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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
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8.a
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24" 3
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43 ' 4
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74"0
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72"0
78"5
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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
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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
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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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https://europepmc.org/articles/pmc4549369?pdf=render
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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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SpringerPlus
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cc-by
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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
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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
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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. Jeon, S., Ko, M., Lee, J., Choi, I., Byun, S.Y., Park, S., Shum, D. and Kim, S. (2020)
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G
d
D E
J
G M
B
h dd
M
X
J
Ob
i
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M J
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PRM2, RNF32, CNTN6, ACSL6, UBQLN3, HNF4G, ANKRD7, MXD1, SLC17A3, IL12RB2,
ANKRD40CL, SLC6A20, SLC5A12, SLC10A2, ACRV1, ALPI, EPPIN, IL17A, AKAP4, HKDC1,
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In-Pile Qualification of a Fast-Neutron-Detection-System
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IEEE transactions on nuclear science
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cc-by
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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
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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
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A Nanoporous Alumina Membrane Based Electrochemical Biosensor for Histamine Determination with Biofunctionalized Magnetic Nanoparticles Concentration and Signal Amplification
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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 =
ோ
ଶగோାଵ+ ܴ,
(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,
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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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Nonlinear amplitude dynamics in flagellar beating
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Royal Society open science
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cc-by
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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
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
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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
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500
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20
40
–1
0
1
250
500
750
0
0.5
1.0
–0.2
0
0.2
250
500
750
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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
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0.2
f
eigenmode
t (ms)
s
(a) 11
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
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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). 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. 12 13
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Nonlinear amplitude dynamics in flagellar beating. Dryad Digital Repository. (doi:10.5061/dryad. qs65q) 26. Werner S, Rink JC, Riedel-Kruse IH, Friederich BM. 2014 Shape mode analysis exposes movement
patterns in biology: flagella and flatworms as case
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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
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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 𝑞and 𝑞such 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 𝑞are all acceptance edges, the incoming edges of 𝑞
are nonacceptance edges (See Figure 1). The outgoing edges
of 𝑞and 𝑞are the same as the outgoing edges of 𝑞, and
the labeling function ant and cons are the same. All such 𝑞𝑠
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 𝑞to 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 𝑞and 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 𝜌of 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
𝜌of 𝑀𝐺be 𝜌= (𝑞0, 0)(𝑞1, 0) ⋅⋅⋅(𝑞𝑙−1, 0)(𝑞𝑙, 1)(𝑞𝑙+1, 1) ⋅⋅⋅. This path 𝜌will visit infinitely often every (𝑞, 1) ∈𝐹if
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
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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
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graphs,” in Proceedings of the 12th Advanced Research Working
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English
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Calculation of Basalt Fiber Reinforced Concrete Beams for Strength
|
International journal of multidisciplinary research and analysis
| 2,022
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cc-by
| 4,269
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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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https://www.qeios.com/read/M4LWSY/pdf
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English
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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
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qualitativa. Porto Alegre: Artmed, 2009. _____________. Constructing Grounded Theory. A Practical Guide Through
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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
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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.
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de máster: Universidade Federal de Sergipe, 2013. HALL, P. M. Interactionism and the study of social organization.
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nº 37, 2006. TRINIDAD, A., CARRERO, V. e SORIANO, R. M. Teoría Fundamentada “Grounded
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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
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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
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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.
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ISSN 2318-3888
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HISOR TOG' TIZMASIDAGI QIZIL DО'LANANING (CRATAEGUS TURKESTANICA POJARK.) TABIIY KО'PAYISHI VA О'RMONCHILIKDAGI AHAMIYATI
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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
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GAMBARAN PEGETAHUAN REMAJA PUTRI TENTANG DISMINORE PADA MAHASISWA TINGKAT I PROGRAM STUDI DIPLOMA III KEPERAWATAN STIKES DIRGAHAY SAMARINDA
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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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PloS one
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cc-by
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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
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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. The
northeast
is
once
again
a
predominantly
forested
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Recursive Partitioning. Available: http://cran.r-project.org/web/packages/
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conditional inference framework. Journal of Computational and Graphical
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restoration strategies for the Mississippi River Alluvial Valley. Forest Ecology
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Georgia Piedmont. American Midland Naturalist 140: 78–89. 57. Orwig D, Foster DR, Mausel DL (2002) Landscape patterns of hemlock decline
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75. Huang C, Yang B, Wylie B, Homer C (2001) A strategy for estimating tree
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Thyroid Abscess: Challenges in Diagnosis and Management
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Journal of investigative medicine high impact case reports
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cc-by
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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
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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.,
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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
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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
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<span>Experimental and Computational Studies Addressed to 1,3-Dipolar Cycloadditions of D-Erythrose 1,3-Dioxane 1,5-Lactone with </span><em>Regio</em><span><em>- and</em><br /></span><em>Stereo-selectivity</em>
|
Proceedings of MOL2NET 2018, International Conference on Multidisciplinary Sciences, 4th edition
| 2,018
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cc-by
| 576
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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.
