brain/advance-article-abstract/doi/10.1093/brain/awaa045/5780426 by UCL, London user on 11 March 2020 Figure 2 CRISPRa delivered with AAV9 increases endogenous Kcna1 expression and reduces CA1 pyramidal neuron excitabil- ity. (A) Schematic representation of the approach for ex vivo quantification of CRISPRa effects. Mice were injected with AAVs at approximately postnatal Day (P)60 and experiments were performed 3 weeks after. (B) Representative live image of Kcna1-dCas9A expression in the hippocam- pus (bregma –2.70; inset = CA1 region, red = tdTomato) (C) Representative traces from CA1 pyramidal neurons, transduced either with Ctrl- dCas9A (sgLacZ, blue) or Kcna1-dCas9A (sg19, red) in pryramidal neurons injected with 280 pA steps in current clamp. (D) Average firing rates in response to different current steps for neurons transduced with Ctrl-dCas9A or Kcna1-dCas9A. Two-way repeated measures ANOVA. (E) Neuronal and action potential (AP) properties in neurons transduced with Ctrl-dCas9A or Kcna1-dCas9A. Maximal firing rate, current threshold to elicit the first action potential, action potential width and resting membrane potential are shown. Student’s t-test. (F) Cumulative frequency (%) of the action potential widths in neurons injected with 280 pA of current. Kolmogorov-Smirnov test for cumulative distributions. (G) Activity clamp protocol to mimic 24 interictal bursts of synaptic input from an epileptic network in neurons transduced with Ctrl-dCas9A or Kcna1-dCas9A (left). Black arrows represent the action potentials missed in the Kcna1-dCas9A neurons. Number of action potentials for each burst showed as mean 6 standard error of the mean (SEM) (middle). Two-way ANOVA. The histogram represents the average number of action potentials for each neuron in the 24 bursts (right). Student’s t-test. To investigate the ability of Kcna1-dCas9 to treat chronic temporal lobe epilepsy we analysed the frequency of generalized tonic-clonic seizures (Racine stage 5) in each animal before and after doxycycline administration using continuous video-EEG recordings (Fig. 3D–F, Supplementary Fig. 7 and Supplementary Video 2). Kcna1-dCas9A decreases CA1 pyramidal cell excitability (H) Average firing rates in response to different current injections for neurons transduced with ctrl- dCas9A or Kcna1-dCas9A. Two-way RM ANOVA. (I) Neuronal and action potential (AP) properties in neurons transduced with ctrl-dCas9A or Kcna1-dCas9A. Student’s t-test. EEG recordings to analyse Kcna1 and dCas9 expression. Injected hippocampi from two mice failed to express dCas9 and were excluded from the analysis. The injected hippo- campi from the remaining nine mice (five Ctrl-dCas9A and four Kcna1-dCas9A) expressed dCas9 and showed a 50% increase in Kcna1 expression in mice transduced with Kcna1-dCas9A compared to the contralateral counterpart, while no difference was detected in Ctrl-dCas9A hippocampi (Fig. 3C). of recovery to allow expression of the constructs, we began continuous video-EEG recording for 2 weeks (baseline) and then continued recording for two further weeks with doxy- cycline administration (Fig. 3A). Immunohistochemistry of the injected hippocampi showed expression of the tdTomato reporter in dentate gyrus granule cells and hippocampal CA3 excitatory neurons, as well as CA1 pyramidal cells (Fig. 3B and Supplementary Fig. 6). We extracted both the ipsilateral and contralateral hippocampi of 11 mice after the Figure 2 CRISPRa delivered with AAV9 increases endogenous Kcna1 expression and reduces CA1 pyramidal neuron excitabil- ity. (A) Schematic representation of the approach for ex vivo quantification of CRISPRa effects. Mice were injected with AAVs at approximately postnatal Day (P)60 and experiments were performed 3 weeks after. (B) Representative live image of Kcna1-dCas9A expression in the hippocam- pus (bregma –2.70; inset = CA1 region, red = tdTomato) (C) Representative traces from CA1 pyramidal neurons, transduced either with Ctrl- dCas9A (sgLacZ, blue) or Kcna1-dCas9A (sg19, red) in pryramidal neurons injected with 280 pA steps in current clamp. (D) Average firing rates in response to different current steps for neurons transduced with Ctrl-dCas9A or Kcna1-dCas9A. Two-way repeated measures ANOVA. (E) Neuronal and action potential (AP) properties in neurons transduced with Ctrl-dCas9A or Kcna1-dCas9A. Maximal firing rate, current threshold to elicit the first action potential, action potential width and resting membrane potential are shown. Student’s t-test. (F) Cumulative frequency (%) of the action potential widths in neurons injected with 280 pA of current. Kolmogorov-Smirnov test for cumulative distributions. (G) Activity clamp protocol to mimic 24 interictal bursts of synaptic input from an epileptic network in neurons transduced with Ctrl-dCas9A or Kcna1-dCas9A (left). Black arrows represent the action potentials missed in the Kcna1-dCas9A neurons. Kcna1-dCas9A decreases CA1 pyramidal cell excitability (C) Quantitative PCR analysis of Kcna1 and dCas9 expression in the ipsilateral hippocampus relative to the contralateral hippo- campus in epileptic mice transduced with either Ctrl-dCas9A or Kcna1-dCas9A at the end of the experiments. Student’s t-test. (D) Left: Raster plot showing all seizures before and after doxycycline administration in 13 mice treated with Ctrl-dCas9A and nine mice treated with Kcna1- dCas9A. Right: Pie charts showing the proportion of animals showing either more or fewer seizures after doxycycline food than during the base- line. Fisher’s exact test. (E) Number of seizures/day before and after doxycycline administration in control-dCas9 (n = 13) and Kcna1-dCas9 (n = 9) treated animals. Two-way ANOVA followed by Bonferroni multiple comparison test. Empty blue circles are animals that died during base- line and excluded from the analysis. (F) Cumulative plot of seizures normalized to the baseline in mice transduced with either ctrl-dCas9A or Kcna1-dCas9A. Two-way ANOVA. stract/doi/10.1093/brain/awaa045/5780426 by UCL, London user on 11 March 2020 period prior to doxycycline administration between the animals treated either with Kcna1-dCas9A or Ctrl- dCas9A (Fig. 3E, P = 0.09 two-way ANOVA followed by Bonferroni multiple comparison test). However, two Ctrl- dCas9A animals, but none of the Kcna1-dCas9A animals, died during the baseline period from severe seizures and were not included in the comparison of seizure frequencies before and after doxycycline (Fig. 3E). We cannot exclude a protective effect of a low level of basal activation of CRISPRa in the Kcna1-dCas9A animals. Other EEG parameters, such as broadband power, seizure duration and EEG power for night-time and day-time periods were not changed by the treatment (Supplementary Figs 5 and 7). Taken together, these results show that Kcna1-dCas9A reduces the probability of tonic-clonic seizure initiation, but otherwise does not change seizure properties recorded in the cortex. observe robust differences in EEG coastline and power in different frequency bands recorded for 1 h after pilocarpine in either group, although there was a non-significant trend for Kcna1-dCas9A animals to show an attenuation in seiz- ure severity (Supplementary Fig. 8). These data argue that the action of Kcna1-dCas9A on experimental epilepsy goes beyond an effect on individual seizures. Kcna1-dCas9A decreases CA1 pyramidal cell excitability Figure 3 CRISPRa-Kcna1 decreases number of seizures in a mouse model of acquired intrac Figure 3 CRISPRa-Kcna1 decreases number of seizures in a mouse model of acquired intractable temporal lobe epilepsy. (A) Schematic representation of the CRISPRa approach used in vivo to treat the intra-amygdala kainic acid (KA) focal model of temporal lobe epi- lepsy. Mice were injected with AAVs 1 week after kainic acid treatment. Baseline EEG recordings were started the following week and continued for 2 weeks. Two further weeks of EEG recordings were performed after doxycycline food supplementation. (B) Representative immunofluores- cence 7 weeks after status epilepticus of neurons transduced with Ctrl-dCas9A 4 weeks after status epilepticus. Scale bars: DG = 250 lm; CA1 = 50 lm. (C) Quantitative PCR analysis of Kcna1 and dCas9 expression in the ipsilateral hippocampus relative to the contralateral hippo- campus in epileptic mice transduced with either Ctrl-dCas9A or Kcna1-dCas9A at the end of the experiments. Student’s t-test. (D) Left: Raster plot showing all seizures before and after doxycycline administration in 13 mice treated with Ctrl-dCas9A and nine mice treated with Kcna1- dCas9A. Right: Pie charts showing the proportion of animals showing either more or fewer seizures after doxycycline food than during the base- line. Fisher’s exact test. (E) Number of seizures/day before and after doxycycline administration in control-dCas9 (n = 13) and Kcna1-dCas9 (n = 9) treated animals. Two-way ANOVA followed by Bonferroni multiple comparison test. Empty blue circles are animals that died during base- line and excluded from the analysis. (F) Cumulative plot of seizures normalized to the baseline in mice transduced with either ctrl-dCas9A or Kcna1-dCas9A. Two-way ANOVA. Figure 3 CRISPRa-Kcna1 decreases number of seizures in a mouse model of acquired intractable temporal lobe epilepsy. (A) Schematic representation of the CRISPRa approach used in vivo to treat the intra-amygdala kainic acid (KA) focal model of temporal lobe epi- lepsy. Mice were injected with AAVs 1 week after kainic acid treatment. Baseline EEG recordings were started the following week and continued for 2 weeks. Two further weeks of EEG recordings were performed after doxycycline food supplementation. (B) Representative immunofluores- cence 7 weeks after status epilepticus of neurons transduced with Ctrl-dCas9A 4 weeks after status epilepticus. Scale bars: DG = 250 lm; CA1 = 50 lm. Kcna1-dCas9A decreases CA1 pyramidal cell excitability Animals treated with Kcna1-dCas9A showed a significant reduction in the number of seizures after doxycycline was added to the food (Fig. 3D–F). The number of seizures after doxycycline administration decreased in eight of nine animals treated with Kcna1-dCas9A compared to only 5 of 13 animals treated with Ctrl-dCas9 (Fig. 3D). Only Kcna1-dCas9A treated animals showed a significant de- crease in the number of seizures per day after doxycycline administration (Fig. 3E and F). There was no significant difference in seizure frequencies during the baseline Downloaded from https://academic.oup.com/brain/advance-article-abstract/doi/10.1093/brain/awaa045/5780426 by UCL, London user on 11 March 2020 BRAIN 2020: Page 10 of 15 G. Colasante et al. 10 Figure 3 CRISPRa-Kcna1 decreases number of seizures in a mouse model of acquired intractable temporal lobe epilepsy. (A) Schematic representation of the CRISPRa approach used in vivo to treat the intra-amygdala kainic acid (KA) focal model of temporal lobe epi- lepsy. Mice were injected with AAVs 1 week after kainic acid treatment. Baseline EEG recordings were started the following week and continued for 2 weeks. Two further weeks of EEG recordings were performed after doxycycline food supplementation. (B) Representative immunofluores- cence 7 weeks after status epilepticus of neurons transduced with Ctrl-dCas9A 4 weeks after status epilepticus. Scale bars: DG = 250 lm; CA1 = 50 lm. (C) Quantitative PCR analysis of Kcna1 and dCas9 expression in the ipsilateral hippocampus relative to the contralateral hippo- campus in epileptic mice transduced with either Ctrl-dCas9A or Kcna1-dCas9A at the end of the experiments. Student’s t-test. (D) Left: Raster plot showing all seizures before and after doxycycline administration in 13 mice treated with Ctrl-dCas9A and nine mice treated with Kcna1- dCas9A. Right: Pie charts showing the proportion of animals showing either more or fewer seizures after doxycycline food than during the base- line. Fisher’s exact test. (E) Number of seizures/day before and after doxycycline administration in control-dCas9 (n = 13) and Kcna1-dCas9 (n = 9) treated animals. Two-way ANOVA followed by Bonferroni multiple comparison test. Empty blue circles are animals that died during base- line and excluded from the analysis. (F) Cumulative plot of seizures normalized to the baseline in mice transduced with either ctrl-dCas9A or Kcna1-dCas9A. Two-way ANOVA. Downloaded from https://academic.oup.com/brain/advance-article-abstract/doi/10.1093 ber of seizures in a mouse model of acquired intractable temporal lobe epilepsy. Discussion Although CRISPR has attracted intense interest as a possible treatment for inherited or acquired genetic disorders, it has, hitherto, received much less attention as a potential tool to treat acquired non-genetic diseases. The overwhelming ma- jority of epilepsy cases, which represent an enormous disease burden, are not thought to be due to single gene mutations but are acquired during life, often secondary to a variety of brain insults such as infections, strokes and injuries (Kwan et al., 2011; Tang et al., 2017). Here we have shown that CRISPRa can be used to increase endogenous Kcna1 expres- sion to modulate neuronal activity, to reduce seizure initi- ation and to rescue behavioural and transcriptomic abnormalities in a mouse model of chronic temporal lobe epilepsy. This approach can potentially be used to regulate the expression of any gene, opening the way to treating many other neurological diseases associated with altered transcription. Several studies have documented extensive changes in gene expression in different mouse seizure models (Motti et al., 2010; Okamoto et al., 2010; Winden et al., 2011; Hansen et al., 2014; Hawkins et al., 2019). We asked if Kcna1- dCas9A was able to rescue transcriptomic changes in the temporal lobe epilepsy model by performing RNAseq ana- lysis on hippocampi from sham control animals injected with Ctrl-dCas9A and epileptic animals injected either with Ctrl-dCas9A or with Kcna1-dCas9A. A comparison of gene expression between sham control (sham control Ctrl- dCas9A group) and epileptic animals (epileptic Ctrl-dCas9A group) revealed 6654 genes whose expression was altered (Fig. 4D). Kcna1-dCas9A treatment (epileptic Kcna1- dCas9A group) (Fig. 4E) rescued expression of 2742 of them (Fig. 4D and E). Consequently, the transcriptional pro- file of treated hippocampi (epileptic Kcna1-dCas9A group) was more similar to sham control mice (sham control Ctrl- dCas9A group), with only 1465 genes altered (Fig. 4D), than to epileptic Ctrl-dCas9A-treated mice. Sample distribu- tion and K-means clustering along the first two principal components (PC1, PC2) failed to discriminate between sham control Ctrl-dCas9A and epileptic Kcna1-dCas9A groups, while the epileptic Ctrl-dCas9A group was distinct (Supplementary Fig. 10A). Gene ontology analysis of the 2742 genes rescued by Kcna1-dCas9A treatment (Fig. 4F) showed that it counteracted changes implicated in neurode- generation and apoptosis (GO ID: negative regulation of anti-apoptotic process, hippocampus development, positive regulation of dendrite development, neuronal development with synapse formation, axon extension, axonogenesis, axon guidance). Kcna1-dCas9A rescues cognitive deficits and mitigates dysregulation of gene expression Cognitive co-morbidities are an important feature of many forms of intractable epilepsy. We therefore asked if Kcna1- dCas9A treatment rescues behavioural deficits in our chronic intra-amygdala kainic acid model of temporal lobe epilepsy. We used two behavioural paradigms, one directly related to hippocampal function (Object Location Test) and the other more related to prefrontal cortex function (Novel Object Recognition Test) as previously described (Cho et al., 2015). In the Object Location Test, no impairment in performance, We complemented the chronic epilepsy study by looking at the effect of Kcna1-dCas9A treatment on acute seizures evoked by a different mechanism, focal pilocarpine injection in the visual cortex before and after doxycycline administra- tion (Lieb et al., 2018; Magloire et al., 2019). We did not BRAIN 2020: Page 11 of 15 | 11 CRISPRa treats temporal lobe epilepsy We compared the pattern of gene expression changes in our model with previously published studies (Motti et al., 2010; Okamoto et al., 2010; Winden et al., 2011; Hansen et al., 2014; Hawkins et al., 2019); however, this failed to reveal a consistent signature across heterogeneous models of epilepsy (Supplementary Fig. 10B). Nevertheless, when restricting the comparison to focal kainic acid injection (Motti et al., 2010), we identified 388 common deregulated genes, 165 of which were rescued in Kcna1-dCas9A treated mice (Supplementary Figs 10C, D and 11). as measured by the discrimination index, before and after doxycycline, were observed in sham control animals treated with either Ctrl-dCas9A or Kcna1-dCas9A (Fig. 4A–C). However epileptic mice showed a deficit compared to con- trol mice (Ctrl-dCas9A sham control versus epileptic, P = 0.01; Kcna1-dCas9A sham control versus epileptic, P = 0.006; two-way ANOVA followed by Bonferroni mul- tiple comparison test). This deficit was completely rescued only by Kcna1-dCas9A treatment (Fig. 4C). In contrast, no significant differences were observed in the Novel Object Recognition Test either between sham control and epileptic animals, or following treatment with either Ctrl-dCas9A or Kcna1-dCas9A (Supplementary Fig. 9) (Cho et al., 2015). These data suggest that while Kcna1-dCas9A does not have adverse effects on hippocampal function in non-epileptic mice, it is able to restore function in epileptic mice. Discussion Several genes associated with neuronal ac- tivity were upregulated (GO ID: ion transport, calcium transmembrane, chloride transmembrane transport, potas- sium ion transport). The treatment also re-established ex- pression of genes implicated in synapse function (GO ID: neurotransmitter secretion, synapse organization, dendritic spine maintenance, long term synaptic potentiation, trans- mission of nerve impulse, receptor recycling) and glutamater- gic transmission (GO ID: positive regulation of glutamatergic synaptic transmission, regulation of NMDA selective glutamate receptor activity). At present, the main obstacles to clinical translation of the CRISPR/Cas9 toolbox are absence of long-term data on po- tential immunogenicity of the bacterial nuclease in humans and possible off-target effects that have not been detected by transcriptomic analysis (Kosicki et al., 2018). Although in this study a non-mammalian nuclease was used, which can evoke an immune response in the primate CNS (Samaranch et al., 2014), CNS disorders attributable to antibodies against nuclear neuronal proteins are rare, and rare forms of autoimmune encephalitis generally involve membrane pro- teins (Platt et al., 2017). The doxycycline transactivator pro- tein could also potentially evoke an immune response, although new inducible systems are in consideration for clin- ical translation (Das