|
https://openalex.org/W4324360405
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https://www.researchsquare.com/article/rs-2097064/latest.pdf
|
English
| null |
An Efficient Method to Prepare Barcoded cDNA Libraries from Plant Callus for Long-Read Sequencing
|
Methods and protocols
| 2,023
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cc-by
| 6,046
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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
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polyadenylated transcription in embryonic stem cells reveals novel non-coding RNA related to
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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
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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
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epitranscriptome in plants using pacbio iso-seq and nanopore-based direct RNA sequencing. Front
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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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https://europepmc.org/articles/pmc4279428?pdf=render
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English
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Retracted: Immunization Coverage: Role of Sociodemographic Variables
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Advances in preventive medicine
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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
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Reward Activates Stimulus-Specific and Task-Dependent Representations in Visual Association Cortices
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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
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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)
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https://openalex.org/W4200490295
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https://www.qeios.com/read/P5VEAL/pdf
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English
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Review of: "ProteinGLUE: A multi-task benchmark suite for self-supervised protein modeling."
| null | 2,021
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cc-by
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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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https://openalex.org/W2988147444
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https://research.rug.nl/files/118018616/s12889_019_7797_7.pdf
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English
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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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BMC public health
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cc-by
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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
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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
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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. BMC Public Health, 19(1), Article
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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
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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–
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in young women. American Journal of Preventive Medicine. 2003;25(2):140–
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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/
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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
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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
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Associated with Obesity. Obesity (Silver Spring). 2009;17(7):1441–7. https://doi.org/10.1038/oby.2009.97. 31. Miech RA, Shanahan MJ, Boardman J, Bauldry S. The sequencing of a
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1Department of Sociology, McGill University, Peterson Hall Building 3460
McTavish Street, Montréal, QC H3A 0E6, Canada. 2Netherlands
Interdisciplinary Demographic Institute, Lange Houtstraat 19, 2511 CV The
Hague, The Netherlands. 3Department of Epidemiology, University Medical
Centre Groningen (UMCG) / University of Groningen, Groningen,
Netherlands. 4Department of Sociology, VU University Amsterdam,
Amsterdam, Netherlands. 5Department of Social and Political Sciences and
Carlo F. Dondena Centre for Research on Social Dynamics and Public
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Constraints; 2010. p. 22–39.
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STUDIES IN COLLOIDAL ARSENIC TRISULPHIDE. PART II. AGEING OF THE SOL
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Zenodo (CERN European Organization for Nuclear Research)
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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~.
|
https://openalex.org/W2414497709
|
https://europepmc.org/articles/pmc7235827?pdf=render
|
English
| null |
Deep Functional Profiling Facilitates the Evaluation of the Antibacterial Potential of the Antibiotic Amicoumacin
|
Antibiotics
| 2,020
|
cc-by
| 9,400
|
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.;
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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
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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.;
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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.;
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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,
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Templates To Find Out Pupils’ Views. Devon: Southgate Publishers. 30 Whitebread, D., Coltman, P., Pasternak, D. P., Sangster, C., Grau, V., Bingham,
S., Almeqdad, Q. & Demetriou, D. (2009). The development of two observational
tools for assessing metacognition and self-regulated learning in young children. Metacognition and Learning, 4(1), 63-85. doi 10.1007/s11409-008-9033-1
Wilson, D. B., & Lipsey, M. W. (2001). Practical meta-analysis (Vol. 49). London:
Sage Publications. Winne, P. H. (2005). A perspective on state-of-the-art research on self-regulated
learning. Instructional Science, 33, 559–565. doi: 10.1007/s11251-005-1280-9 Winne, P.H. (2011). A Cognitive and Metacogntive Analysis of Self-regulated
Learning in B.J. perspectives (pp. 1–37). Mahwah, NJ: Erlbaum. 27
Educational Studies, 59(2), 197-201. doi:10.1080/00071005.2011.584660
Higgins, S., Wall, K., Baumfield, V., Hall, E., Leat, D., Moseley, D. & Woolner, P.
(2007). Learning to Learn in Schools Phase 3 Evaluation: Final Report London,
Campaign for Learning. Retrieved from http://www.campaign-for-
learning.org.uk/cfl/assets/documents/Research/L2Lyr3report.pdf
Higgins, S., Kokotsaki, D. & Coe, R (2011a). Pupil Premium Toolkit: Summary for
Schools London: Sutton Trust. Retrieved from
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Higgins, S. Kokotsaki, D. & Coe, R (2011b). Pupil Premium Toolkit: Technical
Appendices London: Sutton Trust. Retrieved from
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technical-appendices/
Hill, C.J., Bloom, H.S., Black, A.R. & Lipsey, M.W. (2007). Empirical Benchmarks
for Interpreting Effect Sizes in Research MDRC. Working Papers on Research
Methodology New York: MDRC. Retrieved from
http://ww.mdrc.org/publications/459/full.pdf
Ho, R. (2006). Handbook of univariate and multivariate data analysis and
interpretation with SPSS. Florida: Chapman and Hall.
Ioannidis, J.P.A & Trikalinos T.A. (2007). The appropriateness of asymmetry
tests for publication bias in meta-analyses: a large survey. Canadian Medical Zimmerman & D. H. Schunk (eds) Handbook of Self-Regulation
of Learning and Performance Abingdon: Taylor and Francis. Zeidner, M., Boekaerts, M., & Pintrich, P. (2000). Self-regulation: Directions and
challenges for future research. In M. Boekaerts, P. Pintrich, & M. Zeidner (Eds.),
Handbook of self-regulation (pp. 750–768). San Diego, CA: Academic. Zimmerman, B. J. (1986). Becoming a self-regulated learner: Which are the key
subprocesses? Contemporary Educational Psychology, 11(4), 307-313. doi:
10.1016/0361-476X(86)90027-5 Zimmerman, B. J. (2001). Theories of self-regulated learning and academic
achievement: An overview and analysis. In B. J. Zimmerman, & D. H. Schunk
(Eds.), Self-regulated learning and academic achievement: Theoretical 31 perspectives (pp. 1–37). Mahwah, NJ: Erlbaum. Zimmerman, B. J. (2008). Investigating Self-Regulation and Motivation: Historical
Background, Methodological Developments, and Future Prospects American
Educational Research Journal 45. 166. 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
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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
| 2,024
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cc-by
| 1,821
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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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English
| null |
Microstructural evolution and mechanical properties of rheocast Al-7.1WT%Si alloy
|
Metallurgical & Materials Engineering
| 2,016
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cc-by
| 4,701
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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
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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
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PLoS neglected tropical diseases
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cc-by
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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. This paper, however, suggests that these modifiable factors are
more important than sociodemographic attributes and are thus a
direct target for disease prevention and control. 1. Beaver PC, Snyder CH, Carrera GM, Dent JH, Lafferty JW (1952). Chronic
eosinophilia due to visceral larva migrans - report of 3 cases. Pediatrics 9 (1):7–
19. Discussion 9
4.1 (2.0–7.3)
2
22.0 (3.9–56.2)
0.477
Enterobius vermicularis
1
0.4 (0.0 – 2.2)
0
0.0
-
Ascaris lumbricoides
2
0.9 (0.1–2.9)
0
0.0
-
Strongyloides stercoralis
6
3.1 (1.1–5.5)
1
16.0 (0.8–59.1)
0.662
Ancylostoma spp. 4
1.8 (0.5–4.3)
1
25.0 (1.2–75.7)
0.603
Total
33
14.9 (10.6–20.1)
6
18.2 (7.7–34.3)
0.519
{Based on 221 analyzed samples. {{Based on Exact Mid-P. {{{Based on Fisher exact test. doi:10.1371/journal.pntd.0002830.t004
PLOS Neglected Tropical Diseases | www.plosntds.org
8
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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
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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.,
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ROUTE ON THE FORMATION OF FOCAL ZONES FOR
THE DEVELOPMENT OF THE EASTERN ARCTIC Leonov S. N., Zaostrovskikh E. A. Economic Research Institute of Far Eastern Branch of the Russian Academy of Sciences (Khabarovsk, Russian Federation) Leonov S. N., Zaostrovskikh E. A. Economic Research Institute of Far Eastern Branch of the Russian Academy of Sciences (Khabarovsk, Russian Federation) The article was received on October 9, 2020 The article was received on October 9, 2020 Экономика и управление
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ц
р
Леонов Сергей Николаевич, доктор экономических наук, профессор, ведущий научный сотрудник, Инсти-
тут экономических исследований Дальневосточного отделения РАН (680042, Россия, Хабаровск, Тихоокеан-
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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-
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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:
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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
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Inflammopharmacology
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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
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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
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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. Discussion The antiallodynic activity of cebranopadol
(and morphine) in the oxaliplatin model of neuropathic
pain, in particular in the acute phase, together with the
results obtained in the formalin test indicate that both drugs
tested
are
able
to
attenuate
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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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https://www.eurosurveillance.org/deliver/fulltext/eurosurveillance/22/5/eurosurv-22-5-2.pdf?itemId=/content/10.2807/1560-7917.ES.2017.22.5.30454&mimeType=pdf&containerItemId=content/eurosurveillance