et al., 2016; Kundert et al., 2019). Cas9 has already been delivered with AAV in rodents and has been shown to induce a mild cellular response, but this has not been reported in the brain (Chew et al., 2016). CRISPRa is, in principle, less likely to have deleterious off-target effects than CRISPR gene editing because it does not cleave DNA (La Russa and Qi, 2015; Zheng et al., 2018), but fur- ther research is necessary. d d f Among distinct advantages of CRISPRa over exogenous gene delivery is the possibility to select one or more sgRNAs to tailor the exact level of gene expression independently by the number of viral copies effectively entered within each neuron. In addition, several sgRNAs could in principle be BRAIN 2020: Page 12 of 15 G. Colasante et al. 12 Figure 4 CRISPRa-Kcna1 rescues cognitive deficits and mitigates transcriptomic changes in a mouse model of acquired in tractable temporal lobe epilepsy. (A) Experimental plan for behaviour and transcriptomic analysis. Mice were injected with AAVs a wee after kainic acid (KA) treatment. Baseline behavioural tests were performed 2 weeks after AAV injection. Further behavioural tests were pe formed 2 weeks after doxycycline food supplementation. Discussion Further refinements can be consid- ered, such as the use of an inducible promoter to allow the therapy to be switched off (Wykes and Lignani, 2018), which would not be possible with a gene editing strategy. We have shown that is possible to upregulate an endogen- ous ion channel in vitro and in vivo and thereby to signifi- cantly change neuronal excitability (Figs 1 and 2). This result underlines an important paradigm of neuronal net- work, that relatively small changes in gene expression can robustly alter neuronal firing rates. We argue that these small changes in gene activation could be more sustainable long-term for a permanent modification of neuronal net- work activity without triggering adverse side effects such as protein accumulation or widespread transcriptomic compen- sation that could lead to less physiological alterations. Interestingly, the target gene upregulation was slightly lower when two sgRNAs were combined than when either of the sgRNAs was delivered on its own. This might be due to a relative reduction in transduction or transfection efficiency of each single sgRNA. We hypothesize that some cells/neu- rons received just one of the two sgRNAs and therefore the final output is a decrease in the total efficiency. This hypoth- esis is supported by the even smaller upregulation observed when three sgRNAs were combined together. A further un- expected finding was a decrease in input resistance observed in neurons transduced with Kcna1-dCas9A. This change was not observed with exogenous overexpression (Heeroma et al., 2009; Wykes et al., 2012) but potentially contributed to the therapeutic effect. Among possible explanations is that a homeostatic compensation by other channels was trig- gered by the increase of expression of the endogenous chan- nel. We should, however, point out that Wykes et al. (2012) focused exclusively on layer 5 pyramidal neurons. Many studies have reported transcriptomic changes associ- ated with epilepsy in different mouse models (Motti et al., 2010; Okamoto et al., 2010; Winden et al., 2011; Hansen et al., 2014; Hawkins et al., 2019). However, no single gene alteration recurs across all available databases (Supplementary Fig. 9B). This inconsistency can be ascribed to differences in RNA-Seq technologies, epilepsy models, ages of animals, and delay after the insult. Our data add to the available data on gene expression, and provide, to our knowledge, the first evidence that gene therapy for epilepsy can correct dysregulation of a significant subset of genes. Discussion BRAIN 2020: Page 13 of 15 | 1 CRISPRa treats temporal lobe epilepsy 13 Importantly, in the present study we observed not only a decrease of seizure number but also the rescue of a behav- ioural co-morbidity and transcriptomic alterations that ac- company epileptogenesis (Figs 3 and 4). Although the effect of our treatment on the absolute number of seizures was relatively modest, this is the first proof-of-principle that CRISPRa can be used in vivo to decrease spontaneous seiz- ures. Thus far, this effect on seizures was demonstrated only in one model of chronic epilepsy. Further studies will be required to test other models of epilepsy before clinical translation. A single AAV system with a small dCAS9, pos- sibly using multiple injection sites, such as in the contralat- eral hippocampus and amygdala, would potentially further improve the efficacy of suppressing seizures. Nevertheless, even the modest effect on number of seizures produced by this treatment was able to correct a behavioural co-morbid- ity. We tested the effects of epilepsy and dCas9A treatment in two behavioural tests previously studied in epileptic ani- mals (Cho et al., 2015), but several other tests, beyond the scope of this study, could be used to assess the extent of res- cue of co-morbidities (Heinrichs and Seyfried, 2006). used in combination to control the transcription of hetero- multimeric proteins such as GABAA or NMDA receptors, or of multiple genes in a signalling pathway. Finally, whilst ex- ogenous gene delivery is limited by the viral packaging cap- acity, CRISPRa can potentially be applied to control the activity of any gene irrespective of its length (Konermann et al., 2015). used in combination to control the transcription of hetero- multimeric proteins such as GABAA or NMDA receptors, or of multiple genes in a signalling pathway. Finally, whilst ex- ogenous gene delivery is limited by the viral packaging cap- acity, CRISPRa can potentially be applied to control the activity of any gene irrespective of its length (Konermann et al., 2015). Although the present study made use of two AAVs to allow inducible activation of CRISPRa and expression of a fluorescent reporter protein, for clinical translation these fea- tures would not be required, as it should be possible to package both the dCas9 and the sgRNA in a single AAV to simplify clinical delivery. Discussion Although we detected an upregulation of Kcna1 in Kcna1- dCas9A injected hippocampus when compared to Ctrl- dCas9A injected tissue (Fig. 3C), we did not observe a clear increase in the expression levels of Kcna1 using RNA-Seq (Supplementary Fig. 10). This may reflect a limitation of RNA-Seq due to a relatively low number of reads for Kcna1. Importantly, the effect of CRISPRa-mediated Kcna1 upre- gulation on generalized seizures may reflect an overall rescue of the pathology, including co-morbidities and gene expres- sion, and not only an attenuation of acute seizures. In conclusion, CRISPR-mediated control of gene expression can be successfully exploited to modulate neuronal activity and to mitigate seizures and behavioural co-morbidity in an experimental model of intractable temporal lobe epilepsy. When moving to in vivo experiments we had to switch from lentivirus to AAV9 to transduce a larger area (whole hippocampus), which is not possible with lentivirus. We used an hSyn promoter in vivo to ensure specific expression in neurons and also used a dual-AAV system as SpdCas9 does not fit in a single AAV together with the sgRNA. For the same reason, we chose to use dCas9VP64 instead of VP160 used in vitro. Because of all these changes in the constructs, we tested the efficiency of the AAV9 in reducing neuronal fir- ing ex vivo, showing a lower degree of firing reduction but still significantly lower than in control neurons (Fig. 2). Discussion At the end of the experiment hippocampi were collected for transcriptomic analysi (B) Graphical representation of the Object Location Test (OLT) and Novel Object Recognition Test (NORT). (C) Discrimination index for sha control and epileptic animals before and after (green box) doxycycline in mice treated either with Ctrl-dCas9A or Kcna1-dCas9A. Two-wa ANOVA followed by Bonferroni multiple comparison test. (D) Volcano plots showing statistical significance (–log10 P-value) as a function of fold change in gene expression (log2FC), comparing pairs of datasets as indicated above each plot. Genes showing P 5 0.05 difference in expressio (–log10 P-value 4 1.3) are highlighted in red. (E) Venn diagram showing the fraction of differentially regulated genes in epileptic Ctrl-dCas9A treated mice (E Ctrl-dCas9A) which were rescued in Kcna1-dCas9A-treated mice (E Kcna1-dCas9A). (F) Histogram displaying representativ gene ontology (GO) categories functionally enriched among the 2742 genes that were differentially expressed in epileptic control mice (E Ctr dCas9A) versus treated mice (E Kcna1-dCas9A). Downloaded from https://academic.oup.com/brain/advance-article-abstract/doi/10.1093/brain/awaa045/5780426 by UCL, London user on 11 March 2020 Downloaded from https://academic.oup.com/brain/advance-article-abstract/doi/10.1093 Figure 4 CRISPRa-Kcna1 rescues cognitive deficits and mitigates transcriptomic changes in a mouse model of acquired in- tractable temporal lobe epilepsy. (A) Experimental plan for behaviour and transcriptomic analysis. Mice were injected with AAVs a week after kainic acid (KA) treatment. Baseline behavioural tests were performed 2 weeks after AAV injection. Further behavioural tests were per- formed 2 weeks after doxycycline food supplementation. At the end of the experiment hippocampi were collected for transcriptomic analysis. (B) Graphical representation of the Object Location Test (OLT) and Novel Object Recognition Test (NORT). (C) Discrimination index for sham control and epileptic animals before and after (green box) doxycycline in mice treated either with Ctrl-dCas9A or Kcna1-dCas9A. Two-way ANOVA followed by Bonferroni multiple comparison test. (D) Volcano plots showing statistical significance (–log10 P-value) as a function of fold- change in gene expression (log2FC), comparing pairs of datasets as indicated above each plot. Genes showing P 5 0.05 difference in expression (–log10 P-value 4 1.3) are highlighted in red. (E) Venn diagram showing the fraction of differentially regulated genes in epileptic Ctrl-dCas9A- treated mice (E Ctrl-dCas9A) which were rescued in Kcna1-dCas9A-treated mice (E Kcna1-dCas9A). (F) Histogram displaying representative gene ontology (GO) categories functionally enriched among the 2742 genes that were differentially expressed in epileptic control mice (E Ctrl- dCas9A) versus treated mice (E Kcna1-dCas9A). References Kullmann DM, Schorge S, Walker MC, Wykes RC. Gene therapy in epilepsy-is it time for clinical trials? Nat Rev Neurol 2014; 10: 300–4. Adli M. The CRISPR tool kit for genome editing and beyond. Nat Commun 2018; 9: 1911. Agostinho AS, Mietzsch M, Zangrandi L, Kmiec I, Mutti A, Kraus L, et al. Dynorphin-based “release on demand” gene therapy for drug-resistant temporal lobe epilepsy. EMBO Mol Med 2019; 11: e9963. Kundert K, Lucas JE, Watters KE, Fellmann C, Ng AH, Heineike BM, et al. Controlling CRISPR-Cas9 with ligand-activated and ligand- deactivated sgRNAs. 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A randomized, controlled cross-over trial of dermally-applied lavender (Lavandula angustifolia) oil as a treatment of agitated behaviour in dementia
BMC complementary and alternative medicine
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© 2013 O’Connor et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: Daniel.OConnor@monash.edu School of Psychology and Psychiatry, Monash University, Kingston Centre, Warrigal Road, Cheltenham, Victoria 3192, Australia O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 Open Access Open Access A randomized, controlled cross-over trial of dermally-applied lavender (Lavandula angustifolia) oil as a treatment of agitated behaviour in dementia Daniel W O’Connor*, Barbara Eppingstall, John Taffe and Eva S van der Ploeg Daniel W O’Connor*, Barbara Eppingstall, John Taffe and Eva S van der Ploeg Abstract Background: Lavender essential oil shows evidence of sedative properties in neurophysiological and animal studies but clinical trials of its effectiveness as a treatment of agitation in people with dementia have shown mixed results. Study methods have varied widely, however, making comparisons hazardous. To help remedy previous methodological shortcomings, we delivered high grade lavender oil in specified amounts to nursing home residents whose agitated behaviours were recorded objectively. Methods: 64 nursing home residents with frequent physically agitated behaviours were entered into a randomized, single-blind cross-over trial of dermally-applied, neurophysiologically active, high purity 30% lavender oil versus an inactive control oil. A blinded observer counted the presence or absence of target behaviours and rated participants’ predominant affect during each minute for 30 minutes prior to exposure and for 60 minutes afterwards. Results: Lavender oil did not prove superior to the control oil in reducing the frequency of physically agitated behaviours or in improving participants’ affect. Conclusions: Studies of essential oils are constrained by their variable formulations and uncertain pharmacokinetics and so optimal dosing and delivery regimens remain speculative. Notwithstanding this, topically delivered, high strength, pure lavender oil had no discernible effect on affect and behaviour in a well-defined clinical sample. Trial registration: Australian and New Zealand Clinical Trials Registry (ACTRN 12609000569202) Trial registration: Australian and New Zealand Clinical Trials Registry (ACTRN 12609000569202) Keywords: Lavender, Dementia, Behaviour, Mood, Randomised controlled trial Background administered lavender or sunflower oils by vapour for an hour each night to 70 nursing home residents with marked dementia and agitation [13]. After the oils were adminis- tered in random order for three week periods, scores on the Cohen Mansfield Agitation Inventory (CMAI) fell by 7% with lavender compared to 1% with sunflower oil. Optimal dosing regimens and formulations have yet to be established [20]. When lavender oil is diffused via droplets or nebuliser, as happened in five of the six previous studies, the amounts inhaled by an individual nursing home resident are likely to vary according to dispersion rates, room size, ventilation patterns and the person’s respiratory capacity. While central nervous system activity might result from respiratory, olfactory and/or transdermal delivery, a limited evidence base suggests that topical application achieves much higher plasma levels of linalool and linalyl acetate than inhalation (nothing appears to be known of olfactory delivery) [11]. Known volumes of oil can be applied to the skin for specified periods to readily access- ible and relatively absorbent areas [21], leaving residents free to move about as they please. Treatment will fail, how- ever, if the quantities applied are too small to be effective, regardless of mode of administration. When applied topically, lavender is typically prescribed in Australia as a 2.5% lotion but there is no evidence to support this choice. Doses should therefore be substantial so that treatment effects are not missed. h d d d dd h h Four other controlled trials were less positive. Snow et al. pinned sachets infused with lavender, thyme or neutral oils for several hours daily to the collars of seven dementia unit residents [14]. Treatments were rotated every two weeks while nurses completed the CMAI on alternate days. There were no significant changes in scores with either lavender or thyme oil. Smallwood et al. randomly allocated 21 severely demented hospital patients to a lavender oil massage, neutral oil massage or vaporised lavender oil coupled with conversation [15]. Treatments were adminis- tered twice weekly at different times of day for unspecified periods. Videotapes made four times daily revealed no significant differences in behaviour. Gray and Clair studied 13 confused nursing home residents who were reluctant to swallow medications [16]. They were randomly rotated through exposures on four occasions each to lavender oil, sweet orange oil, tea tree oil and no aroma. Background properties are significant with 3% inspired linalool reducing spontaneous motor activity to a greater degree than 1 mg/kg of parenteral diazepam [6]. In humans, lavender has been shown to lower plasma cortisol levels [7,8]; reduce the need for post-operative analgesia [9], and exert anti- anxiety effects equivalent to those of lorazepam 0.5 mg daily [10]. It has no known adverse effects in customary, and even substantial, doses [11]. The fragrant oil extracted from lavender (Lavandula) is used as a relaxant in many aged care facilities. While evidence in support of its use is mixed, its potential for sedative and anticonvulsant activity is clear. After rapid absorption via skin or respiration [1], lavender and its principal components, linalool and linalyl acetate, enter the brain where they act as depressants, inhibiting glutamate and GABA receptor binding [2,3]. In an effort to find alternatives to psychotropic medica- tions, there have been several controlled trials of lavender oil as a treatment of agitated behaviours in nursing home residents with dementia. Two trials had promising results. In the first, Holmes et al. sprayed a dementia ward with either 2% lavender oil or water for two hours daily on alternating days [12]. All 15 participants had severe From a behavioural perspective, lavender reduces conflict between mice; dampens their motor behaviour, and coun- ters the stimulating effects of caffeine [4,5]. Its sedative O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 Page 2 of 7 Page 2 of 7 condition should involve a similar amount of time and physical contact with participants [20]. dementia and daily agitation. When an observer wearing a nose clip rated participants’ behaviours on the Pittsburgh Agitation Scale, scores were 20% lower when exposed to lavender compared to water. In the second study, Lin et al. administered lavender or sunflower oils by vapour for an hour each night to 70 nursing home residents with marked dementia and agitation [13]. After the oils were adminis- tered in random order for three week periods, scores on the Cohen Mansfield Agitation Inventory (CMAI) fell by 7% with lavender compared to 1% with sunflower oil. dementia and daily agitation. When an observer wearing a nose clip rated participants’ behaviours on the Pittsburgh Agitation Scale, scores were 20% lower when exposed to lavender compared to water. In the second study, Lin et al. Background Cotton balls infused with the oils were pinned to subjects’ collars 20 minutes before their medications were dispensed. Videotapes of residents’ responses to medication adminis- tration showed no significant differences in behaviour during any condition. Finally, Fujii et al. found no changes in blinded behaviour ratings after lavender oil was applied three times daily over a four