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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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Euro surveillance/Eurosurveillance
| 2,017
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cc-by
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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) !(
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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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https://openalex.org/W3204094951
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https://link.springer.com/content/pdf/10.1007/JHEP02(2022)143.pdf
|
English
| null |
Axion emission from supernova with axion-pion-nucleon contact interaction
|
The Journal of high energy physics/The journal of high energy physics
| 2,022
|
cc-by
| 10,329
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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
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degenerate system [32]. Figure 6 shows the emissivity ratio of the C2
aπN contribution with
respect to the other contributions from the axion-nucleon couplings for µn > mn. Actually,
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degenerated limit and that for non-degenerate case, the proton chemical potential is taken
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https://openalex.org/W4287898881
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https://zenodo.org/records/4311411/files/WJARR-2020-0018.pdf
|
English
| null |
Some aspects of associations and interactions between biomedical variables
|
Zenodo (CERN European Organization for Nuclear Research)
| 2,020
|
cc-by
| 5,926
|
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]
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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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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
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42
49
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Time (Days)
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Vector + Vehicle
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BAD
a
b
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e
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Time (Days)
Percent survival
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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
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Time (Days)
% Cell Death
Vector
BAD
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* * *
b b a Vector
BAD
d
Vehicle Docetaxel Vector
BAD
c
d
Vector + Vehicle
Vector + Dtxl
BAD + Vehicle
BAD + Dtxl
0
150
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Tumor Volume (mm3)
*
*
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Docetaxel treated Vector
d
Vehicle Docetaxel e
0
20
40
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25
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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
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16 24 48
0.0
0.5
1.0
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Docetaxel (Hours)
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BAD
*
*
Input
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_ +
_ +
IP: BAD
_ + _ +
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_ + _ +
Dtxl, 24 hrs
BAD
Bcl-XL
pBcl-XL-S62
Vector BAD Vector BAD
a
b 0
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BAD
*
* Input
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_ +
_ +
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_ + _ +
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Bcl-XL
pBcl-XL-S62
Vector BAD Vector BAD
b Dtxl (hrs)
Vector cells
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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
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0
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d
Vector BAD L114A
0
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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
-
-
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Days
Days
Control
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% Annexin-V +/PI -
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(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
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PARP
Tubulin
Tubulin
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+
+
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Input
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-
+
+
-
-
+
+
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). Uncoupling with stepwise titra-
tion to an optimal concentration of the protonophore carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone,
FCCP, induced maximal noncoupled respiration as a measure of electron transfer system capacity (ET state). Statistical analysis. GraphPad Prism Software was used for all statistical tests. A Student’s t-test was used
for comparisons between two groups. For comparisons between greater than two groups with one variable,
one-way ANOVA followed by a Dunnett’s (compared to one control group) or Tukey’s (comparing all groups to
each other) multiple comparisons tests were performed. A two-way ANOVA was used when two variables were
present. All data are presented as ± standard error of the mean (SEM). Experimental replicates are indicated and
were performed at least three times. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. References 1. Yu, K. D et al. Identification of prognosis-relevant subgroups in patients with chemoresistant triple-negative breast cancer, Clin
Cancer Res., https://doi.org/10.1158/1078-0432.CCR-12-2986 (2013). p
g
2. Bonnefoi, H. et al. TP53 status for prediction of sensitivity to taxane versus non-taxane neoadjuvant chemotherapy in breast cancer
(EORTC 10994/BIG 1-00): A randomised phase 3 trial, Lancet Oncol., https://doi.org/10.1016/S1470-2045(11)70094-8 (2011). g
2. Bonnefoi, H. et al. TP53 status for prediction of sensitivity to taxane versus non-taxane neoadjuvant chemotherapy in breast cancer
(EORTC 10994/BIG 1-00): A randomised phase 3 trial, Lancet Oncol., https://doi.org/10.1016/S1470-2045(11)70094-8 (2011). 2. Bonnefoi, H. et al. TP53 status for prediction of sensitivity to taxane versus non-taxane neoadjuvant chemotherapy in breast ca
(EORTC 10994/BIG 1-00): A randomised phase 3 trial, Lancet Oncol., https://doi.org/10.1016/S1470-2045(11)70094-8 (2011)
3. Murray, S. et al. Taxane resistance in breast cancer: Mechanisms, predictive biomarkers and circumvention strategies, Cancer T
Rev https://doi org/10 1016/j ctrv 2012 02 011 (2012) (EORTC 10994/BIG 1-00): A randomised phase 3 trial, Lancet Oncol., https://doi.org/10.1016/S1470-2045(11)70094-8 (2011)
3. Murray, S. et al. Taxane resistance in breast cancer: Mechanisms, predictive biomarkers and circumvention strategies, Cancer T
Rev., https://doi.org/10.1016/j.ctrv.2012.02.011 (2012). (EORTC 10994/BIG 1 00): A randomised phase 3 trial, Lancet Oncol., https://doi.org/10.1016/S1470 2045(11)70094 8 (2011). 3. Murray, S. et al. Taxane resistance in breast cancer: Mechanisms, predictive biomarkers and circumvention strategies, Cancer Treat. Rev., https://doi.org/10.1016/j.ctrv.2012.02.011 (2012). p
p
g
3. Murray, S. et al. Taxane resistance in breast cancer: Mechanisms, predictive biomarkers and circumvention strategies, Cancer Treat. Rev., https://doi.org/10.1016/j.ctrv.2012.02.011 (2012). g
j
4. Harris, L. N. et al. Use of biomarkers to guide decisions on adjuvant systemic therapy forwomenwith early-stage invasive breas
cancer: American Society of clinical Oncology clinical practice guideline, J. Clin. Oncol., https://doi.org/10.1200/JCO.2015.65.2289
(2016). 4. Harris, L. N. et al. Use of biomarkers to guide decisions on adjuvant systemic therapy forwomenwith early-stage invasive breast
cancer: American Society of clinical Oncology clinical practice guideline, J. Clin. Oncol., https://doi.org/10.1200/JCO.2015.65.2289
(2016).h 5. Craik, A. C. et al. The BH3-only protein bad confers breast cancer taxane sensitivity through a nonapoptotic mechanism, Oncogene. https://doi.org/10.1038/onc.2010.272 (2010). p
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(
)
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1. Yang, N., Weinfeld, M., Lemieux, H., Montpetit, B. & Goping, I. S. Photo-activation of the delocalized lipophilic cation D112
potentiates cancer selective ROS production and apoptosis, Cell Death Dis., https://doi.org/10.1038/cddis.2017.19 (2017). 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
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Determination of Optimal Line-Heating Conditions for Flatness Control of Wind Tower Blocks Using Strain as Direct Boundary Method
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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
ሺሻ+ ℎ௬ሺ𝑦ሻ
൱𝑑𝑧ൡ𝑑𝑦
ሻ+ ℎ௬ሺ𝑦ሻ
2
, ℎଶ= −ℎ
ሺሻ+ ℎ௬ሺ𝑦ሻ
2
+ ൫ℎ
ሺሻ+ ℎ
by pass ℎሺ𝑢ሻ: 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
ሺሻ+ ℎ௬ሺ𝑦ሻቁ∙𝑏
൝න
൭ℎ
+ ℎ௬ሺ𝑦ሻ
ℎ
∓
4𝑧
ℎ
ሺሻ+ ℎ௬ሺ𝑦ሻ
భ
∙
ℎ
ℎ
ሺሻ+ ℎ௬ሺ𝑦ሻ
൱𝑑𝑧ൡ𝑑𝑦
ℎଵ= −ℎ
ሺሻ+ ℎ௬ሺ𝑦ሻ
2
, ℎଶ= −ℎ
ሺሻ+ ℎ௬ሺ𝑦ሻ
2
+ ൫ℎ
ሺሻ+ ℎିଵ
ሺሻ൯
where 𝑏ሺ𝑢ሻ: Weld width by pass ℎሺ𝑢ሻ: 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
ሺሻ+ ℎ௬ሺ𝑦ሻቁ∙𝑏
൝න
൭
௬ሺ𝑦ሻ
ℎ
∓
ℎ
ሺሻ+ ℎ௬ሺ𝑦ሻ
భ
∙
ℎ
ℎ
ሺሻ+ ℎ௬ሺ𝑦ሻ
൱𝑑𝑧ൡ𝑑𝑦
ℎଵ= −ℎ
ሺሻ+ ℎ௬ሺ𝑦ሻ
2
, ℎଶ= −ℎ
ሺሻ+ ℎ௬ሺ𝑦ሻ
2
+ ൫ℎ
ሺሻ+ ℎିଵ
ሺሻ൯
where 𝑏ሺ𝑢ሻ: Weld width by pass ℎሺ𝑢ሻ: 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) 𝑇௧௧
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. ฬሼ𝑓௪ௗሽ +
𝑚∙൛𝑓
௧ൟ
ୀଵ
ฬ≤𝑇𝑜𝑙𝑒𝑟𝑎𝑛𝑐𝑒
ฬሼ𝛿௪ௗሽ+
𝑚∙൛𝛿
௧ൟ
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. ฬሼ𝑓௪ௗሽ +
𝑚∙൛𝑓
௧ൟ
ୀଵ
ฬ≤𝑇𝑜𝑙𝑒𝑟𝑎𝑛𝑐𝑒
ฬሼ𝛿௪ௗሽ+
𝑚∙൛𝛿
௧ൟ
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.
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https://www.frontiersin.org/articles/10.3389/fmars.2020.574790/pdf
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English
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Reproductive Strategies of the Seagrass Zostera japonica Under Different Geographic Conditions in Northern China
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Frontiers in marine science
| 2,020
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cc-by
| 9,786
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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
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Zostera marina L. populations across gradients of latitude and temperature.