week period to the collars of 14 out of 28 long-term patients with dementia [17]. The current study was designed to address these meth- odological issues by means of a single-blinded, randomised, controlled cross-over trial of high strength, certified pure lavender oil with proven neurophysiological activity. It was administered topically to nursing home residents with significant, persistent physically agitated behaviours that were counted by trained observers before and after application. Physically agitated behaviours were chosen in preference to physically aggressive and verbally agitated behaviours because of their higher frequency and greater ease of measurement. It was hypothesised that lavender would be associated with a significantly greater reduction in selected behaviours than a neutral control oil. Since lavender appears to act as a sedative, its calming effects on behaviour should be accompanied by an evident improve- ment in emotional well-being. Measures of positive and negative affect were therefore added as outcomes. Differences in lavender formulation, delivery methods, participant numbers, selection criteria, outcome measures and time frames make it almost impossible to compare reports [18]. It is not clear, therefore, why lavender has proved effective in two earlier studies but not in four others. Such a varied evidence base concerning a safe, inexpensive and readily implemented treatment makes it important to conduct further trials using optimal research methods [18]. Measures d A discretely positioned research assistant recorded if the selected target behaviour was present (one point) or absent (zero points) during each minute giving a maximum score of one point per minute of observation. Results were computed as mean counts over the three 30-minute observation periods. Inter-rater reliability was high with an average Kappa of 0.98 over five training sessions. This time-sampling method has been used successfully in our previous studies [24]. Materials Four samples of 100% pure lavender oil (Lavandula angustifolia) were submitted for electrophysiological ana- lysis to the Department of Pharmacology and Toxicology, Otago University, New Zealand, using established methods [3]. A sample sourced from the Ukraine out-performed others in reducing synaptic activity and membrane excit- ability in cultured rat embryo pyramidal cells and was therefore selected for use in this trial (Prof. George Lees, personal communication). On preliminary testing, 1 ml of 30% lavender in jojoba oil proved to be the maximum feasible volume and concentration that could be delivered topically. Jojoba oil, which had no electrophysiological activity, was used as a control. Observers also used the Philadelphia Geriatric Center Affect Rating Scale to record the predominant type of affect (pleasure, contentment, interest, anger, sadness, anxiety/ fear) evident during every minute [25]. The predominant affect was rated as one and all others were rated zero giving a maximum score of one point per minute of obser- vation. Results were averaged as described above. The mean Kappa for inter-rater reliability was 0.92. For reasons of brevity, results for pleasure, contentment and interest are categorized here as positive affects and those for anger, sadness and anxiety/fear as negative affects. Study design h f ll The full protocol of this randomized, placebo-controlled, single-blind cross-over trial has been described previously [23]. Participants were allocated randomly by the project manager using an Excel random number generator to either a lavender or control study condition with no pre-set blocking. They were later crossed over to the other condition. To capture any cumulative effects, each of the two experimental conditions comprised three exposures over a one week period with a four-day washout period between them. Treatments were administered at times when nursing staff reported that the selected physically agitated behaviour was most likely to be present, excluding times of personal nursing care. To minimise confounding by intercurrent illness and medication changes, the trial was limited to a two-week period and nursing and medical staff were asked not to alter participants’ psychotropic medications if possible. Other measures included the Mini-Mental State Examin- ation (MMSE) and the Cohen-Mansfield Agitation Inven- tory (CMAI) [26,27]. To identify a suitable behaviour for use as an outcome measure, the CMAI was completed at baseline by nursing staff who rated physically agitated behaviours (e.g. pacing) on a seven-point scale ranging from “occurs never” to “occurs several times an hour” in the previous fortnight. Participants Study participants were recruited from eight specialist psychogeriatric nursing homes and three private nursing homes in Melbourne, Australia, between 2009 and 2011. The following criteria were required for inclusion: (i) a rating by nursing staff of at least mild dementia on the Clinical Dementia Rating scale [22]; (ii) a physically agitated behaviour that occurred at least several times each day in daylight hours, at times other than during nursing care, to a degree that required staff intervention; (iii) an assessment by the nursing staff, visiting medical practitioner and/or psychiatrist that the behaviour was not due primarily to Ideally, studies in this field require that: (i) participants demonstrate one or more behavioral symptom to a spe- cified degree and frequency; (ii) treatments are deployed in time frames and settings when the behavioural symptom is most evident; (iii) behaviours are counted directly to limit the bias inherent in staff-completed rating scales; (iv) observers are blinded as much as possible, (v) and allowance is made for non-specific placebo effects [19]. For studies involving fragrance, researchers should wear nose clips or masks to maintain blinding and the control O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 Page 3 of 7 O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 Page 3 of 7 observed for 30 minutes before and 60 minutes after application [1]. To maintain observer blinding, nurses applying the oil wore a nose clip and research assistants, who completed the observations, applied a mixture of essential oils to their upper lip to disguise lavender’s fragrance. It was not considered practicable or desirable to attempt to blind participants, all of whom had marked cognitive impairment, to the treatment condition. pain, physical illness, depression or psychosis; (iv) residence in the facility for at least three months, and (v) consent to study participation by the next of kin or guardian. Exclu- sion criteria included: (i) an acute, life-threatening illness; (ii) a variable psychotropic medication regime, and (iii) a medical condition that precluded the use of topical oils. No attempt was made to distinguish one type of dementia from another in this severely disabled group. The trial was approved by all relevant committees including the Monash University Human Research Ethics Committee. Written consent was obtained from the next of kin or legal guardians of all participants. Analysis l Sample size was calculated with respect to the primary outcome measure of physically agitated behaviour for a two-sided hypothesis test with a Type I error rate of 0.05 and power set at 90%, based on data from an earlier repeated measures study of simulated family presence versus music therapy in a similar population and setting [24]. In that study, simulated family presence had an effect size of 0.45 relative to the control condition. However, given the mixed evidence supporting lavender, it seemed prudent to apply the smaller effect size of 0.32 for music therapy. It was estimated that 77 participants might be required to detect a difference of this magnitude. At the same time, there were 18 data points for every participant (6 applications x 3 summary behaviour and affect counts The lavender and control oils were stored in identical vials, marked as A or B. Only a single researcher, who had no other involvement in the study, was aware of the allocation. A nursing staff member then massaged 1 ml of either the lavender or control oil into both forearms for one minute each, giving a total of 2 mls per session. Since lavender plasma levels peak after 20 minutes, and are barely detectable after 90 minutes, participants were Page 4 of 7 O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 Assessed for eligibility (n= 440) Excluded (n=374) Inclusion criteria not met (n= 361) Died (n=10) Relocated (n=2) Analysed (n=37) Refused (n=1) Allocated to lavender first (n=38) Received lavender (n=38) Died (n=1) Allocated to placebo first (n=28) Received placebo (n=28) Analysed (n=27) Allocation Analysis Follow-Up Randomized (n=66) Enrollment Figure 1 Study Flow Chart. Assessed for eligibility (n= 440) Allocated to lavender first (n=38) Received lavender (n=38) Allocated to placebo first (n=28) Received placebo (n=28) Figure 1 Study Flow Chart. observation (with numbers) included pacing (55), repetitive mannerisms (5), general restlessness (2), disrobing (1) and intrusiveness (1). per application) which generated considerable statistical power for only a limited number of analyses. The behaviour and affect scores were over-dispersed relative to Poisson expectations and were therefore mod- elled using random effects negative binomial regression, taking account of participants’ age, gender and dementia severity. The results of the analyses are shown as mean scores and incidence rate ratios for completed cases. Analysis l Ratios over one signify an increase in a mean value, and ratios below one signify a decrease in that value, with respect to a reference condition. Ratios are displayed for treatment (lavender versus the control reference condition), time (post-exposure versus the pre-exposure reference condition) and treatment-time interaction. For reasons of clarity, raw results are listed in Table 1 and shown graphically in Figures 2a and 2b. Findings of the binomial regression analyses are presented in Table 2. Table 1 and Figure 2a show that agitated behaviour counts were lower in the lavender condition than in the control condition. While this difference approached statistical significance (p = 0.057), the lower counts were Table 1 Mean (SD) behaviour and affect scores before and after exposure to lavender and control oils Behaviour Affect Positive Negative Pre- exposure Lavender 16.1 (10.6) 7.2 (10.2) 0.9 (3.5) Control 16.9 (10.0) 6.7 (9.7) 1.2 (4.5) First 30 minutes post-exposure Lavender 14.5 (10.8) 7.0 (10.1) 0.9 (3.9) Control 16.0 (10.4) 6.4 (9.9) 1.0 (3.9) Second 30 minutes post-exposure Lavender 14.4 (10.6) 6.7 (10.2) 0.7 (3.9) Control 15.5 (10.7) 6.3 (9.6) 0.9 (3.8) Table 1 Mean (SD) behaviour and affect scores before and after exposure to lavender and control oils Table 1 Mean (SD) behaviour and affect scores before and after exposure to lavender and control oils Results Figure 1 shows the processes of participant selection, enrolment and follow-up. Finding 66 people suitable for randomization entailed screening 440 residents, most of whom failed to demonstrate sufficient behavioural dis- turbance. Once the trial began, all but two participants completed both study conditions. Most participants were female (59%); very old (mean age 77.6 years, S.D. 9.4); markedly cognitively impaired (mean MMSE score 6.1, S.D. 7.0), and physically agitated (mean CMAI score 40.3, S.D. 18.8). The behaviours selected for O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 Page 5 of 7 Behaviour score Positive affect score b a Figure 2 Mean scores before and after exposure to lavender and control oils. (a) Behaviour. (b) Positive Affect. Behaviour score Behaviour score Positive affect score than control periods but not to significantly different degree. Counts then fell with time, regardless of treat- ment condition, but this difference was also not significant. evident prior to exposure to lavender, pointing away from a true treatment effect. Similarly, behavior counts fell with time to a statistically significant degree but this occurred regardless of condition. There were no significant treat- ment-time interactions. No dermatological or other possible or probable adverse effects were noted as a result of exposure to either the lavender or control oils. Results were similar for affect ratings (Table 1, Figure 2b). O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 Page 5 of 7 http://www.biomedcentral.com/1472-6882/13/315 Discussion results while five others, including the present one, have not. Without a consistent proprietary formulation and sound pharmacokinetic data, of the kind now reported routinely for new pharmaceutical products, there may be little value in further clinical trials. Counts of agitated behaviours were lower during exposure to lavender than to the control oil but this occurred before and after exposure. The observed differences were most probably due to chance. Counts of agitated behaviours also declined over the 90-minute observation period, irre- spective of experimental condition, consistent with a spon- taneous remission in symptom severity. Treatment was delivered at the time of day when agitated behaviours were expected to peak to maximise the chances of identifying any benefit. Since symptoms wax and wane over the 24-hour period, behaviours were likely to remit regardless of intervention, representing a simple regression to the mean rather than a genuine therapeutic effect. Results Counts of positive affect were higher in all lavender Table 2 Incidence Rate Ratios (IRR) with 95% confidence intervals for effects of treatment, time and treatment-time interaction on behaviour and affect scores after taking account of age, gender and dementia severity Behaviour Affect Table 2 Incidence Rate Ratios (IRR) with 95% confidence intervals for effects of treatment, time and treatment-time interaction on behaviour and affect scores after taking account of age, gender and dementia severity Behaviour Affect Table 2 Incidence Rate Ratios (IRR) with 95% confidence intervals for effects of treatment, time and treatment-time interaction on behaviour and affect scores after taking account of age, gender and dementia severity Behaviour Affect Positive Negative IRR p value IRR p value IRR p value Treatment Lavender compared to control 0.884 (0.778-1.004) 0.057 1.072 (0.848-1.355) 0.56 0.891 (0.504-1.573) 0.690 Time First 30 minutes post-exposure compared to pre- exposure 0.899 (0.793-1.020) 0.097 0.900 (0.706-1.147) 0.393 0.960 (0.550-1.675) 0.887 Second 30 minutes post-exposure compared to pre- exposure 0.858 (0.755-0.974) 0.018 0.865 (0.678-1.106) 0.248 0.641 (0.348-1.179) 0.153 Treatment- time interactions Lavender x first 30 min. post-exposure 0.961 (0.798-1.157) 0.672 1.020 (0.726-1.433) 0.910 0.848 (0.371-1.938) 0.696 Lavender x second 30 min post-exposure 1.045 (0.869-1.259) 0.636 0.954 (0.675-1.348) 0.790 0.687 (0.269-1.750) 0.431 O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 Page 6 of 7 Page 6 of 7 Authors’ contributions All h h i i All the authors participated in the preparation of the manuscript and have read and approved the final draft. DO’C conceptualised and designed the study; EvdP and BE conducted the study and analysed the results, and JT helped with statistical analysis. All authors have read and approved the manuscript. Acknowledgements We thank participants, family members and nursing staff for their support; Mr Ron Guba of Essential Therapeutics for supplying certified pure lavender oil, and Prof. George Lees, Otago University, for testing lavender samples. To ensure statistical power, we sought 77 subjects based on data from an earlier controlled trial of a psychosocial intervention using identical methods in a similar clinical sample. We succeeded in recruiting and retaining only 64 people, despite doubling the study period from one to two years. It seems very unlikely, however, that our negative results are due to inadequate power as there is no sugges- tion in the raw data of any improvement in behaviour or affect during exposure to lavender relative to the control oil based on a limited number of analyses of a very large number of data points. Competing interests Competing interests The authors do not have competing interests. In devising the protocol, we had sought to avoid the methodological pitfalls identified in previous systematic reviews of treatments of the behavioural and psychological symptoms of dementia [19,28]. Participants had proven, frequent behavioural symptoms; high strength lavender oil with demonstrable neurophysiological activity was administered in a uniform fashion to nursing home resi- dents who were reasonably typical of those with marked behavioural disturbances, and behaviour and affect were measured reliably over a plausible time frame by raters who were blinded to treatment condition. The authors do not have competing interests. Funding h d g The study was funded by the National Health and Medical Research Council (Grant #545843) and the Dementia Collaborative Research Centre administered through the University of New South Wales. Neither funding body, nor the supplier of materials, played any part in analysing data and preparing this report. Received: 16 November 2012 Accepted: 1 November 2013 Published: 13 November 2013 Received: 16 November 2012 Accepted: 1 November 2013 Published: 13 November 2013 References 1. Jäger W, Buchbauer G, Jorovetz L, Fritzer M: Percutaneous absorption of lavender oil from a massage oil. J Soc Cosmet Chem 1992, 43:49–54. 2. Elisabetsky E, Marschner J, Souza DO: Effects of linalool on glutamatergic system in the rat cerebral cortex. Neurochem Res 1995, 20:461–465. The most significant limitation to research of essential oils concerns product variability and the limited knowledge base of lavender’s pharmacological properties. There are very few studies of its absorption, metabolism and central nervous system availability in animals and healthy humans, let alone frail older people with dementia. Lavender appears to be well absorbed and to have sedative and anticonvulsant potential but information is otherwise limited with the result that dosing strategies are based largely on traditional practice. We settled on a topical dose of 1 ml 30% certified pure lavender oil applied to each forearm only because a higher strength was so viscous that application became impracticable. This was still much higher than the doses delivered by vapour in most previous studies. Future researchers should consider measuring plasma levels of linalool and linalyl acetate as a check on absorption. 3. Huang L, Abuhamdah S, Howes MJR, et al: Pharmacological profile of essential oils derived from Lavandula angustifolia and Melissa officinalis with anti-agitation properties: focus on ligand-gated channels. J Pharm Pharmacol 2008, 60:1515–1522. 4. Buchbauer G, Jirovetz L, Jager W, Plank C, Dietrich H: Fragrance compounds and essential oils with sedative properties. J Pharm Sci 1993, 82:660–664. 