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the macrobenthic community structure in Swan Lake, Northern China? Mar. Biodivers. 48, 1337–1344. doi: 10.1007/s12526-016-0601-3 Conflict of Interest: XW was employed by the company Mashan Group Co., Ltd. York, P. H., Carter, A. B., Chartrand, K., Sankey, T., Wells, L., and Rasheed, M. A. (2015). Dynamics of a deep-water seagrass population on the Great Barrier Reef:
annual occurrence and response to a major dredging program. Sci. Rep. 5:13167. doi: 10.1038/srep13167 The remaining 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. Zhang, X. M., Lin, H. 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. References Flinn EM, Busch CM, Wright AP (1998) Myc boxes, which are conserved in
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specific modulation of c-Myc cotransformation by residues phosphorylated
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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
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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:
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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
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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
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identified using HGTector v0.2.164 with the following cut-off values: e-value = 1e–20, 9 NATURE COMMUNICATIONS | (2019) 10:5529 | https://doi.org/10.1038/s41467-019-13499-2 | www.nature.com/naturecommunicati Author contributions T.S. collected samples, established a culture, performed light and electron microscopic
observations, FISH experiments, genome sequencing and molecular phylogenetic analyses. S.S. performed molecular phylogenetic analyses, genome assembly and genome analyses. Y.K. performed light and electron microscopic observations. K.I. designed sampling trip and
experiments. T.S. and S.S. wrote the paper. All authors edited and approved the paper. 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
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W350–W355 (2014). 10 NATURE COMMUNICATIONS | (2019) 10:5529 | https://doi.org/10.1038/s41467-019-13499-2 | www.nature.com/naturecommunications ARTICLE Correspondence and requests for materials should be addressed to T.S. Peer review information Nature Communications thanks John A Fuerst, Simon
Johnston, Olga Lage and Tâm Mignot for their contribution to the peer review of
this work. 65. Cherry, J. M. et al. Saccharomyces Genome Database: the genomics resource
of budding yeast. Nucleic Acids Res. 42, D700–D705 (2012). Reprints and permission information is available at http://www.nature.com/reprints Additional information 63. Sharma, A. K., Kumar, S. K. H., Dhakan, D. B. & Sharma, V. K. Prediction of
peptidoglycan hydrolases- a new class of antibacterial proteins. BMC
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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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Descriptive Method in Spatiotemporal Simulation Observation
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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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English
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Soil Properties Responding to <em>Pinus tabulaeformis</em> Forest Thinning in Mountainous Areas, Beijing
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Advance journal of food science & technology/Advance journal of food science and technology
| 2,014
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cc-by
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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
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capacities of soils with major forest types in
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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
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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
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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:
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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
P. Mengucci, G. Barucca, A. Gatto, E. Bassoli, L. Denti, F. Fiori, E. Girardin, P. Bastianoni, B. Rutkowski, A. Czyrska-Filemonowicz
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DOI:
http://dx.doi.org/10.1016/j.jmbbm.2015.12.045
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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:
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DOI:
http://dx.doi.org/10.1016/j.jmbbm.2015.12.045
Reference:
JMBBM1760 PII:
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DOI:
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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,
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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. Characterization of Co-Cr-Mo alloy after a thermal
treatment for high wear resistance. Mat. Sci. Eng. C 32, 1868-1877. Barucca, G., Santecchia, E., Majni, G., Girardin, E., Bassoli, E., Denti, L., Gatto, A., Iuliano, L.,
Moskalewicz, T., Mengucci, P., 2015. Structural characterization of biomedical Co–Cr–Mo
components produced by direct metal laser sintering. Mat. Sci. Eng. C 48, 263–269. Bassoli, E., Gatto, A., Iuliano, L., 2012. Joining mechanisms and mechanical properties of PA
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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. The estimated lattice parameter of the γ-Co phase in the powder is reported for
comparison. Table 8
Results of STEM-EDX analysis performed on matrix and precipitates. Element
concentration in wt%. Figure 1
Geometry of samples for mechanical tests: a) flexural (FLEX), b) tensile (TENS). Figure 2
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. Figure 4
XRD patterns of the as sintered (AS) and heat treated (CHT) samples. 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. Average value (AV) and standard deviation (SD) of Rockwell A hardness (HRA) and
transverse rupture strength (TRS) of the flexural specimens. p-values resulting from the t-test for UTS, εb, HRA and TRS among the groups AS, 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. RHT and CHT. Records below the level of significance of 0.05 are underlined. 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
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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
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https://www.qeios.com/read/JF6SCT/pdf
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English
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Review of: "Growing Confidence and Remaining Uncertainty About Animal Consciousness"
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cc-by
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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
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https://openalex.org/W3039712416
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https://link.springer.com/content/pdf/10.1007/s00440-020-00984-9.pdf
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English
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On the number of excursion sets of planar Gaussian fields
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Probability theory and related fields
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cc-by
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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
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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/. 123 D. Beliaev et al. 698 References 1. Adler, R.J., Taylor, J.E.: Random Fields and Geometry. Springer, Berlin (2009) 2. Azaïs, J.M., Wschebor, M.: Level Sets and Extrema of Random Processes and Fields. Wiley, Hoboken
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2083–2091 (1977) 5. Cheng, D., Schwartzman, A.: Expected number and height distribution of critical points of smoo
isotropic Gaussian random fields. Bernoulli 24(4B), 3422–3446 (2018) 6. Dennis, M.R.: Nodal densities of planar Gaussian random waves. Eur. Phys. J. Spec. Top. 145(1
191–210 (2007) 7. Farb, B., Margalit, D.: A Primer on Mapping Class Groups (pms-49). Princeton University Press,
Princeton (2011) 8. Handron, D.G.: Generalized billiard paths and Morse theory for manifolds with corners. Topol. Its
Appl. 126(1–2), 83–118 (2002) 9. Ingremeau, M., Rivera, A.: A lower bound for the Bogomolny–Schmit constant for random monochro-
matic plane waves. Math. Res. Lett. 26(4), 1179–1186 (2019). https://doi.org/10.4310/MRL.2019.v26. n4.a9 10. Kurlberg, P., Wigman, I.: Variation of the Nazarov–Sodin constant for random plane waves and arith-
metic random waves. Adv. Math. 330, 516–552 (2018) 11. Lax, P.D., Phillips, R.S.: The asymptotic distribution of lattice points in Euclidean and non-Euclidea
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(2016) 14. Nicolaescu, L.: On the Kac–Rice formula. Unpublished (2014) 15. Sodin, M.: Lectures on random nodal portraits. Probab. Stat. Phys. St. Petersburg 91, 395–422 (201 ,
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and institutional affiliations. 123
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Über das Isochinolin im Steinkohlenteer
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Berichte der Deutschen Chemischen Gesellschaft
| 1,914
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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
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Journal of mathematical biology
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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
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g
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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
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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
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162(1):1–32 123 123
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|
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How Can We Provide Lifelong Support for People with Epilepsy to Reduce Their Self-stigma?
|
IntechOpen eBooks
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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
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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
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i
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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
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UMR7095 CNRS U i
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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
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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
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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,
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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. 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. 8. Conclusions and perspectives C., et al. 1996, ApJ, 467, 38
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CNRS/INSU with INP and IN2P3, co-funded by CEA and CNES. We acknowl-
edge support from the Spanish Programa Nacional de Astronomía y Astrofísica
under grant AYA 2006-02358 and the Swiss National Science Foundation. Par-
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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
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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
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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-
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has been provided by NSF grants AST95-09298, AST-0071048, AST-0071198,
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Part of this work was supported by the French CNRS,
the French Programme National de Cosmologie et Galaxies (PNCG) of
CNRS/INSU with INP and IN2P3, co-funded by CEA and CNES. We acknowl-
edge support from the Spanish Programa Nacional de Astronomía y Astrofísica
under grant AYA 2006-02358 and the Swiss National Science Foundation. Par-
tially funded by the Spanish MEC under the Consolider-Ingenio 2010 Pro-
gram grant CSD2006-00070: First Science with the GTC (http://www.iac.
es/consolider-ingenio-gtc/). 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
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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
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Test sensitivity is secondary to frequency and turnaround time for COVID-19 screening
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†daniel.larremore@colorado.edu
‡mmina@hsph.harvard.edu
§roy.parker@colorado.edu .
CC-BY-NC-ND 4.0 International license
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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
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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
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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
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18
20
days since exposure
2
3
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9
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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
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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)
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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
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this version posted September 8, 2020. . https://doi.org/10.1101/2020.06.22.20136309
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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
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medRxiv preprint . CC-BY-NC-ND 4.0 International license
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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
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20
days since exposure
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3
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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
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self-isolation
LOD 103
LOD 105
D 1
infectiousness removed
C 1
d
C A days since exposure
0
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16
18
20
days since exposure
0.00
0.25
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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
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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
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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
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1
1
1
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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
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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
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0
0
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0
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1
1
1
1
1
2
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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
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1
1
1
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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
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1
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1
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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
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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
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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
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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
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edRxiv preprint 0
2
4
6
8
weeks after surveillance begins
0%
2%
4%
6%
8%
10%
12%
prevalence
fully mixed model
12%
38%
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100%
A
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0%
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agent based model
13%
37%
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B
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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
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B no intervention
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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)
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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
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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
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this version posted September 8, 2020. . org/10.1101/2020.06.22.20136309 0
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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
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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)
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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
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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
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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
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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
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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
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0
2000
4000
6000
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no surveillance
A
infected
test-based isolation
symptom-based isolation
0
50
100
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250
300
350
Day
0
200
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800
Count
LOD 105, every 7 days
C
0
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350
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0
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LOD 103, every 7 days
B
0
50
100
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250
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350
Day
0
10
20
30
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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
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is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
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edRxiv preprint Contents CC-BY-NC-ND 4.0 International license
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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
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effective repr. num. R
fully mixed model
C
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0
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B
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0.0
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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
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LOD 105
LOD 106
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fully mixed model
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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
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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:
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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. J.H. is the developer of the EPaS programme
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and the first five years of life. London: Department of Health. Ethical Approval Ethical approval was granted by Bangor University
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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
| 1,961
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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.
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https://www.qeios.com/read/MINY0N/pdf
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English
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Review of: "In-Vitro Antibacterial Activity of some Ganoderma Species: A Review"
| null | 2,023
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cc-by
| 73
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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
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https://openalex.org/W2041623144
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https://europepmc.org/articles/pmc3305035?pdf=render
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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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cc-by
| 519
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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.
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https://molecularneurodegeneration.biomedcentral.com/track/pdf/10.1186/s13024-019-0353-1
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Longitudinal and nonlinear relations of dietary and Serum cholesterol in midlife with cognitive decline: results from EMCOA study
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Molecular neurodegeneration
| 2,019
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cc-by
| 12,590
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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. Consent for publication
All h
h
d All the co-authors and participants have given their consent for publication
in Molecular Neurodegeneration. 4.