5. Umezu T: Behavioral effects of plant-derived essential oils in the Geller type conflict test in mice. Jpn J Pharmacol 2000, 83:150–153. 5. Umezu T: Behavioral effects of plant-derived essential oils in the Geller type conflict test in mice. Jpn J Pharmacol 2000, 83:150–153. 6. Linck VM, da Silva AL, Figueiró M, et al: Inhaled linalool-induced sedation in mice. Phytomed 2009, 16:303–307. 5. Umezu T: Behavioral effects of plant-derived essential oils in the Geller type conflict test in mice. Jpn J Pharmacol 2000, 83:150–153. 6. Linck VM, da Silva AL, Figueiró M, et al: Inhaled linalool-induced sedation in mice. Phytomed 2009, 16:303–307. 7. Shiina Y, Funabashi N, Lee K, et al: Relaxation effects of lavender aromatherapy improve coronary flow velocity reserve in healthy men evaluated by transthoracic Doppler echocardiography. Int J Cardiol 2008, 129:193–197. 6. Linck VM, da Silva AL, Figueiró M, et al: Inhaled linalool-induced sedation in mice. Phytomed 2009, 16:303–307. 7. Conclusion Studies of essential oils are constrained by their variable formulations and uncertain pharmacokinetics and so optimal dosing and delivery regimens remain speculative. Notwithstanding this, topically delivered, high strength, pure lavender oil had no discernible effect on affect and behaviour in a well-defined clinical sample. 8. Field T, Field T, Cullen C, et al: Lavender bath oil reduces stress and crying and enhances sleep in very young infants. Early Hum Dev 2008, 84:399–401. O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 10. Kaspar S, Gastpar M, Müller WE, et al: Efficacy and safety of silexan, a new orally administered lavender oil preparation, in subthreshold anxiety disorder – evidence from clinical trials. Wien Med Wochenschr 2010, 160:547–556. 11. Bickers D, Calow P, Greim H, et al: A toxicogic and dermatologic assessment of linalool and related esters when used as fragrance ingredients. Food Chem Toxicol 2003, 41:919–942. 12. Holmes C, Hopkins V, Hensford C, et al: Lavender oil as a treatment for agitated behaviour in severe dementia: a placebo controlled study. Int J Geriatr Psychiatry 2007, 17:305–308. 12. Holmes C, Hopkins V, Hensford C, et al: Lavender oil as a treatment for agitated behaviour in severe dementia: a placebo controlled study. Int J Geriatr Psychiatry 2007, 17:305–308. 13. Lin PW, Chan W, Ng BF, Lam LC: Efficacy of aromatherapy (Lavandula angustifolia) as an intervention for agitated behaviours in Chinese older persons with dementia: a cross-over randomised trial. Int J Geriatr Psychiatry 2007, 22:405–410. y y 14. Snow AL, Hovanec L, Brandt J: A controlled trial of aromatherapy of for agitation in nursing home patients with dementia. J Altern Complem Med 2004, 10:431–437. 15. Smallwood J, Brown R, Coulter F, Irvine E: Aromatherapy and behaviour disturbances in dementia. Int J Geriatr Psychiatry 2001, 16:1010–1013. 16. Gray SG, Clair AA: Influence of aromatherapy on medication administration to residential care residents with dementia and behavioral challenges. Am J Alzheimers Dis 2002, 17:169–174. g 17. Fujii M, Hatakeyama R, Fukuoka Y, et al: Lavender aroma therapy for behavioral and psychological symptoms in dementia patients. Geriatr Gerontol Int 2008, 8:136–138. 18. Holt FE, Birks TPH, Thorgrimsen LM, et al: Aroma therapy for dementia. Cochrane Database Syst Rev 2003. 10.1002/14651858.CD003150. 19. O’Connor DW, Ames D, Gardner B, King M: Psychosocial treatments of behavior symptoms in dementia: a systematic review of reports meeting quality standards. Int Psychogeriatr 2009, 21:225–240. 20. Holmes C, Ballard C: Aromatherapy in dementia. Adv Psychiat Treat 2004, 10:296–300. 21. Kaestli LZ, Wasilewski-Rasca AF, Bonnabry P, Vogt-Ferrier N: Use of transder- mal drug formulations in the elderly. Drugs Ageing 2008, 25:269–280. 22. Hughes CP, Berg L, Danziger WL, Coben LA, Martin RL: A new clinical scale for the staging of dementia. Brit J Psychiat 1982, 140:566–572. 23. References Shiina Y, Funabashi N, Lee K, et al: Relaxation effects of lavender aromatherapy improve coronary flow velocity reserve in healthy men evaluated by transthoracic Doppler echocardiography. Int J Cardiol 2008, 129:193–197. 8. Field T, Field T, Cullen C, et al: Lavender bath oil reduces stress and crying and enhances sleep in very young infants. Early Hum Dev 2008, 84:399–401. 8. Field T, Field T, Cullen C, et al: Lavender bath oil reduces stress and crying and enhances sleep in very young infants. Early Hum Dev 2008, 84:399–401. 9. Kim JT, Ren CJ, Fielding GA, et al: Treatment of lavender aromatherapy in the post-anesthesia care unit reduces opioid requirements of morbidly obese patients undergoing laparoscopic adjustable gastric banding. Obes Surg 2007, 17:920–925. 9. Kim JT, Ren CJ, Fielding GA, et al: Treatment of lavender aromatherapy in the post-anesthesia care unit reduces opioid requirements of morbidly obese patients undergoing laparoscopic adjustable gastric banding. Obes Surg 2007, 17:920–925. The current lack of information also makes it difficult to understand why two studies have returned positive Page 7 of 7 doi:10.1186/1472-6882-13-315 Cite this article as: O’Connor et al.: A randomized, controlled cross-over trial of dermally-applied lavender (Lavandula angustifolia) oil as a treatment of agitated behaviour in dementia. BMC Complementary and Alternative Medi- cine 2013 13:315. O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 Van der Ploeg ES, Eppingstall B, O’Connor DW: The study protocol of a blinded, randomised controlled trial of lavender oil as a treatment of behavioural symptoms of dementia. BMC Geriatr 2010, 10:49. 24. Garland K, Beer E, Eppingstall B, O’Connor DW: A comparison of two treatments of agitated behavior in nursing home residents with behaviour: simulated family presence and preferred music. Am J Geriatr Psychiat 2007, 15:514–521. 25. Lawton MP, van Haitsma K, Klapper J: Observed affect in nursing home residents with Alzheimer’s disease. J Gerontol 1996, 51:3–14. 26. Folstein M, Folstein S, McHugh P: Mini-Mental State: a practical method 26. Folstein M, Folstein S, McHugh P: Mini-Mental State: a practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res 1975, 12:189–198. for grading the cognitive state of patients for the clinician. J Psychiatr Res 1975, 12:189–198. 27. Cohen-Mansfield J: Agitated behaviors in the elderly: preliminary results in the cognitively deteriorated. J Am Geriatr Soc 1986, 34:722–727. 28. O’Connor DW, Ames D, Gardner B, King M: Psychosocial treatments of psychological symptoms in dementia: a systematic review of reports meeting quality standards. Int Psychogeriatr 2009, 21:241–251. doi:10.1186/1472-6882-13-315 Cite this article as: O’Connor et al.: A randomized, controlled cross-over trial of dermally-applied lavender (Lavandula angustifolia) oil as a treatment of agitated behaviour in dementia. BMC Complementary and Alternative Medi- cine 2013 13:315. O’Connor et al. BMC Complementary and Alternative Medicine 2013, 13:315 http://www.biomedcentral.com/1472-6882/13/315 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission
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https://www.nature.com/articles/srep27091.pdf
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miR-146a is essential for lipopolysaccharide (LPS)-induced cross-tolerance against kidney ischemia/reperfusion injury in mice
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miR-146a is essential for lipopolysaccharide (LPS)-induced cross-tolerance against kidney ischemia/reperfusion injury in mice received: 28 October 2015 accepted: 09 May 2016 Published: 02 June 2016 Yan Dai1,2,3,*, Ping Jia1,2,3,*, Yi Fang1, Hong Liu1, Xiaoyan Jiao1,2,3, John C. He4,5 & i i i 1 2 3 MicroRNA-146a is one of most important microRNAs involved in development of endotoxin tolerance via (toll-like receptors) TLRs/ NF-κB pathway. In this study, we sought to identify the mechanistic role of miR-146a in mediating the protective effect of lipopolysaccharide (LPS) pretreatment on kidney ischemia/reperfusion injury. A locked nucleic acid–modified anti-miR-146a given before LPS treatment knocked down miR-146a expression and completely negated LPS-mediated protection against kidney ischemia/reperfusion injury. Knockdown of miR-146a resulted in significantly higher histopathological scores for tubular damage, expression of proinflammatory cytokines and chemokines, and neutrophil and macrophage infiltration. Furthermore, knockdown of miR-146a greatly up-regulated the protein levels of IL-1 receptor-associated kinase (IRAK-1) and tumor-necrosis factor (TNF) receptor-associated factor 6 (TRAF6), which are known target genes of miR-146a, leading to activation of NF-κB. Finally, elevation of nuclear translocation of NF-κB p65/p50 and caspase-3 expression, degradation of cytosolic IkBα and BcL-xL, and substantially exacerbation of tubular cell apoptosis were inversely correlated with miR-146a expression. Taken together, our results identify that miR146a exerts a kidney protective effect through negative regulation of acute inflammatory response by suppressing NF-κB activation and proinflammatory genes expression. Acute kidney injury (AKI) is a major clinical problem with a high degree of morbidity and mortality1. Kidney ischemia/reperfusion (I/R) injury is the major cause of acute kidney injury after major surgery or kidney trans- plantation2,3. I/R injury may activate innate immunity by endogenous ligands through the engagement of Toll-like receptors (TLRs), particularly TLR4 and TLR24–6. TLR4 expressed within the kidney is a potential mediator of innate activation and inflammation7. Uncontrolled inflammation results in extensive tissue damage, and therefore this important inflammatory reaction must be tightly regulated; this regulatory response is seen in the manifes- tation of endotoxin tolerance8,9.i Endotoxin tolerance is claimed to be a specific phenomenon, where animals injected with subtoxic LPS dose show an increased survival rate against inflammation damage in vivo and endotoxin-primed cells show reduced responsiveness toward repeated endotoxin stimulation in vitro10–12. Cross-tolerance, which means following endotoxin exposure, endotoxin-primed cells are hyporesponsive to stimulation with other TLR ligands has also been described13. LPS is a classical ligand for TLR4 and mediates TLR4/MyD88-dependent signal transduction to activate NF-κ​B, leading to an increase in inflammatory cytokine expression such as IL-1β​ and TNF-α​14–16. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 28 October 2015 accepted: 09 May 2016 Published: 02 June 2016 Results K kd Knockdown of miR-146a negates the kidney protection conferred by LPS. To determine the functional role of miR-146a in the kidney protection conferred by LPS pretreatment, locked nucleic acid (LNA)– modified anti-scrambled or anti-miR-146a oligonucleotides (10 mg/kg) were administered to mice through the tail vein just 2 hour prior to LPS (3 mg/kg, ip) or vehicle treatment. At 24 h after LPS injection, the kid- neys were subjected to I/R procedures and then harvested. As a first step to investigate the role of miR-146a in cross-tolerance, we measured its expression profile in the kidney activated with LPS. As shown in Fig. 1a, the miR-146a levels of LPS +​ I/R +​ Scrambled control group increased upon LPS stimulation at all time points compared with LPS +​ I/R +​ anti-miR-146a group and I/R +​ anti-miR-146a group (P <​ 0.001), whereas there was no change compared with LPS +​ Sham group (P >​ 0.05). In contrast, miR-146a levels in LPS +​ I/R +​ anti-miR- 146a and I/R +​ anti-miR-146a group were markedly decreased after its knockdown (Fig. 1a). To analyze the role of miR-146a in injury and repair responses following kidney I/R, we studied additional time course of miR- 146a expression and compared those of miR-21, which is also known to contribute to I/R injury. MicroRNA-21 was immediately up-regulated following I/R injury compared to sham samples at day 0 with a peak at day3. However, miR-146a was up-regulated after day3 and achieved its peak at day7 (Supplementary Figure 1a). Serum creatinine measured at 24 h after I/R injury was also significantly elevated in LPS +​ I/R +​ anti-miR-146a group and I/R +​ Scrambled control group as compared to LPS +​ I/R +​ Scrambled control group (159.1 ±​ 11.6 umol/l vs. 183.2 ±​ 9.1 umol/l vs. 54.25 ±​ 3.96 umol/l, P <​ 0.001, Fig. 1b). There was no statistical difference in serum creatinine levels between LPS +​ Sham and LPS +​ I/R +​ Scrambled control groups. However, both I/R +​ anti- miR-146a group and I/R +​ Scrambled control group mice displayed a similar elevation in serum creatinine (183.2 ±​ 9.1 umol/l vs. 156.8 ±​ 13.32 umol/l). To confirm the robustness of the observed effect of anti-miR-146a, we measured serum creatinine levels at 0, 1, 2, 3, 5, or 7 days following the I/R injury in additional groups of mice with LPS +​ I/R +​ anti-miR-146a and LPS +​ I/R +​ Scrambled control. Results K kd Mice treated with LPS +​ I/R +​ Scrambled control were largely protected from the I/R injury (Fig. 1c). g y p j y g The functional data correlated with histological evidence in periodic acid-Schiff-stained sections. As shown in Fig. 1d and Supplementary Figure 1c, mice receiving LPS +​ I/R +​ anti-miR-146a exhibited severe tubular damage, as evidenced by widespread tubular necrosis, loss of the brush border, cast formation, cellu- lar swelling and serious inflammatory infiltration at the corticomedullary junction, maximal at 24 h, whereas LPS +​ I/R +​ Scrambled control group showed significantly less tubular damage and mild cellular swelling as com- pared with LPS +​ I/R +​ anti-miR-146a group and I/R +​ Scrambled control group at 24 h after I/R injury (Fig. 1e). LPS +​ Sham group incurred no tubular injury. It is important to note that both I/R +​ anti-miR-146a group and I/R +​ Scrambled control group mice showed a similar morphological damage score (P >​ 0.05, Supplementary Figure 1d). These results suggest that LPS pretreatment increases miR-146a expression and protects mice from kidney I/R injury, which is negated by knockdown of miR-146a in vivo. Induction of miR-146a in mouse kidneys by LPS suppresses I/R injury-induced inflammatory cytokine production. It has been reported that NF-κ​B-dependent proinflammatory cytokines, such as IL-1β​, TNF-α​, IL-6, are down-regulated, whereas anti-inflammatory cytokines, such as IL-10, TGF- β​, and IL-1RA, are up-regulated in endotoxin tolerance models21. IL-1β​ and TNF-α​ mRNAs examined by real-time PCR in LPS +​ I/R +​ anti-miR-146a group was significantly higher than LPS +​ I/R +​ Scrambled control group at 6 h and 24 h after I/R injury, peaking at 24 h (Fig. 2a), then declined at 48 h. Analysis of protein levels of IL-1β​ and TNF-α​ by Enzyme-linked immunosorbent assay (ELISA) showed levels consistent with the mRNA data, and induction of IL-1β​ and TNF-α​ in LPS +​ I/R +​ anti-miR-146a group was significant elevated compared with LPS +​ I/R +​ Scrambled control group (Fig. 2b). As shown in Fig. 3, we also measured the mRNA levels of cytokines (IL-10 and IL-6). Lower mRNA level of IL-10 was detected in mice receiving LPS +​ I/R +​ anti-miR- 146a group while significantly increased in LPS +​ I/R +​ Scrambled control group. We found a slightly increase of IL-10 mRNA in I/R +​ Scrambled control group. miR-146a is essential for lipopolysaccharide (LPS)-induced cross-tolerance against kidney ischemia/reperfusion injury in mice However, sepsis/endotoxin and inflammation are frequently observed complications during kidney I/R injury. Endotoxin can play a role in kidney I/R injury induced during clinical procedures. Thus, preconditional LPS treatment provokes cross-tolerance and protects kidney against a subsequent, sustained ischemic result17. 1Division of Nephrology, Zhongshan Hospital, Fudan University, Shanghai, China. 2Kidney and Dialysis Institute of Shanghai, Shanghai, China. 3Kidney and Blood Purification Laboratory of Shanghai, Shanghai, China. 4Department of Medicine/Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York, USA. 5Kidney Section, James J Peter Veteran Administration Medical Center at Bronx, NY, United States. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to X.D. (email: ding.xiaoqiang@zs- hospital.sh.cn) Scientific Reports | 6:27091 | DOI: 10.1038/srep27091 1 www.nature.com/scientificreports/ MicroRNAs (miRNAs), a family of small noncoding RNAs which regulate gene expression by base-pairing to the 3′​ untranslated region of their target genes, have recently emerged as a novel class of posttranscriptional regu- lators in the immune system, including endotoxin tolerance18–20. Previously, a number of different miRNA (miR- 146a, miR-98, miR-155) have been found to be involved in regulation of endotoxin tolerance21. Among them, miR-146a attracted our attention: previous studies show that LPS-induced up-regulation of miR-146a is indeed NF-κ​B dependent22; Importantly, two key adapter molecules in the TLR4/NF-κ​B pathway, TRAF6 and IRAK-1 are identified as direct targets of miR-146a, and they also promote inflammation stimulated by proinflammatory cytokines including TNF-a23. Above finding suggests the role of miR-146a in TLR4/ NF-κ​B signaling through a negative feedback regulatory loop, which can suppress acute inflammatory response24. Experiments with THP-1 cells have shown that miR-146a participates in transcriptional and translational modifications associated with gene reprogramming during induction of endotoxin tolerance13,25. This leads to the speculation that increased miR-146a expression during an LPS-primed state might play a part in a negative feedback pathway for other ligand-TLRs interactions such as TLR4/Myd88-dependent signaling implicated in a mouse model of ischemic AKI26. Additionally, increased miR-146a expression also has been confirmed during kidney I/R injury27. Thus, we focused on miR-146a for further study. In this study, with highly effective in vivo knockdown of miR-146a, we explored the role of miR-146a in mediating the protective effect of LPS pretreatment on kidney ischemia/ reperfusion injury. Results K kd For IL-6, mRNA levels were strongly up-regulated in LPS +​ I/R +​ anti-miR-146a group at 24 h after kidney I/R injury, and that of I/R +​ Scrambled control group also increased but to a greater extent compared with LPS +​ I/R +​ Scrambled control group (Fig. 3).l Scientific Reports | 6:27091 | DOI: 10.1038/srep27091 2 www.nature.com/scientificreports/ Figure 1. LPS pretreatment protects mice from kidney I/R injury, which is negated by knockdown of miR- 146a. (a) Locked nucleic acid (LNA) anti-miR-146a (10 mg/kg) or anti-scrambled was administrated 2 hour prior to LPS injection. At 24 h after exposure to either normal saline (NS) or lipopolysaccharide (LPS; 3 mg/kg, intraperitoneal injection), mice were subjected to kidney ischemia surgery. miR-146a expression was examined and normalized to u6 at 6, 24, 48 h after kidney I/R by TaqMan-based real-time PCR, fold changes were calculated against the mean value of LPS +​ Sham group at each time point. (n =​ 6 mice per group for each time point. #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control, respectively. NS, not significant). (b) Serum creatinine was measured and data was expressed as mean ±​ SEM at 24 h after kidney I/R injury. (n =​ 6 mice per group, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). (c) Time course of serum creatinine levels after I/R in mice exposed to LPS with or without miR-146a knockdown. (n =​ 4 mice per group for each time point, *​P <​ 0.05, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). (d) Representative periodic acid–Schiff (PAS)-stained kidney sections from mice receiving anti-miR-146a or anti-scrambled oligonucleotides at 24 h after pretreatment with either vehicle +​ I/R or LPS +​ I/R procedures (original magnification x200, Bar =​ 50 um. The black arrow indicates damaged tubules). (e) Abnormalities based on PAS-stained sections were graded by a semiquantitative histomorphological scoring system from 0 to 4. *​P <​ 0.05 vs. LPS +​ I/R +​ Scrambled control. Figure 1. LPS pretreatment protects mice from kidney I/R injury, which is negated by knockdown of m Figure 1. LPS pretreatment protects mice from kidney I/R injury, which is negated by knockdown of miR- 146a. (a) Locked nucleic acid (LNA) anti-miR-146a (10 mg/kg) or anti-scrambled was administrated 2 hour prior to LPS injection. Results K kd At 24 h after exposure to either normal saline (NS) or lipopolysaccharide (LPS; 3 mg/kg, intraperitoneal injection), mice were subjected to kidney ischemia surgery. miR-146a expression was examined and normalized to u6 at 6, 24, 48 h after kidney I/R by TaqMan-based real-time PCR, fold changes were calculated against the mean value of LPS +​ Sham group at each time point. (n =​ 6 mice per group for each time point. #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control, respectively. NS, not significant). (b) Serum creatinine was measured and data was expressed as mean ±​ SEM at 24 h after kidney I/R injury. (n =​ 6 mice per group, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). (c) Time course of serum creatinine levels after I/R in mice exposed to LPS with or without miR-146a knockdown. (n =​ 4 mice per group for each time point, *​P <​ 0.05, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). (d) Representative periodic acid–Schiff (PAS)-stained kidney sections from mice receiving anti-miR-146a or anti-scrambled oligonucleotides at 24 h after pretreatment with either vehicle +​ I/R or LPS +​ I/R procedures (original magnification x200, Bar =​ 50 um. The black arrow indicates damaged tubules). (e) Abnormalities based on PAS-stained sections were graded by a semiquantitative histomorphological scoring system from 0 to 4. *​P <​ 0.05 vs. LPS +​ I/R +​ Scrambled control. Figure 1. LPS pretreatment protects mice from kidney I/R injury, which is negated by knockdown of miR- 146a. (a) Locked nucleic acid (LNA) anti-miR-146a (10 mg/kg) or anti-scrambled was administrated 2 hour prior to LPS injection. At 24 h after exposure to either normal saline (NS) or lipopolysaccharide (LPS; 3 mg/kg, intraperitoneal injection), mice were subjected to kidney ischemia surgery. miR-146a expression was examined and normalized to u6 at 6, 24, 48 h after kidney I/R by TaqMan-based real-time PCR, fold changes were calculated against the mean value of LPS +​ Sham group at each time point. (n =​ 6 mice per group for each time point. #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control, respectively. NS, not significant). (b) Serum creatinine was measured and data was expressed as mean ±​ SEM at 24 h after kidney I/R injury. (n =​ 6 mice per group, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). Results K kd (a) mRNA of IL-1β​ and TNF-α​ in the kidney medulla was determined by real-time PCR and was normalized to GAPDH at 6, 24, 48 h after kidney I/R. (n =​ 6 mice per group, *​*​P <​ 0.01, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). (b) IL-1β​ and TNF-α​ protein levels at each time point were evaluated by Enzyme-linked immunosorbent assay (ELISA) at each time point. n =​ 6 mice/group, *​P <​ 0.05, *​*​P <​ 0.01, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). All data were expressed as mean ±​ SEM. with LPS +​ I/R +​ anti-miR-146a group or I/R +​ Scrambled control group, LPS +​ I/R +​ Scrambled control had significantly less interstitial macrophages during I/R injury (Fig. 4c,d). gi y p g g j y ( g ) Tubular cell apoptosis has been shown to contribute to the pathogenesis of ischemic kidney injury29; we therefore investigated the role of miR146a in ischemia–reperfusion induced tubular epithelial cell apoptosis. Our results showed that there was a significant increase in the number of tubular apoptotic cells as assessed by terminal transferase dUTP nickend labeling (TUNEL) in LPS +​ I/R +​ anti-miR-146a group (Fig. 5a,b). LPS pre- treatment attenuated kidney tubular cell apoptosis 24 h after I/R (n =​ 6 mice per group), suggesting a critical role of miR-146a in LPS mediated renoprotection. Caspase-3 is the final effector caspase that mediates apoptotic cell death. As such, immunostaining demonstrated that cleaved caspase-3 protein expressed by tubular epithelial cells (TECs) particularly at the corticomedullary junction. The amount of caspase-3 detected in LPS +​ I/R +​ Scrambled control group was substantially less compared with that seen in LPS +​ I/R +​ anti-miR-146a and I/R +​ Scrambled control group (Fig. 5c,d). Knockdown of miR-146a enhances TRAF6 and IRAK-1 protein expression. MicroRNA-146a down-regulates the expression of IRAK-1 and TRAF-6 signaling molecules involved in LPS/TLR4 mediated acti- vation of innate immune response24. To further demonstrate the expression of known miR-146a target genes in kidney medulla, TRAF6 and IRAK-1, were determined by real-time PCR and western blot analysis in LPS or normal saline treated animals in a time course (6 h, 24 h, 48 h) following I/R injury.f ( ) g j y As shown in Fig. 6a, mRNA transcript levels of TRAF6 and IRAK-1 were not markedly different between mice with and without miR-146a knockdown at all time points after I/R injury. Results K kd (c) Time course of serum creatinine levels after I/R in mice exposed to LPS with or without miR-146a knockdown. (n =​ 4 mice per group for each time point, *​P <​ 0.05, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). (d) Representative periodic acid–Schiff (PAS)-stained kidney sections from mice receiving anti-miR-146a or anti-scrambled oligonucleotides at 24 h after pretreatment with either vehicle +​ I/R or LPS +​ I/R procedures (original magnification x200, Bar =​ 50 um. The black arrow indicates damaged tubules). (e) Abnormalities based on PAS-stained sections were graded by a semiquantitative histomorphological scoring system from 0 to 4. *​P <​ 0.05 vs. LPS +​ I/R +​ Scrambled control. I/R +​ Scrambled control group, compared with LPS +​ I/R +​ Scrambled control group (Fig. 3). Similarly, signifi- cant differences were found for intercellular adhesion molecule-1 (Icam1), a key regulator of leukocyte adhesion and transendothelial migration (Fig. 3). There was no statistically difference between LPS +​ I/R +​ Scrambled control group and LPS +​ Sham group (P >​ 0.05). However, there was no statistically difference between I/R +​ Scrambled control group and I/R +​ anti-miR-146a group mice at 24 h after reperfusion (6 h and 48 h data not shown, P >​ 0.05, Supplementary Figure 1b). Consistent with the histological data, knockdown of miR- 146a in mice receiving only I/R procedures showed a similar increase of the kidney proinflammatory cytokines production after reperfusion. Taken together, these results indicate that miR-146a contributes to LPS induced cross-tolerance against kidney I/R injury by inhibiting proinflammatory pathways. Knockdown of miR-146a promotes inflammatory cell infiltration and apoptosis. Inflammatory cells have been shown to have a critical role in the pathogenesis of I/R injury28. To further characterize the role of miR-146a in the inflammatory cells, immunoperoxidase labeling for CD68 (a macrophage marker) and mye- loperoxidase (MPO) (a neutrophil marker) on paraffin-embedded kidney sections were performed at 24 h after I/R injury. Polymorphonuclear (PMN) leukocyte infiltration was significantly less in LPS +​ I/R +​ Scrambled con- trol group compared to that of LPS +​ I/R +​ anti-miR-146a group and I/R +​ Scrambled control group (Fig. 4a,b). Consistent with the neutrophil accumulation, staining of CD68 localized in the outer medulla showed that LPS +​ I/R +​ anti-miR-146a group induced a progressive increase in interstitial at 24 h after I/R injury. Compared Scientific Reports | 6:27091 | DOI: 10.1038/srep27091 3 www.nature.com/scientificreports/ Figure 2. Results K kd Proinflammatory cytokine (IL-1β and TNF-α) production in the LPS-challenged kidney following I/R injury. (a) mRNA of IL-1β​ and TNF-α​ in the kidney medulla was determined by real-time PCR and was normalized to GAPDH at 6, 24, 48 h after kidney I/R. (n =​ 6 mice per group, *​*​P <​ 0.01, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). (b) IL-1β​ and TNF-α​ protein levels at each time point were evaluated by Enzyme-linked immunosorbent assay (ELISA) at each time point. n =​ 6 mice/group, *​P <​ 0.05, *​*​P <​ 0.01, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). All data were expressed as mean ±​ SEM. Figure 2. Proinflammatory cytokine (IL-1β and TNF-α) production in the LPS-challenged kidney following I/R injury. (a) mRNA of IL-1β​ and TNF-α​ in the kidney medulla was determined by real-time PCR and was normalized to GAPDH at 6, 24, 48 h after kidney I/R. (n =​ 6 mice per group, *​*​P <​ 0.01, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). (b) IL-1β​ and TNF-α​ protein levels at each time point were evaluated by Enzyme-linked immunosorbent assay (ELISA) at each time point. n =​ 6 mice/group, *​P <​ 0.05, *​*​P <​ 0.01, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). All data were expressed as mean ±​ SEM. Figure 2 Proinflammatory cytokine (IL 1β and TNF α) production in the LPS challenged kidney Figure 2. Proinflammatory cytokine (IL-1β and TNF-α) production in the LPS-challenged kidney Figure 2. Proinflammatory cytokine (IL-1β and TNF-α) production in the LPS-challenged kidney following I/R injury. (a) mRNA of IL-1β​ and TNF-α​ in the kidney medulla was determined by real-time PCR and was normalized to GAPDH at 6, 24, 48 h after kidney I/R. (n =​ 6 mice per group, *​*​P <​ 0.01, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). (b) IL-1β​ and TNF-α​ protein levels at each time point were evaluated by Enzyme-linked immunosorbent assay (ELISA) at each time point. n =​ 6 mice/group, *​P <​ 0.05, *​*​P <​ 0.01, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). All data were expressed as mean ±​ SEM. Figure 2. Proinflammatory cytokine (IL-1β and TNF-α) production in the LPS-challenged kidney following I/R injury. Results K kd There was a slightly increase of mRNA levels of TRAF6 in I/R +​ Scrambled control group at 6 h time points. In contrast, a significant increase of TRAF6 protein levels occurred at 6 h (Fig. 6b,c) and remained at later time up to 48 h (Fig. 6d) from LPS +​ I/R +​ anti-miR- 146a and I/R +​ Scrambled control mice but not from LPS +​ I/R +​ Scrambled control mice. However, induction of IRAK-1 was also observed from LPS +​ I/R +​ anti-miR-146a mice at 6 h and sharply reduced at 24 h, whereas mice in LPS +​ I/R +​ Scrambled control group expressed negligible levels of IRAK-1 at all time points (Fig. 6d). Our data confirmed that miR-146a mediated down-regulation of TLR4/ NF-κ​B signaling via targeting TRAF6 and IRAK-1 in LPS-challenged kidney following I/R injury. Knockdown of miR-146a activates nuclear translocation of NF-κB p65/p50. TLR4/ NF-κ​B pathway is involved in the mechanism of LPS induced endotoxin and cross-tolerance21,30. Signaling through MyD88 leads to phosphorylation-mediated degradation of inhibitors of NF-κ​B (Iκ​Bs), nuclear translocation of NF-κ​B p65/p50 and transcriptional activation of proinflammatory genes31. To determine the link of miR-146a and NF-κ​B activation, we measured protein levels of phosphorylated P65 (p-p65) in nuclear extracts and p65, Scientific Reports | 6:27091 | DOI: 10.1038/srep27091 4 www.nature.com/scientificreports/ ture.com/scientificreports/ Figure 3. Inflammatory mediators mRNA profile in the LPS-challenged kidney following I/R injury. Kidney expression of the proinflammatory cytokines and chemokines were quantified by real-time PCR at 24 h after kidney I/R injury. (n =​ 6 mice per group, *​P <​ 0.05, *​*​P <​ 0.01, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). Figure 3. Inflammatory mediators mRNA profile in the LPS-challenged kidney following I/R injury. Kidney expression of the proinflammatory cytokines and chemokines were quantified by real-time PCR at 24 h after kidney I/R injury. (n =​ 6 mice per group, *​P <​ 0.05, *​*​P <​ 0.01, #P <​ 0.001 vs. LPS +​ I/R +​ Scrambled control). IkBα​ in cytoplasm at 6, 24 and 48 h after reperfusion. To confirm the nuclear translocation of NF-κ​B p50, immu- nohistochemical localization of p50 was carried out at 24 h after I/R injury (Fig. 7a,b, 6 h and 48 h data not shown). LPS pretreatment completely blocked the nuclear localization of p50 induced by I/R injury, which can be reversed by knockdown of miR-146a. Likewise, knockdown of miR-146a increased the expression of nuclear NF-κ​B p65. Results K kd As compared to the mice in LPS +​ I/R +​ Scrambled control group, mice in the LPS +​ I/R +​ anti-miR-146a group had approximately five-fold increase of nuclear NF-κ​B p65 expression at 24 h (4.8-fold; Fig. 7c,d) and at 6 h (4.7-fold; Supplementary Figure 2a,c). A 2.5-fold increase was also observed at 48 h (Supplementary Figure 2b,d). Mice in the I/R +​ Scrambled control group showed a similar elevation of NF-κ​B p65 nuclear translocation at all time points. Either normal saline (NS) or LPS treated Sham scrambled control mice showed the same NF-κ​B activity baseline (data not shown). Furthermore, the increased NF-κ​B activation was accompanied by a simul- taneous decrease in cytosolic IkBα​, an inhibitory protein that prevents translocation of NF-κ​B into the nucleus (Fig. 7c,d, Supplementary Figure 2).h ( g , , pp y g ) The balance between pro- and anti-apoptotic forces determines the fate of tubule cells underwent an ischemic insult, as evidenced by studies both in rodents32,33 and in kidney epithelial cells in vitro34. As such, we examined the effect of miR-146a knockdown on Bcl-extralarge (BcL-xL) protein expression in the kidney. As results indi- cated above, there is a general inverse correlation between miR-146a expression and kidney tubular cell apoptosis assessed by TUNEL staining. This observation correlated with decreased protein level of antiapoptotic BcL-xL, which can be reversed in LPS +​ I/R +​ Scrambled control group (Fig. 7c, Supplementary Figure 2a,b). Collectively, these findings support the hypothesis that elevation of nuclear translocation of NF-κ​Bp65/p50, degradation of cytosolic IkBα​ and BcL-xL, and substantially exacerbation of tubular cell apoptosis are inversely associated with miR-146a expression. MicoRNA-146a can function as a key regulator in the development of LPS-induced cross-tolerance by inhibition of NF-κ​B. www.nature.com/scientificreports/ All data were expressed as mean ±​ SEM. NF-κ​B by its direct targets of TRAF6 and IRAK-1. (3) As such, miR-146a could serve as a key negative regulator of inflammation.l l Inflammation and recruitment of leukocytes during epithelial injury are now recognized as major mediators of all phases of endothelial and tubular cell injury during AKI29. Lack of miR-146a expression results in hyperres- ponsiveness of macrophages to LPS and leads to an exaggerated inflammatory response in endotoxin-challenged mice. In contrast, overexpression of miR-146a in monocytes has the opposite effect. Our data revealed the pathological correlation between the expression of miR-146a and proinflammatory cytokines. In our study, those of inflammatory mediators e.g., TNF-α​, IL-1β​, IL-6; chemokines responsible for neutrophil (MIP-2), mac- rophage (MCP-1), RANTES (monocytes and T cells), leukocyte adhesion molecules (Icam1) altered, thus pro- motes interstitial infiltration, all features of I/R injury. These findings are consistent with previous reports in the endotoxin-tolerant THP-1 sepsis cell models23. p According to previous cellular experiments, endotoxin tolerance is associated with reduced TLR4-MyD88 complex formation, impairment of TRAF6 and IRAK-1 activity, down-regulation of NF-κ​B activity, disrup- tion of chromatin remodeling15. Promoter analyses have shown that transcriptional up-regulation of miR-146a induced by TLR4 is NF-κ​B dependent22; miR-146a targets the 3′​-UTR region of TRAF6 and IRAK-1 by the post-transcriptional silencing11,25. Above evidence in vitro suggests mir-146a is important in controlling TLR signaling in innate immunnity7,24. However, our results identify a direct functional link between miR-146a, TRAF6 and IRAK-1 accumulation, and tubular damage in mouse tissue in vivo, implying that TLR signaling is impaired due to the translation inhibition of miR-146a targeted adaptor kinases during innate immune acti- vation, giving rise to endotoxin tolerance. Consistent findings that miR-146a mediates translational repression of IRAK-1 and protects innate immune tolerance in the neonate intestine has been reported35. In current study, miR-146a-mediated down-regulation of TRAF6 and IRAK-1 is time-dependent and underlying molecular mech- anisms is not clear. In addition, we also found that miR-146a knockdown didn’t alter its upstream TLR4 expres- sion (Supplementary Figure S3).l y g Considering the regulation of TRAF6 and IRAK-1 in TLR4/NF-κ​B signaling and associated inflammation in endotoxin tolerance36, we used an anti-miR-146a oligonucleotides approach to block miR-146a and determine the interrelationship between miR-146a and the NF-κ​B system. We demonstrated that up-regulation of miR-146a in LPS/IR stimulated kidneys was inversely correlated with activation of NF-κ​B p65/p50, degradation of Iκ​Bα​. Our results are consistent with previous studies36,37. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Inflammatory cell infiltration. (a) Representative kidney sections immunostained for myeloperoxidase (MPO) to show polymorphonuclear (PMN) leukocyte infiltration (original magnification x200, Bar =​ 50 um, the black arrow indicates positive area). (b) PMN infiltration was scored in mouse kidney. (n =​ 6 mice per group, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control). (c) Representative kidney sections immunostained for CD68 to show macrophage accumulation within the interstitium of the kidney (original magnification x200, Bar =​ 50 um). (d) Analysis of macrophage infiltration in mouse kidney. (n =​ 6 mice per group, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control). All data were expressed as mean ±​ SEM. Figure 4. Inflammatory cell infiltration. (a) Representative kidney sections immunostained for myeloperoxidase (MPO) to show polymorphonuclear (PMN) leukocyte infiltration (original magnification x200, Bar =​ 50 um, the black arrow indicates positive area). (b) PMN infiltration was scored in mouse kidney. (n=6 mice per group, #P<0 001, vs LPS+I/R+Scrambled control) (c) Representative kidney sections Figure 4. Inflammatory cell infiltration. (a) Representative kidney sections immunostained for i Figure 4. Inflammatory cell infiltration. (a) Representative kidney sections immunostained for myeloperoxidase (MPO) to show polymorphonuclear (PMN) leukocyte infiltration (original magnification x200, Bar =​ 50 um, the black arrow indicates positive area). (b) PMN infiltration was scored in mouse kidney. (n =​ 6 mice per group, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control). (c) Representative kidney sections immunostained for CD68 to show macrophage accumulation within the interstitium of the kidney (original magnification x200, Bar =​ 50 um). (d) Analysis of macrophage infiltration in mouse kidney. (n =​ 6 mice per group, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control). All data were expressed as mean ±​ SEM. Figure 4. Inflammatory cell infiltration. (a) Representative kidney sections immunostained for myeloperoxidase (MPO) to show polymorphonuclear (PMN) leukocyte infiltration (original magnification x200, Bar =​ 50 um, the black arrow indicates positive area). (b) PMN infiltration was scored in mouse kidney. (n =​ 6 mice per group, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control). (c) Representative kidney sections immunostained for CD68 to show macrophage accumulation within the interstitium of the kidney (original magnification x200, Bar =​ 50 um). (d) Analysis of macrophage infiltration in mouse kidney. (n =​ 6 mice per group, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control). Discussion Although miR-146a has been recognized to function in the endotoxin tolerance for a long time12,13,25, the mech- anistic role of miR-146a underlying LPS-induced cross-tolerance against kidney I/R injury remains unclear. The present study has revealed a novel role of miR146a in vivo that induction of miR146a by LPS pretreatment pro- tects kidney from I/R injury. We postulate that miR-146a could function as a key regulator in the development of LPS-induced cross-tolerance through one of three potential mechanisms: (1) elevation of nuclear translocation of NF-κ​Bp65/p50 and substantially exacerbation of tubular cell apoptosis are inversely correlated with miR-146a expression. (2) miR-146a is an important member of the negative feedback loop that controls (TLRs) signaling to Scientific Reports | 6:27091 | DOI: 10.1038/srep27091 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ examined that LPS application induced biphasic renal NF-κ​B binding activity in wild-type animals with a first peak in the proinflammatory induction phase at 3 and 6 h after LPS injection and a second peak during the resolu- tion period of the inflammatory process at 48 h40. In current study, miR-146a expression was inversely correlated with NF-κ​B activation and histology score. Knockdown of miR-146a caused a stronger nuclear translocation of NF-κ​B p65 at 6 h and 24 h compared with 48 h after I/R injury in LPS +​ I/R +​ anti-miR-146a group. Based on these results, miR-146a is an important member of the negative feedback loop that controls (TLRs) signaling to NF-κ​B. translocation of p50/p50 homodimers and decreased levels of the active p65/p50 heterodimers38,39. However, many studies have showed that NF-κ​B participates in both the generation of proinflammatory mediators and the antiapoptotic pathway, and the balance between these determines the fate of I/R injured kidney cells40. Our previous studies confirmed that administration of NF-κ​B decoy oligodeoxynucleotides in vivo and curtailed the production of inflammatory cytokine genes, thereby attenuate kidney I/R injury41,42. The effects of NF-κ​B block- ade are highly dependent on the time course of inflammation and the experimental conditions. Ulf Panzer et al. examined that LPS application induced biphasic renal NF-κ​B binding activity in wild-type animals with a first peak in the proinflammatory induction phase at 3 and 6 h after LPS injection and a second peak during the resolu- tion period of the inflammatory process at 48 h40. In current study, miR-146a expression was inversely correlated with NF-κ​B activation and histology score. Knockdown of miR-146a caused a stronger nuclear translocation of NF-κ​B p65 at 6 h and 24 h compared with 48 h after I/R injury in LPS +​ I/R +​ anti-miR-146a group. Based on these results, miR-146a is an important member of the negative feedback loop that controls (TLRs) signaling to NF-κ​B. Given the importance of microRNAs (miRNAs) in AKI, Godwin et al. performed microarray to determine the differentially expressed miRNAs following unilateral renal I/R and miR-146a was one of them27. Therefore, we determined whether miR-146a played a role in the development of LPS-induced cross-tolerance against kidney I/R injury. We observed an up-regulation of miR-146a after day3 following IRI and achieved its peak after day5. This result was consistent with the results reported by Godwin25,27. www.nature.com/scientificreports/ Endotoxin tolerance is linked to overexpression of NF-κ​B, nuclear Scientific Reports | 6:27091 | DOI: 10.1038/srep27091 6 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Inhibiting miR-146a activity in the LPS-challenged kidney exacerbates apoptosis during I/R injury. (a) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in kidney sections, photographed at original magnification x400, Bar =​ 50 um. The arrow indicates apoptotic tubular cell. (b) Mean value of TUNEL-positive cells in kidney sections. Knockdown of miR-146a increased kidney cell apoptosis. (n =​ 6 mice per group, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control). (c) Representative micrographs showing immunohistochemical staining for cleaved caspase-3 among different groups as indicated. (original magnification x200 Bar =​ 50 um). (d) Analysis of cleaved caspase-3 accumulation in mouse kidney. (n =​ 6 mice per group, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control). All data were expressed as mean ±​ SEM. Figure 5. Inhibiting miR-146a activity in the LPS-challenged kidney exacerbates apoptosis during I/R l d l d l f d d d k d l b l ll Figure 5. Inhibiting miR-146a activity in the LPS-challenged kidney exacerbates apoptosis during I/R injury. (a) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in kidney sections, photographed at original magnification x400, Bar =​ 50 um. The arrow indicates apoptotic tubular cell. (b) Mean value of TUNEL-positive cells in kidney sections. Knockdown of miR-146a increased kidney cell apoptosis. (n =​ 6 mice per group, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control). (c) Representative micrographs showing immunohistochemical staining for cleaved caspase-3 among different groups as indicated. (original magnification x200 Bar =​ 50 um). (d) Analysis of cleaved caspase-3 accumulation in mouse kidney. (n =​ 6 mice per group, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control). All data were expressed as mean ±​ SEM. translocation of p50/p50 homodimers and decreased levels of the active p65/p50 heterodimers38,39. However, many studies have showed that NF-κ​B participates in both the generation of proinflammatory mediators and the antiapoptotic pathway, and the balance between these determines the fate of I/R injured kidney cells40. Our previous studies confirmed that administration of NF-κ​B decoy oligodeoxynucleotides in vivo and curtailed the production of inflammatory cytokine genes, thereby attenuate kidney I/R injury41,42. The effects of NF-κ​B block- ade are highly dependent on the time course of inflammation and the experimental conditions. Ulf Panzer et al. Scientific Reports | 6:27091 | DOI: 10.1038/srep27091 www.nature.com/scientificreports/ (a) mRNA of TRAF6 and IRAK-1 in the kidney medulla was determined by real-time RT-PCR and normalized to GAPDH at all time points. (n =​ 6 mice per group, P >​ 0.05 vs. LPS +​ I/R +​ Scrambled control group). (b) Kidney lysates of 6 h groups were subject to Western blot analysis for TRAF6 and IRAK-1. (c) The Western blots from all 6 h experiments were quantified by densitometry analysis. The ratios of TRAF6/ β​-actin and IRAK-1/ β​-actin were calculated; the fold changes relative to LPS +​ Sham protein are shown. (n =​ 6 mice per group, *​P <​ 0.05, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control group). (d) Time course of TRAF6 and IRAK-1 expression was confirmed by Western-blot in the LPS-challenged kidney following I/R injury. Figure 6. Knockdown of miR-146a promotes TRAF6 and IRAK-1 protein expression. LNA-anti-miR-146a or anti-scrambled oligonucleotides (10 m/kg) were administered 2 h before LPS treatment. After 24 h LPS exposure, the kidneys were subjected to I/R or Sham procedures followed by 6 h, 24 h, 48 h of reperfusion and then harvested. (a) mRNA of TRAF6 and IRAK-1 in the kidney medulla was determined by real-time RT-PCR and normalized to GAPDH at all time points. (n =​ 6 mice per group, P >​ 0.05 vs. LPS +​ I/R +​ Scrambled control group). (b) Kidney lysates of 6 h groups were subject to Western blot analysis for TRAF6 and IRAK-1. (c) The Western blots from all 6 h experiments were quantified by densitometry analysis. The ratios of TRAF6/ β​-actin and IRAK-1/ β​-actin were calculated; the fold changes relative to LPS +​ Sham protein are shown. (n =​ 6 mice per group, *​P <​ 0.05, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control group). (d) Time course of TRAF6 and IRAK-1 expression was confirmed by Western-blot in the LPS-challenged kidney following I/R injury. by LPS-induced NF-κ​B and hypoxia-inducible factor-2a (HIF-2a) signaling17. The current study indicates that TLR4/ NF-κ​B signaling regulation by miR-146a represents another important mechanism. In summary, our studies indicate that miR-146a contributes to the establishment of LPS induced cross-tolerance based on the inverse correlation with proinflammatory cytokine production and the repressed protein levels of target genes (IRAK-1and TRAF6) and NF-κ​B activation. The role of miR-146a in controlling cytokine and TLR signaling is through a negative feedback regulatory loop. www.nature.com/scientificreports/ Why knockdown of miR-146a in mice under- went ischemia procedures is not further exacerbate renal injury after reperfusion remains unclear. One plausible explanation is that induction of miR-146a occurred at 6 h after LPS whereas up-regulation of miR-146a by I/R injury occurred at day3 (peaking after day7, Supplementary Figure S1). Therefore, miR-146a induced by LPS is significantly earlier than by I/R injury, suggesting that the protective effect of miR-146a might depend on LPS induction instead of I/R injury.h j y Multiple microRNA (miR-155, let-7e, miR-98) are involved in development of tolerance21. The expression of particular microRNA has been shown to be limited to particular cell types. As a multifactorial process, the kidney protection conferred by LPS pretreatment is likely mediated by several mechanisms. Kang He et al. demonstrated LPS exposure prior to ischemia–reperfusion ameliorated AKI in mice (cross-tolerance), which was mediated Scientific Reports | 6:27091 | DOI: 10.1038/srep27091 7 www.nature.com/scientificreports/ Figure 6. Knockdown of miR-146a promotes TRAF6 and IRAK-1 protein expression. LNA-anti-miR-146a or anti-scrambled oligonucleotides (10 m/kg) were administered 2 h before LPS treatment. After 24 h LPS exposure, the kidneys were subjected to I/R or Sham procedures followed by 6 h, 24 h, 48 h of reperfusion and then harvested. (a) mRNA of TRAF6 and IRAK-1 in the kidney medulla was determined by real-time RT-PCR and normalized to GAPDH at all time points. (n =​ 6 mice per group, P >​ 0.05 vs. LPS +​ I/R +​ Scrambled control group). (b) Kidney lysates of 6 h groups were subject to Western blot analysis for TRAF6 and IRAK-1. (c) The Western blots from all 6 h experiments were quantified by densitometry analysis. The ratios of TRAF6/ β​-actin and IRAK-1/ β​-actin were calculated; the fold changes relative to LPS +​ Sham protein are shown. (n =​ 6 mice per group, *​P <​ 0.05, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control group). (d) Time course of TRAF6 and IRAK-1 expression was confirmed by Western-blot in the LPS-challenged kidney following I/R injury. Figure 6. Knockdown of miR-146a promotes TRAF6 and IRAK-1 protein expression. LNA-anti-miR-14 Figure 6. Knockdown of miR-146a promotes TRAF6 and IRAK-1 protein expression. LNA-anti-miR-146a or anti-scrambled oligonucleotides (10 m/kg) were administered 2 h before LPS treatment. After 24 h LPS exposure, the kidneys were subjected to I/R or Sham procedures followed by 6 h, 24 h, 48 h of reperfusion and then harvested. www.nature.com/scientificreports/ Thus, miR-146a may be a viable strat- egy to prevent acute inflammatory response and improve the outcome of AKI. Methods A i l Animal models. A warm kidney I/R model was induced in 8-week-old male C57BL/6 mice (Animal Center of Fudan University, shanghai, China) and as described previously43. In brief, anesthesia was induced with intra- peritoneal sodium pentobarbital (80 mg/kg body weight) and kidney I/R was induced by bilateral kidney pedicle clamping for 30 min, followed by reperfusion for the indicated time. Sham controls underwent all the same sur- gical procedures except vascular occlusion. This study was approved by the International Animal Care and Use Committee of Fudan University and adhered strictly to the National Institutes of Health Guide For the Care and Use of Laboratory Animals. Experimental design. LNA-modified anti-scrambled or anti-miR-146a oligonucleotides (Exiqon, Woburn, MA) were diluted in saline (5 mg/ml), and administered into the tail vein (10 mg/kg) 2 hour prior to LPS injection. At 24 h after exposure to either normal saline (NS) or lipopolysaccharide (LPS, from E. coli sero- type 055:B5, 3 mg/kg body weight), mice were subjected to kidney ischemia or sham surgery. The animals were divided into following groups (n =​ 6 per group): (1) I/R +​ anti-miR-146a +​ I/R and I/R +​ scrambled control; (2) LPS +​ I/R +​ anti-miR-146a and LPS +​ I/R +​ scrambled control; (3) LPS +​ Sham; (4) NS (normal saline) +​ Sham. Blood and kidney samples were examined at 6, 24 and 48 h after the surgery. For time course experiments, sam- ples were collected at 0, 1, 2, 3, 5, 7 days following the I/R or sham injury in additional groups of mice (n =​ 4 per group). Plasma creatinine was measured using the improved Jaffe method (Quantichrom creatinine Assay Kit, BioAssay Systems, Hayward, CA). Concentrations of cytokines in tissue homogenate were examined by Scientific Reports | 6:27091 | DOI: 10.1038/srep27091 8 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 7. The link of miR-146a and NF-κB activation. (a) Immunohistochemistry shows positive nuclear NF-κ​B p50 staining 24 h after I/R in mice exposed to LPS with or without miR-146a knockdown (original magnification x200, Bar =​ 50 um) (b) Nuclear NF-κ​B p50 expression was scored in mouse kidney. (n =​ 6 mice per group, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control group). Methods A i l The specificity of the staining was further demonstrated by the absence of signals in LPS +​ Sham group (c) Kidney lysates of 24 h groups were probed with specific antibody against phosphorylated P65 (p-p65) (nuclear extracts), and IkBα​, BcL-xL, T-P65 (cytosolic extracts). Co-detection of Histone H3 (nuclear) and β​-actin (cytoplasm) were performed to assess equal loading. A significant increase in nuclear NF-κ​B p65 expression, cytosolic IkBα​ and BcL-xL degradation at 24 h was observed. (d) The Western blots from all experiments were quantified by densitometry analysis. The ratios of phosphor-protein to total protein for p65 were calculated. The fold changes relative to LPS +​ Sham protein are shown. (n =​ 3, #P <​ 0.001, *​*​P <​ 0.01, vs. LPS +​ I/R +​ Scrambled control group). Figure 7. The link of miR-146a and NF-κB activation. (a) Immunohistochemistry shows positive nuclear NF-κ​B p50 staining 24 h after I/R in mice exposed to LPS with or without miR-146a knockdown (original magnification x200, Bar =​ 50 um) (b) Nuclear NF-κ​B p50 expression was scored in mouse kidney. (n =​ 6 mice per group, #P <​ 0.001, vs. LPS +​ I/R +​ Scrambled control group). The specificity of the staining was further demonstrated by the absence of signals in LPS +​ Sham group (c) Kidney lysates of 24 h groups were probed with specific antibody against phosphorylated P65 (p-p65) (nuclear extracts), and IkBα​, BcL-xL, T-P65 (cytosolic extracts). Co-detection of Histone H3 (nuclear) and β​-actin (cytoplasm) were performed to assess equal loading. A significant increase in nuclear NF-κ​B p65 expression, cytosolic IkBα​ and BcL-xL degradation at 24 h was observed. (d) The Western blots from all experiments were quantified by densitometry analysis. The ratios of phosphor-protein to total protein for p65 were calculated. The fold changes relative to LPS +​ Sham protein are shown. (n =​ 3, #P <​ 0.001, *​*​P <​ 0.01, vs. LPS +​ I/R +​ Scrambled control group). commercially available enzymelinked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) for interleukin (IL)-1β​, and tumor necrosis factor (TNF)-α​, according to the manufacturer’s protocol. commercially available enzymelinked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) for interleukin (IL)-1β​, and tumor necrosis factor (TNF)-α​, according to the manufacturer’s protocol. The number of TUNEL-positive cells from 10 areas of randomly selected kidney cortex was counted under a light microscope. Real-time RT-PCR. Quantitative RT-PCR for gene expression were carried out using cDNA reverse tran- scribed from RNA extracted from harvested kidney tissue as described using primers corresponding to the gene of interest. Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA), followed by quantification. Extracted RNA was reverse-transcribed to complementary DNA (PrimeScript RT reagent Kit; TaKaRa, Kyoto, Japan). PCR was performed using SYBR Green Master Mix (Premix Ex TaqTM TaKaRa and the Applied Biosystems 7500 Real-time PCR system). PCR primers were designed using Primer-Blast (NCBI) to span at least one intron of the targeted gene. Ct values of target genes were normalized to GAPDH and presented as fold increase compared to the reference experimental group using the 2−​△​△​CT method. PCR primers used are listed (Table 1). RT- and TaqMan-based real-time PCR. Total RNA from kidneys samples was reverse-transcribed using miRNA-specific stemloop RT primers, RTase, RT buffer, dNTPs, and RNase inhibitor according to the manufac- turer’s instructions (Applied Biosystems; ABI, Foster City, CA). Expression level of miR-146a was quantified using real-time reverse transcription-PCR with the Taqman chemistry (ABI) and miRNA-specific TaqMan primers (Table 1), as described previously45. U6 small nuclear RNA was used as endogenous housekeeping control for data normalization of miRNA levels. Western blot analysis. The kidneys were lysed with RIPA solution containing 1% NP40, 0.1% SDS, 100 mg/ml PMSF, Complete Protease Inhibitor Cocktail Tablets (Roche, Indiana, USA) on ice. Nuclear extracts were iso- lated from harvested kidney medulla using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Inc, USA). The purity of nuclear proteins was verified by western blot analysis to confirm the absence of β​-actin (cytosolic protein) and the presence of Histone H3 (nuclear protein). Equal amounts of protein samples were electrophoretically separated on sodium dodecyl sulfate polyacrylamide gel, transferred to polyvi- nylidene fluoride membranes (Millipore), and probed with primary antibodies as follows: anti-BcL-xL (#2764, rabbit monoclonal 1:1000), anti-p65 (#6956, mouse monoclonal 1:1000) and anti-phosphorylated P65 (#3036, mouse monoclonal 1:1000) were from Cell Signaling Technology (Danvers, MA); anti-TRAF6 (sc-7221, rabbit polyclonal 1:1000) anti-IRAK-1(sc-7883, rabbit polyclonal 1:1000), anti-Iκ​B-α​ (sc-371, rabbit polyclonal 1:1000), anti- β​-actin(sc-8432, mouse monoclonal 1:1000), GAPDH (sc-365062, mouse monoclonal 1:1000) were from Santa Cruz Biotechnology (Santa Cruz, CA); Histone H3 (ab1791, rabbit polyclonal 1:1000) was from abcam. commercially available enzymelinked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) for interleukin (IL)-1β​, and tumor necrosis factor (TNF)-α​, according to the manufacturer’s protocol. Assessment of kidney injury. Kidneys were fixed in 10% formalin and embedded in paraffin. Histopathological changes were assessed on periodic acid–Schiffstained 4-mm-thick sections in 10 nonoverlap- ping fields (original magnification x200) of the corticomedullary junction and outer medulla. Tissue damage was assessed in a blinded manner and scored using a tubular damage score, as previously described44. Briefly, injury was scored according to the percentage of damaged tubules (loss of brush border, shedding of both necrotic and viable epithelial cells into the tubular lumen, tubular dilation, cast formation, and cell lysis): (i) less than 25% damaged; (ii) 25–50% damaged; (iii) 50–75% damaged; and (iv) more than 75% damaged. Immunohistochemistry. Kidney sections from mice were prepared as described A citrate-based antigen retrieval solution was used. Sections were exposed to 3% H2O2 in methanol for 5 minutes to quench endogenous peroxidases. Endogenous mouse Ig staining was blocked with M.O.MTM mouse IgG blocking regent (Vector Laboratories) for 1 h. Sections were incubated with the CD68 (ED-1, mouse monoclonal; Abcam, Cambridge, MA) or MPO (mouse monoclonal, 1:200; Novus) or anti-P50 (sc-114, rabbit polyclonal 1:5000), anti-cleaved Caspase-3 (#9661, rabbit polyclonal 1:500; cell signaling Technology, Danvers, MA), TLR4 (mouse monoclo- nal, 1:200; Abcam, Cambridge, MA) overnight at 4 °C for overnight. After washing, sections were incubated with an M.O.MTM biotinylated anti-mouse IgG or anti-rabbit biotinylated secondary antibody at room tempera- ture for 30 min, and then with the avidin–biotin–peroxidase complex (Vector M.O.M.TM Immunodetection Kit, Vector Laboratories). The reaction products were developed using the 3, 3′​-diaminobenzidine substrate from Vector Laboratory, mounted with a glass coverslip, and photographed using a Zeiss Axioplan2 microscope with a Scientific Reports | 6:27091 | DOI: 10.1038/srep27091 9 www.nature.com/scientificreports/ Gene Forward Reverse IRAK1 5′​-GAGACCCTTGCTGGTCAGAG-3′​ 5′​-GCTACACCCACCCACAGAGT-3′​ TRAF6 5′​-GCCCAGGCTGTTCATAATGT-3′​ 5′​-CGGATCTGATGGTCCTGTCT-3′​ IL-6 5′​-CCTCTCTGCAAGAGACTTCCATCCA-3′​ 5′​-AGCCTCCGACTTGTGAAGTGGT-3' MCP-1 5′​-TTAAAAACCTGGATCGGAACCAA-3′​ 5′​-GCATTAGCTTCAGATTTACGGGT-3′​ RANTES 5′​-GAGTGACAAACACGACTGCAAGAT-3′​ 5′​-CTGCTTTGCCTACCTCTCCCT-3′​ MIP-2 5′​-GCCCCCAGGACCCCA-3′​ 5‘-CTTTTTGACCGCCCTTGAGA-3′​ ICAM-1 5′​-GTGATGCTCAGGTATCCATCCA-3′​ 5′​-CACAGTTCTCAAAGCACAGCG-3′​ IL-1β​ 5′​-CCTTCCAGGATGAGGACATGA-3' 5′​-TCATCCCATGAGTCACAGAGGAT-3′​ TNF-α​ 5′​-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC-3′​ 5′​-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-3′​ Il-10 5′​-GCCTTCAGTATAAAAGGGGGACC-3′​ 5′​-GTGGGTGCAGTTATTGTCTTCCCG-3′​ GAPDH 5′​-GCCATCAACGACCCCTTCAT-3′​ 5′​-ATGATGACCCGTTTGGCTCC-3′​ Gene name MiRBaseID Sequences mmu-miR-146a MIMAT0000158 UGAGAACUGAAUUCCAUGGGUU Table 1 Real-time PCR primers and miR-146a TaqMan primers Table 1. Real-time PCR primers and miR-146a TaqMan primers. Q-imaging MP3.3 RTV camera under 200 or 400 magnification. The area labeled was quantified using Image-Pro Plus software. TUNEL assay. DeadEnd Colometric TUNEL System from Promega (Madison, WI) was used to detect apop- totic cells on formalin-fixed, paraffinembedded kidney sections. Manufacturer’s protocol was used to processes the sections. References 1. Bellomo, R., Kellum, J. A. & Ronco, C. Acute kidney injury. Lancet 380, 756–66 (2012). 2. Bonventre, J. V. & Zuk, A. Ischemic acute renal failure: an inflammatory disease? Kidney Int 66, 480–5 (2004). 3. Chapman, J. R., O’Connell, P. J. & Nankivell, B. J. Chronic renal allograft dysfunction. J Am Soc Nephrol 16, 3015–26 (2005). commercially available enzymelinked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) for interleukin (IL)-1β​, and tumor necrosis factor (TNF)-α​, according to the manufacturer’s protocol. Then the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody, and devel- oped by chemiluminescent Horseradish Peroxidase Substrate (Millipore, Billerica, MA). All values were normal- ized to the loading control and expressed as fold increase relative to control. Statistical analysis. Data are expressed as mean ±​ SEM. For comparison of means with more than two subgroups, the nonparametric Kruskal-Wallis ANOVA followed by Dunnett with significance cut off <​0.05 was used. All analyses were performed using GraphPad Prism Software. J y j y ( ) 2. Bonventre, J. V. & Zuk, A. Ischemic acute renal failure: an inflammatory disease? Kidney Int 66, 480–5 (2004). 3. Chapman, J. R., O’Connell, P. J. & Nankivell, B. J. Chronic renal allograft dysfunction. J Am Soc Nephrol 16, 3015–26 (2005). www.nature.com/scientificreports/ Identification of a microRNA signature of renal ischemia reperfusion injury. Proc Natl Acad Sci USA 10 14339–44 (2010). 28. Bonventre, J. 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Ichii, O. et al. Altered expression of microRNA miR-146a correlates with the development of chronic renal inflammation. Kidney In 81, 280–92 (2012). References Scientific Reports | 6:27091 | DOI: 10.1038/srep27091 10 Acknowledgementsh g This work is supported by the National Natural Science Foundation of China grant 81270779 and 81430015 (to X.D.), Science and Technology Commission of Shanghai 14DZ2260200 (to X.D.). Author Contributions www.nature.com/scientificreports/ Lu, Y. C., Yeh, W. C. & Ohashi, P. S. LPS/TLR4 signal transduction pathway. Cytokine 42, 145–51 (2008). 17. He, K. et al. Lipopolysaccharide-induced cross-tolerance against renal ischemia-reperfusion injury is mediated by hypoxia-inducible factor-2alpha-regulated nitric oxide production. Kidney Int 85, 276–88 (2014). p g p y 8. Baltimore, D., Boldin, M. P., O’Connell, R. M., Rao, D. S. & Taganov, K. D. MicroRNAs: new regulators of immune cell developmen and function. Nat Immunol 9, 839–45 (2008).i 19. Pritchard, C. 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Author Contributions ut o Co t but o s Y.D., P.J. and X.J. performed the experiments, H.L. scored the histology and Y.D. and Y.F. performed the data analysis. X.D. and J.C.H. designed the experimental protocol and Y.D. wrote the paper. Scientific Reports | 6:27091 | DOI: 10.1038/srep27091 11 www.nature.com/scientificreports/ Scientific Reports | 6:27091 | DOI: 10.1038/srep27091 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Dai, Y. et al. miR-146a is essential for lipopolysaccharide (LPS)-induced cross-tolerance against kidney ischemia/reperfusion injury in mice. Sci. Rep. 6, 27091; doi: 10.1038/srep27091 (2016). How to cite this article: Dai, Y. et al. miR-146a is essential for lipopolysaccharide (LPS)-induced cross-tolerance against kidney ischemia/reperfusion injury in mice. Sci. Rep. 6, 27091; doi: 10.1038/srep27091 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports | 6:27091 | DOI: 10.1038/srep27091 12
https://openalex.org/W4377833255
https://jurnal.ukdc.ac.id/index.php/BIP/article/download/396/274
Indonesian
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Analisis Pengaruh Disiplin Dan Motivasi Kerja Terhadap Produktivitas Kerja Karyawan Hotel Goodrich Suites Jakarta
BIP'S/BIP'S: Jurnal Bisnis dan Perspektif
2,023
cc-by-sa
4,681
p-ISSN 1979-4932 e-ISSN 2715-2596 p-ISSN 1979-4932 e-ISSN 2715-2596 p-ISSN 1979-4932 e-ISSN 2715-2596 Jurnal Bisnis Perspektif (BIP’s) Volume 15, Nomor 1, Januari 2023 Halaman 65-77 http://jurnal.ukdc.ac.id/index.php/BIP ABSTRACT The existence of hotels in Indonesia is increasing every year. In line with the increasing number of new hotels, the competition in the hotel industry is also getting tougher. Every hotel should be able to provide the best to the visitors. In an effort to realize this, it is very important to have employees who have good work productivity in an effort to achieve the goals set by the company. The purpose of this study was to determine the effect of discipline and work motivation on work productivity of the employees of the Hotel Goodrich Suites Jakarta. The population of this study were all employees of Hotel Goodrich Suites Jakarta, totaling 60 people and all populations were taken as samples. So the sample is a saturated sample or census. Based on the results of data processing with SPSS, it was found that all statement items for each variable were declared valid and all variables used in the study were declared reliable. The results of the classic assumption test consisting of the normality test, multicollinearity test, and heteroscedasticity test show that all of them fulfill. The multiple linear regression equation obtained is Y = 6.795 + 0.211 X1 + 0.189 X2. The Adjusted R Square value is 0.116 (11.6%), so it can be said that the contribution of work discipline and work motivation variables to work productivity for employees at the Goodrich Suites Hotel Jakarta is very low. Based on the results of the t test, it can be said that work discipline has a significant effect on the work productivity of Goodrich Suites Jakarta hotel employees, while work motivation has no effect on employee work productivity. Keywords: work discipline, work motivation, work productivity. ANALISIS PENGARUH DISIPLIN DAN MOTIVASI KERJA TERHADAP PRODUKTIVITAS KERJA KARYAWAN HOTEL GOODRICH SUITES JAKARTA Wendy Shelma Santika1, Thyophoida W. S. Panjaitan2* 1, 2Program Studi Manajemen, Fakultas Ekonomi, Universitas Katolik Darma Cendika Jalan Dr. Ir. H. Soekarno 201, Surabaya *Corresponding Author: thyophoidapanjaitan@gmail.com Kata kunci: disiplin kerja, motivasi kerja, produktivitas kerja. Kata kunci: disiplin kerja, motivasi kerja, produktivitas kerja. ABSTRAK Keberadaan hotel di Indonesia semakin meningkat setiap tahunnya. Sejalan dengan semakin banyaknya hotel baru maka semakin ketat juga persaingan yang ada pada industri hotel. Setiap hotel harus dapat memberikan yang terbaik kepada para pengunjung. Dalam upaya mewujudkan hal tersebut maka sangat penting memiliki karyawan yang mempunyai produktivitas kerja yang baik dalam upaya mencapai tujuan yang telah ditetapkan perusahaan. Tujuan dari penelitian ini 65 Analisis Pengaruh Disiplin dan Motivasi Kerja Terhadap Produktivitas Kerja Karyawan Hotel Goodrich Suites Jakarta Wendy Shelma Santika dan Thyophoida W. S. Panjaitan untuk mengetahui pengaruh disiplin dan motivasi kerja terhadap produktivitas kerja pada karyawan Hotel Goodrich Suites Jakarta. Populasi dari penelitian ini adalah seluruh karyawan Hotel Goodrich Suites Jakarta yang berjumlah 60 orang dan semua populasi diambil sebagai sampel. Jadi sampelnya merupakan sampel jenuh atau sensus. Berdasarkan hasil pengolahan data dengan SPSS diperoleh hasil bahwa semua item pernyataan dari setiap variabel dinyatakan valid dan semua variabel yang digunakan dalam penelitian dinyatakan reliabel. Hasil uji asumsi klasik yang terdiri dari uji normalitas, uji multikolinearitas, dan uji heteroskedastisitas menunjukkan bahwa semua memenuhi. Adapun Persamaam regresi linier berganda yang diperoleh adalah Y = 6,795 + 0,211 X1 + 0,189 X2. Nilai Adjusted R Square sebesar 0,116 (11,6%), sehingga dapat dikatakan kontribusi varibel disiplin kerja dan motivasi kerja terhadap produktivitas kerja pada karyawan Hotel Goodrich Suites Jakarta sangat rendah. Berdasarkan hasil uji t dapat dikatakan disiplin kerja berpengaruh signifikan terhadap produktivitas kerja karyawan Hotel Goodrich Suites Jakarta, sedangkan motivasi kerja tidak berpengaruh terhadap produktivitas kerja karyawan. PENDAHULUAN Industri yang bergerak dalam usaha perhotelan di Indonesia dewasa ini mengalami perkembangan yang sangat pesat. Perkembangan yang terjadi pada suatu hotel dapat dilihat dari pertumbuhan tingkat hunian kamar, di mana data pertumbuhan tingkat hunian kamar pada hotel berbintang yang ada di Indonesia pada tahun 2020-2022 dapat dilihat pada Gambar 1. Sumber: Javier, 2022 Gambar 1 Tingkat Hunian Kamar Hotel Berbintang di Indonesia Tahun 2020 - 2022 Gambar 1 Tingkat Hunian Kamar Hotel Berbintang di Indonesia Tahun 2020 - 2022 Tingkat Hunian Kamar Hotel Berbintang di Indonesia Tahun 2020 - 2022 66 Jurnal Bisnis Perspektif (BIP’s), Volume 15, Nomor 1, Januari 2023 Dari Gambar 1 terlihat bahwa di Indonesia pada bulan Juni 2022 jumlah tingkat hunian kamar hotel berbintang sebesar 50,28%, di mana jumlah tersebut mengalami peningkatan sebesar 11,73% dibandingkan pada bulan Juni 2021. Menurut Chaer (2020) dengan semakin meningkatnya jumlah hotel yang ada maka akan berdampak semakin meningkatnya persaingan yang terjadi pada industri perhotelan. Salah satu hotel yang juga menghadapi persaingan adalah Hotel Goodrich Suites Jakarta. Dalam upaya menghadapi persaingan yang ada, maka sangat penting bagi Hotel Goodrich Suites untuk memiliki sumber daya manusia yang mempunyai produktivitas kerja yang baik. Oleh karena itu, sangat penting bagi manajemen hotel untuk mengelola sumber daya manusia yang ada dengan baik dalam upaya menghasilkan sumber daya yang berkualitas, karena dengan memiliki karyawan yang memiliki kinerja baik maka dapat meningkatkan pertumbuhan hotel. Menurut Lande et al. (2016) dalam Baiti et al. (2020) terdapat beberapa faktor yang dapat mempengaruhi produktivitas kerja dari seorang karyawan yaitu motivasi, disiplin kerja, dan lingkungan kerja. Kemudian ada beberapa hal yang dapat mempengaruhi kedisiplinan karyawan dalam bekerja yaitu kurang optimalnya memanfaatkan waktu dalam bekerja sehingga berdampak pada tidak dapatnya menyelesaikan pekerjaan tepat waktu, serta kehadiran kerja yang tidak tepat waktu. Tingkat disiplin karyawan yang rendah akan berdampak terhadap produktivitas kerja yang dihasilkan dan pada akhirnya akan berdampak pada kinerja hotel. Santoso dan Ramadhan (2020) menyatakan disiplin dan motivasi kerja memiliki pengaruh yang positif dan signifikan terhadap produktivitas karyawan. Oleh karena itu, selain memperhatikan disiplin kerja, suatu perusahaan juga perlu memberikan motivasi kepada para karyawan. Motivasi kerja dapat menjadi pendorong bagi karyawan dalam melaksanakan pekerjaannya. Adapun bentuk motivasi yang dapat diberikan seperti kompensasi yang adil, penghargaan, dan lain-lain. Menurut Yohanson et al. (2022) terdapat beberapa permasalahan mengenai disiplin kerja antara lain datang terlambat dengan alasan jarak yang ditempuh jauh atau jalanan yang macet, karyawan yang tidak dapat menyelesaikan pekerjaan dengan tepat waktu. TINJAUAN PUSTAKA Disiplin Kerja p j Wau et al. (2021) menyatakan disiplin kerja merupakan suatu sikap menghormati, menghargai, taat terhadap peraturan yang berlaku baik secara tertulis maupun tidak tertulis serta mampu menjalankan dan menerima sanksi apabila melanggar peraturan yang telah dibuat. Sutrisno (2019) dalam Djaya (2021) menyatakan disiplin kerja merupakan suatu alat yang digunakan manajer dalam berkomunikasi dengan para karyawan dalam upaya merubah perilaku serta meningkatkan kesadaran dan kesediaan seseorang untuk mentaati peraturan dan norma-norma sosial yang ada. Menurut Hasibuan (2016) dalam Mamanna et al. (2022) kedisiplinan adalah adanya kesadaran serta kesediaan dari setiap karyawan untuk selalu mentaati setiap peraturan dan norma sosial yang berlaku. Oleh karena itu dapat dikatakan bahwa disiplin kerja sangat penting bagi suatu perusahaan dalam upaya mencapai tujuan yang diinginkan. Selain itu, disiplin kerja juga memiliki keterkaitan yang erat dengan setiap kegiatan manajemen di mana berhubungan dengan standar kerja yang harus dicapai suatu perusahaan dalam menjalankan tujuannya secara efektif dan efisien (Mamanna et al., 2022). Menurut Pratiwi (2018) terdapat beberapa jenis disiplin kerja yaitu: a. Disiplin preventif, merupakan kegiatan yang dilakukan untuk mendorong pegawai mengikuti aturan yang berlaku sesuai dengan Undang-Undang. b. Disiplin korektif, merupakan kegiatan yang diambil untuk menangani pelanggaran terhadap aturan yang sudah ditetapkan. c. Disiplin waktu, merupakan disiplin yang mudah dilihat dan dikontrol baik dari pihak manajemen maupun masyarakat. d. Disiplin kerja, terdiri atas metode pengerjaan, prosedur kerjanya, waktu, dan jumlah unit yang telah ditetapkan. Adapun indikator disiplin kerja menurut Pranitasari dan Khotimah (2021) antara lain: a. Mentaati peraturan waktu yang dapat dilihat dari jam masuk kerja, jam pulang kerja, dan jam istirahat sesuai aturan yang berlaku di perusahaan. b. Mentaati peraturan perusahaan yaitu peraturan tentang cara berpakaian, sikap, dan tingkah laku dalam pekerjaan. c. Mentaati peraturan perilaku dalam bekerja yang dapat ditunjukkan dengan cara bekerja sesuai dengan jabatan, tugas, dan tanggung jawab, serta cara berhubungan dengan unit kerja lain. d. Mentaati peraturan perusahaan tentang apa yang boleh dan tidak boleh dilakukan oleh pegawai. PENDAHULUAN Produktivitas kerja karyawan dapat ditingkatkan dengan melalui beberapa faktor. Menurut Gumelar (2017) terdapat 3 variabel yang mempengaruhi produktivitas kerja karyawan, yaitu kedisiplinan, motivasi kerja yang tinggi, dan penerapan SOP (Standard Operating Procedure). Irawan et al. (2021) menyatakan bahwa disiplin kerja tidak berpengaruh terhadap kinerja pegawai, sedangkan Kuswibowo (2020) menyatakan disiplin berpengaruh positif dan signifikan terhadap produktivitas kerja. Kemudian Hidayat (2021) menyatakan bahwa motivasi tidak berpengaruh terhadap kinerja karyawan, sedangkan Kuswibowo (2020) menyatakan motivasi kerja berpengaruh positif dan signifikan terhadap produktivitas kerja. Berdasarkan latar belakang dan adanya beberapa hasil penelitian yang tidak konsisten, maka dilakukan penelitian yang bertujuan untuk mengetahui apakah disiplin dan motivasi kerja berpengaruh terhadap produktivitas kerja karyawan Hotel Goodrich Suites Jakarta. 67 Analisis Pengaruh Disiplin dan Motivasi Kerja Terhadap Produktivitas Kerja Karyawan Hotel Goodrich Suites Jakarta Wendy Shelma Santika dan Thyophoida W. S. Panjaitan Analisis Pengaruh Disiplin dan Motivasi Kerja Terhadap Produktivitas Kerja Karyawan Hotel Goodrich Suites Jakarta Wendy Shelma Santika dan Thyophoida W. S. Panjaitan Analisis Pengaruh Disiplin dan Motivasi Kerja Terhadap Produktivitas Kerja Karyawan Hotel Goodrich Suites Jakarta Wendy Shelma Santika dan Thyophoida W. S. Panjaitan Motivasi Kerja Sutrisno (2016) dalam Djaya (2021) menyatakan bahwa motivasi adalah sebuah faktor yang mendorong seseorang untuk melakukan aktivitas tertentu. Widodo (2015) dalam Ma’ruf dan Chair (2020) menyatakan motivasi merupakan kekuatan yang berasal dari diri sendiri yang mendorong perilakunya untuk melakukan suatu kegiatan. Hasibuan (2010) dalam Kurniasari (2018) menyatakan tujuan dari motivasi kerja adalah: 68 Jurnal Bisnis Perspektif (BIP’s), Volume 15, Nomor 1, Januari 2023 a. Untuk meningkatkan moral dan kepuasan kerja. b. Untuk mendorong semangat kerja karyawan. c. Untuk meningkatkan produktivitas karyawan. d. Untuk meningkatkan kedisiplinan karyawan. e. Untuk meningkatkan hubungan kerja yang baik. f. Untuk meningkatkan kesejahteraan karyawan . g. Untuk meningkatkan kreativitas karyawan. h. Untuk mempertinggi rasa tanggung jawab terhadap tugasnya. i. Untuk meningkatkan kinerja karyawan. Menurut Sanjaya (2018) terdapat beberapa bentuk motivasi kerja yang ada yaitu kompensasi dalam bentuk uang, pengarahan dan pengendalian, serta kebijakan yang telah diambil dengan sengaja oleh pihak manajemen untuk mempengaruhi sikap karyawan. Adapun indikator motivasi kerja menurut George (2005) dalam Halim dan Andreani (2017) adalah: 1. Perilaku Karyawan, cara karyawan memilih berperilaku dalam bekerja di perusahaan. Apabila karyawan berperilaku baik menandakan bahwa karyawan tersebut termotivasi untuk bekerja. 2. Usaha Karyawan, semakin karyawan bekerja keras dalam melakukan usaha maka menandakan semakin tinggi motivasinya dalam bekerja. 3. Kegigihan Karyawan, karyawan mempunyai keinginan untuk mau terus belajar walaupun ada rintangan dan masalah. Semakin tinggi kegigihan karyawan menunjukkan bahwa karyawan memiliki motivasi yang tinggi. Jurnal Bisnis Perspektif (BIP’s), Volume 15, Nomor 1, Januari 2023 Hipotesis Penelitian Pada penelitian ini ada 2 hipotesis yang akan diujikan, yaitu: Pada penelitian ini ada 2 hipotesis yang akan diujikan, yaitu: H1 : Disiplin kerja berpengaruh signifikan terhadap produktivitas kerja. 2 : Motivasi kerja berpengaruh signifikan terhadap produktivitas kerja. H2 : Motivasi kerja berpengaruh signifikan terhadap produktivitas kerja. Produktivitas Kerja Manullang dan Munthe (2013) dalam Ernawati (2017) menyatakan produktivitas kerja adalah sikap semangat yang dimiliki seseorang untuk bekerja keras dan ingin melakukan perbaikan. Hafid et al. (2018) menyatakan produktivitas kerja merupakan kemampuan karyawan dalam memproduksi suatu barang atau jasa sesuai dengan tujuan yang sudah ditetapkan. Sedarmayanti (2012) dalam Ernawati (2017) menyatakan terdapat beberapa sumber dalam produktivitas kerja yaitu: a. Penggunaan pikiran, dalam upaya memberikan hasil yang maksimal dengan menggunakan cara kerja yang mudah. a. Penggunaan pikiran, dalam upaya memberikan hasil yang maksimal dengan menggunakan cara kerja yang mudah. b. Penggunaan tenaga fisik atau jasmani, dalam mengerjakan suatu hal untuk memperoleh hasil dengan jumlah yang banyak dan mutu yang baik. c. Penggunaan waktu yang tepat, dalam mengerjakan suatu hal membutuhkan waktu yang cepat. d. Penggunaan ruangan, menggunakan ruang dengan luas yang wajar. e. Penggunaan bahan dan uang, dalam menggunakan bahan dan uang tidak terlalu banyak sehingga bisa berhemat. y gg Indikator produktivitas kerja menurut Hafid (2018) adalah sebagai berikut: a. Keteladanan pemimpin, memberikan teladan kepada karyawan merupakan salah satu cara memotivasi karyawan untuk bekerja dengan lebih baik lagi. b. Lingkungan kerja, dengan adanya lingkungan yang baik maka membuat karyawan lebih senang dan berkonsentrasi dalam bekerja sehingga meningkatkan kinerja pada karyawan. 69 Analisis Pengaruh Disiplin dan Motivasi Kerja Terhadap Produktivitas Kerja Karyawan Hotel Goodrich Suites Jakarta Wendy Shelma Santika dan Thyophoida W. S. Panjaitan Analisis Pengaruh Disiplin dan Motivasi Kerja Terhadap Produktivitas Kerja Karyawan Hotel Goodrich Suites Jakarta Wendy Shelma Santika dan Thyophoida W. S. Panjaitan c. Kemampuan, merupakan bakat yang dimiliki oleh seseorang secara sengaja atau natural untuk melakukan tugas tertentu dengan sebaik-baiknya. Analisis Pengaruh Disiplin dan Motivasi Kerja Terhadap Produktivitas Kerja Karyawan Hotel Goodrich Suites Jakarta Wendy Shelma Santika dan Thyophoida W. S. Panjaitan f. Uji t f. Uji t Sugiyono (2018: 223) menyatakan uji t merupakan uji hipotesis parsial yang digunakan untuk mengetahui pengaruh antar variabel bebas dengan variabel terikat. Hasil dari uji t dilihat dari nilai t tabel terhadap t hitung serta tingkat signifikannya. Indikator yang membandingkan t tabel dengan t hitung dengan signifikansi 0,05 yaitu: f. Uji t Sugiyono (2018: 223) menyatakan uji t merupakan uji hipotesis parsial yang digunakan untuk mengetahui pengaruh antar variabel bebas dengan variabel terikat. Hasil dari uji t dilihat dari nilai t tabel terhadap t hitung serta tingkat signifikannya. Indikator yang membandingkan t tabel dengan t hitung dengan signifikansi 0,05 yaitu: a. Apabila t hitung > t tabel dan nilai sig. < 0,05, maka variabel independen berpengaruh signifikan terhadap variabel dependen. b. Apabila t hitung ≤ t tabel dan nilai sig. ≥ (0,05), maka variabel independen tidak berpengaruh terhadap variabel dependen. METODE PENELITIAN Penelitian ini merupakan penelitian kuantitatif. Variabel yang digunakan dibagi menjadi 2, yaitu variabel bebas dan variabel terikat. Variabel bebas dalam penelitian ini yaitu disiplin kerja dan motivasi kerja. Sedangkan variabel terikatnya adalah produktivitas kerja. Populasi pada penelitian ini adalah semua karyawan Hotel Goodrich Suites Jakarta yang berjumlah 60 karyawan, dan semua populasi digunakan sebagai sampel maka sampel dikatakan sampel jenuh. Metode dan teknik analisis data dalam penelitian ini adalah sebagai berikut: 1. Uji Validitas Ghozali (2018: 51) menyatakan uji validitas bertujuan untuk mengukur valid atau tidaknya suatu kuesioner. Kriteria yang digunakan untuk mengukur valid atau tidaknya yaitu r hitung ≥ r tabel (uji dua sisi dengan signifikan < 0,05). 2. Uji Reliabilitas Menurut Ghozali (2018: 45) uji reliabilitas merupakan alat untuk mengukur variabel yang akan diteliti. Kuesioner dapat dikatakan reliabel jika jawaban responden terhadap pernyataan adalah konsisten dari waktu ke waktu. Adapun kriteria uji reliabilitas adalah apabila nilai Cronbach`s Alpha ≥ 0,60. 3. Uji Asumsi Klasik a. Uji Normalitas Untuk menguji data normal atau tidaknya menggunakan P-P Plot of Regression Standardized Residual. Dinyatakan berdistribusi normal apabila data menyebar di sekitar garis diagonal dan mengikuti arah garis diagonal. b. Uji Multikolinearitas Ghozali (2018: 107) menyatakan uji multikolineritas bertujuan untuk mengetahui ada atau tidak adanya korelasi antar variabel independen dalam regresi. Cara medeteksi multikolinearitas dapat melihat nilai tolerance dan varian infation factor (VIF) dengan kriteria jika nilai VIF < 10 maka data tersebut tidak terdapat multikolinearitas. c. Uji Heteroskedastisitas Ghozali (2018: 135) menyatakan uji heteroskedastisitas bertujuan untuk mengetahui apakah dalam sebuah model regresi terjadi ketidaksamaan varian dari residual suatu pengamatan ke pengamatan lainnya. Untuk melakukan uji digunakan Rank Spearman test yaitu mengolerasikan variabel bebas terhadap nilai absolut dari residual. Cara mengetahui adanya gejala adanya heteroskedastisitas yaitu apabila data terdapat pola tertentu yang teratur seperti gelombang, melebar kemudian menyempit maka data dapat dikatakan mengalami heteroskedastisitas. 70 Jurnal Bisnis Perspektif (BIP’s), Volume 15, Nomor 1, Januari 2023 d. Regresi Linear Berganda Regresi merupakan uji yang digunakan untuk memeriksa hubungan antara semua variabel. Rumus melihat persamaaan regresi linear berganda yaitu: d. Regresi Linear Berganda Regresi merupakan uji yang digunakan untuk memeriksa hubungan antara semua variabel. Rumus melihat persamaaan regresi linear berganda yaitu: Hasil Penelitian Uji Validitas dan Reliabilitas Y = a + b1 X1 + b2 X2 + b3 X3 + e Y = a + b1 X1 + b2 X2 + b3 X3 + e e. Koefisien Determinasi (R2) Koefisien determinasi (R2) bertujuan untuk mengetahui pengaruh suatu variabel independen terhadap variabel dependen. Jika nilai R2 kecil maka kemampuan variabel bebas berkolerasi dengan variabel terikat terbatas. Interpretasi koefisien kolerasi menurut Sugiyono (2017: 257) terlihat pada Tabel 1. Tabel 1 Interpretasi Keofisien Korelasi Interval Koefisien Tingkat Hubungan 0,00 – 0,199 Sangat rendah 0,20 – 0,399 Rendah 0,40 - 0599 Sedang 0,60 – 0,7999 Kuat 0,80 – 1,000 Sedang kuat Sumber : Sugiyono (2017: 57) Uji Validitas dan Reliabilitas Hasil pengolahan data dengan program SPSS untuk uji validitas seperti terlihat pada Tabel 2. Tabel 2 Hasil Uji Validitas Variabel Item r hitung Sig Disiplin (X2) X2.1 0,800 0,000 X2.2 0,728 0,000 X2.3 0,757 0,000 X2.4 0,804 0,000 Tabel 2 Hasil Uji Validitas Variabel Item r hitung Sig Disiplin (X2) X2.1 0,800 0,000 X2.2 0,728 0,000 X2.3 0,757 0,000 X2.4 0,804 0,000 71 Analisis Pengaruh Disiplin dan Motivasi Kerja Terhadap Produktivitas Kerja Karyawan Hotel Goodrich Suites Jakarta Wendy Shelma Santika dan Thyophoida W. S. Panjaitan Analisis Pengaruh Disiplin dan Motivasi Kerja Terhadap Produktivitas Kerja Karyawan Hotel Goodrich Suites Jakarta Wendy Shelma Santika dan Thyophoida W. S. Panjaitan Motivasi Kerja (X3) X3.1 0,875 0,000 X3.2 0,860 0,000 X3.3 0,871 0,000 Produktivitas Kerja (Y) Y1.1 0,866 0,000 Y1.2 0,818 0,000 Y1.3 0,761 0,000 Data pada Tabel 2 menunjukkan bahwa semua item pernyataan variabel dinyatakan valid karena nilai signifikansi < 0,05. Data pada Tabel 2 menunjukkan bahwa semua item pernyataan variabel dinyatakan valid karena nilai signifikansi < 0,05. Hasil uji reliabilitas seperti terlihat pada Tabel 3, di mana diperoleh nilai Cronbach’s Alpha > 0,6, sehingga semua variabel dinyatakan reliabel. Tabel 3 Hasil Uji Reliabilitas Uji Asumsi Klasik a. Uji Normalitas Hasil uji normalitas seperti terlihat pada Gambar 2. Gambar 2 Uji Normalitas Data pada Gambar 2 menunjukkan bahwa titik-titik menyebar pada sekitar garis diagonal dan penyebarannya mengikuti arah garis diagonal, maka dapat dikatakan model regresi memenuhi asumsi normalitas. b. Uji Multikolinieritas Variabel Cronbach’s Alpha Disiplin (X2) 0,806 Motivasi Kerja (X3) 0,852 Produktivitas Kerja (Y) 0,833 Tabel 3 Hasil Uji Reliabilitas Variabel Cronbach’s Alpha Disiplin (X2) 0,806 Motivasi Kerja (X3) 0,852 Produktivitas Kerja (Y) 0,833 Uji Asumsi Klasik a. Uji Normalitas Hasil uji normalitas seperti terlihat pada Gambar 2. a. Uji Normalitas Hasil uji normalitas seperti terlihat pada Gambar 2. Gambar 2 Uji Normalitas Data pada Gambar 2 menunjukkan bahwa titik-titik menyebar pada sekitar garis diagonal dan penyebarannya mengikuti arah garis diagonal, maka dapat dikatakan model regresi memenuhi asumsi normalitas. b ji l ik li i i Data pada Gambar 2 menunjukkan bahwa titik-titik menyebar pada sekitar garis diagonal dan penyebarannya mengikuti arah garis diagonal, maka dapat dikatakan model regresi memenuhi asumsi normalitas. Data pada Gambar 2 menunjukkan bahwa titik-titik menyebar pada sekitar garis diagonal dan penyebarannya mengikuti arah garis diagonal, maka dapat dikatakan model regresi memenuhi asumsi normalitas. b. Uji Validitas dan Reliabilitas Uji Multikolinieritas Hasil uji multikolinearitas ditentukan oleh nilai tolerance dan VIF yang dapat dilihat pada Tabel 4. 72 Jurnal Bisnis Perspektif (BIP’s), Volume 15, Nomor 1, Januari 2023 Tabel 4 Hasil Uji Multikoliniaritas Variabel Tolerance VIF Disiplin (X2) 0,981 1,019 Motivasi Kerja (X3) 0,981 1,019 Data hasil uji multikolinearitas seperti terlihat pada Tabel 4 menunjukkan bahwa seluruh variabel memiliki nilai tolerance > 0,1 dan nilai VIF < 10,00. Hal tersebut menunjukkan bahwa model regresi ini tidak mengalami multikolinearitas. c. Uji Heteroskedastisitas Hasil uji heteroskedastisitas seperti terlihat pada Gambar 3. j Hasil uji heteroskedastisitas seperti terlihat pada Gambar 3. Gambar 3 Hasil Uji Heteroskedastisitas Hasil uji heteroskedastisitas menunjukkan bahwa penyebaran plot terletak di bagian bawah dan atas angka 0 pada sumbu Y. Plot yang tidak membentuk pola yang jelas ini dapat memberikan hasil bahwa model regresi pada penelitian ini tidak mengalami gejala heteroskedastisitas. Hasil uji heteroskedastisitas menunjukkan bahwa penyebaran plot terletak di bagian bawah dan atas angka 0 pada sumbu Y. Plot yang tidak membentuk pola yang jelas ini dapat memberikan hasil bahwa model regresi pada penelitian ini tidak mengalami gejala heteroskedastisitas. Analisis Regresi Linear Berganda Hasil analisis regresi linear berganda seperti terlihat pada Tabel 5. Dari hasil analisis linear berganda pada Tabel 5 dapat dibuat persamaan model regresi sebagai berikut: Y = 6,795 + 0,211 X1 + 0,189 X2 Tabel 5 Hasil Analisis Regresi Linear Berganda No Model Unstandardized Coefficients Standardized Coefficients B Std Error Beta Constant 6,795 1,826 1 Disiplin Kerja (X1) 0,211 0,092 0,282 2 Motivasi Kerja (X2) 0,189 0,105 0,222 a Dependent Variable: Y Tabel 5 Hasil Analisis Regresi Linear Berganda 73 Analisis Pengaruh Disiplin dan Motivasi Kerja Terhadap Produktivitas Kerja Karyawan Hotel Goodrich Suites Jakarta Wendy Shelma Santika dan Thyophoida W. S. Panjaitan Analisis Pengaruh Disiplin dan Motivasi Kerja Terhadap Produktivitas Kerja Karyawan Hotel Goodrich Suites Jakarta Wendy Shelma Santika dan Thyophoida W. S. Panjaitan Analisis Pengaruh Disiplin dan Motivasi Kerja Terhadap Produktivitas Kerja Karyawan Hotel Goodrich Suites Jakarta Wendy Shelma Santika dan Thyophoida W. S. Panjaitan Uji t j Uji t dilakukan dengan melihat nilai t hitung dan tingkat signifikansinya. Hasil uji t seperti terlihat pada Tabel 7. Data hasil uji t pada Tabel 7 menunjukkan nilai signifikansi X1 sebesar 0,026 < 0,05 yang berarti variabel X1 berpengaruh secara signifikan terhadap Y. Kemudian pada Tabel 7 juga nampak nilai signifikansi X2 sebesar 0,077 > 0,05 yang berarti variabel X2 tidak berpengaruh terhadap variabel Y. Tabel 7 Hasil Uji t Model t Sig. 1 (Constant) 3.720 .000 X1 2.284 .026 X2 1.799 .077 Pembahasan Koefisien Determinasi Hasil analisis koefisien determinasi (R2) seperti terlihat pada Tabel 6. Data yang ada di Tabel 6 menunjukkan nilai adjusted R Square sebesar 0,116, yang berarti bahwa variabel independen yaitu disiplin (X1), dan motivasi kerja (X2) memberikan kontribusi sangat rendah kepada produktivitas kerja (Y) yaitu hanya sebesar 0,116 (11,6%). Sedangkan sisanya sebesar 0,884 (88,4%) berasal dari kontribusi variabel lain. Tabel 6 Hasil Koefisien Determinasi Model R R Square Adjusted R Square Std. Error of the estimate 1 0,383 0,146 0,116 1,80213 Tabel 6 Pembahasan Pembahasan SARAN Dalam upaya meningkatkan disiplin kerja dari setiap karyawan Hotel Goodrich Suites yang ada, maka pihak manajemen hotel menerapkan ketentuan dan kebijakan bagi setiap karyawan untuk mentaati setiap peraturan yang berlaku, misalnya kebijakan terhadap jam kehadiran, di mana apabila karyawan terlambat akan ada sanksi seperti memberlakukan pemotongan uang kehadiran. Pihak manajemen dari Hotel Goodrich Suites Jakarta juga harus secara terus-menerus secara berkesinambungan memberikan motivasi kepada seluruh karyawannya dalam upaya timbulnya motivasi kerja dalam diri setiap karyawan dari Hotel Goodrich Suites Jakarta. Pengaruh Motivasi Kerja terhadap Produktivitas Kerja Karyawan Dari data pada Tabel 7 diperoleh nilai t hitung sebesar 1,799 dengan nilai signifikansi sebesar 0,077, yang berarti bahwa motivasi kerja tidak berpengaruh terhadap produktivitas kerja karyawan pada Hotel Goodrich Suites Jakarta. Hal ini menunjukkan bahwa hipotesis H2 ditolak. Hasil penelitian ini sejalan dengan hasil penelitian Hidayat (2021) yang menyatakan bahwa motivasi kerja tidak berpengaruh terhadap produktivitas karyawan. p g SIMPULAN Hasil penelitian ini menunjukkan bahwa disiplin kerja berpengaruh signifikan terhadap produktivitas kerja karyawan Hotel Goodrich Suites. Hasil penelitian juga menunjukkan bahwa motivasi kerja tidak berpengaruh terhadap produktivitas kerja karyawan Hotel Goodrich Suites. Pengaruh Disiplin Kerja terhadap Produktivitas Kerja Karyawan Pengaruh Disiplin Kerja terhadap Produktivitas Kerja Karyawa Hasil uji t pada Tabel 7 menunjukkan nilai t hitung sebesar 2,283 dengan nilai signifikansi sebesar 0,026, yang berarti disiplin kerja berpengaruh signifikan terhadap produktivitas kerja karyawan pada Hotel Goodrich Suites Jakarta. Hal ini menunjukkan bahwa hipotesis H1 diterima. Hasil penelitian ini sejalan dengan hasil penelitian Kuswibowo (2020) yang menyatakan bahwa disiplin berpengaruh signifikan terhadap kinerja karyawan. Dalam suatu perusahaan sangat penting memiliki karyawan yang memiliki disiplin bekerja dalam upaya mencapai tujuan yang telah ditetapkan perusahaan. Tidak terkecuali pada Hotel Goodrich Suites Jakarta, di mana apabila karyawan yang ada pada Hotel Goodrich Suites tidak memiliki disiplin dalam melaksanakan 74 Jurnal Bisnis Perspektif (BIP’s), Volume 15, Nomor 1, Januari 2023 pekerjaannya maka akan sulit bagi manajemen hotel dalam upaya memperoleh hasil yang optimal. Dengan semakin meningkatkan disiplin kerja karyawan yang ada pada Hotel Goodrich Suites, maka diharapkan akan meningkatkan produktivitas kerja dari para karyawannya dalam melayani setiap konsumen yang menginap di hotel. Dengan semakin meningkatnya disiplin kerja dari para karyawan maka diharapkan akan efektif dan efisien karyawan dalam bekerja, misalnya dengan karyawan bagian housekeeping datang tepat waktu sesuai dengan jam kerja yang berlaku di hotel maka pihak hotel akan dapat menyediakan kamar yang siap untuk dijual serta kerapian dan kebersihan kamar akan tetap terjaga karena tidak perlu terburu-buru dalam menyiapkan. Chaer, V. P. 2020. 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