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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
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apioside from Glycyrrhiza glabra L. TOXICOL IN VITRO 2009;23:680-6. [22] Asl MN, Hosseinzadeh H. Review of pharmacological effects of Glycyrrhiza sp. and its bioactive
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suppresses inflammation and reduces the increased glucose levels induced by the combination of
Porphyromonas gulae and ligature placement in diabetic model mice. INT IMMUNOPHARMACOL
2018;68:30-8. [24] Fan B, Jiang B, Yan S, Xu B, Huang H, Chen G. Anti-Inflammatory 18β-Glycyrrhetinin Acid
Derivatives Produced by Biocatalysis. PLANTA MED 2018;85:56-61. INTEGR MED 2013;19:826-35. [25] Kamisli S, Ciftci O, Taslidere A, Basak TN, Ozcan C. The beneficial effects of 18beta-
glycyrrhetinic acid on the experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mouse model. Immunopharmacol Immunotoxicol 2018;40:344-52. [26] Liu X, Ma X, Huo C, Yu S, Wang Q. [Microbiological transformation of paeoniflorin and albiflorin]. Zhongguo Zhong Yao Za Zhi 2010;35:872-5. [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).
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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.
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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
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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).
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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.
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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.
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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.
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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
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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.
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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. Tanto en radiografía simple como en TC se observaron
bronquiectasias en el 44% de los pacientes con cultivo positivo para
micobacterias atípicas, aunque no se detectaron diferencias significativas ni en
radiografía simple (p=0.0551) ni en TC (p=0.0677) frente a los pacientes con
tuberculosis pulmonar.
182
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198
ANEXO 1:
FORMULARIO DE RECOGIDA DE DATOS:
Id. Paciente:
Año: 2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
1. HALLAZGOS
A. 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
|
|
https://openalex.org/W2383412397
|
https://hal.science/hal-01206067v2/file/transHumUs_icra.pdf
|
English
| null |
transHumUs: A poetic experience in mobile robotics
| null | 2,016
|
cc-by-sa
| 6,025
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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
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scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
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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
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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
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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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https://openalex.org/W3085895781
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https://ijip.in/wp-content/uploads/2020/05/B00315V2I42015.pdf
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Relational Dynamics of MSM Population
|
International journal of Indian psychology
| 2,015
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cc-by
| 2,648
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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
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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
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(ISSN)1520-6394. 24. Tolin DF, Maltby N, Diefenbach GJ, Hannan SE, Worhunsky P. Cognitive–behavioral therapy for medication nonresponders with
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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
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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
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SR, Nakamae T, et al. Brain circuitry of compulsivity. Eur
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https://openalex.org/W2343004781
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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
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https://openalex.org/W4289753846
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https://ccsenet.org/journal/index.php/ijsp/article/download/0/0/39033/40095
|
English
| null |
An Improved Bound for Security in an Identity Disclosure Problem
|
arXiv (Cornell University)
| 2,018
|
cc-by
| 8,440
|
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)! Copyrights Copyright for this article is retained by the author(s), with first publication rights granted to the journal Copyright for this article is retained by the author(s), with first publication rights granted to the journal. This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/). This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/). 31
|
https://openalex.org/W2978421384
|
https://hal.archives-ouvertes.fr/hal-02308851/file/wlb.00561.pdf
|
English
| null |
Habitat fragmentation and anthropogenic factors affect wildcat Felis silvestris silvestris occupancy and detectability on Mt Etna
|
Wildlife biology
| 2,019
|
cc-by
| 12,997
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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
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Submitted on 8 Oct 2019 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
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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
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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
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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
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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
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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
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Washoff of cypermethrin residues from slabs of external building material surfaces using simulated rainfall
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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
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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-
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Screening for potential prophylactics targeting sporozoite motility through the skin
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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://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 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://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. 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
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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. Skin penetration and dermal tolerability of acrylic nanocapsules:
influence of the surface charge and a chitosan gel used as vehicle. Int J
Pharm. 2016;507:12–20.
88. Schulz R, Yamamoto K, Klossek A, Flesch R, Honzke S, Rancan F, et al. Data-
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Comparison and evaluation of digital signature schemes employed in ndn network
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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”,
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(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
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[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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https://openalex.org/W2047119503
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https://www.jstage.jst.go.jp/article/abas/14/1/14_35/_pdf
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English
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A Missing Piece of Mutual Learning Model of March (1991)
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Annals of business administrative science
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cc-by
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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
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(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
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https://journals.uni-lj.si/aas/article/download/15568/13502
|
English
| null |
Endosperm protein polymorphism of common and tartary buckwheat
|
Acta agriculturae Slovenica
| 2,004
|
cc-by
| 3,718
|
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
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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
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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
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de
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Warum wird das DINI-Zertifikat immer wieder angepasst?
|
ABI-Technik
| 2,022
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cc-by
| 472
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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://openalex.org/W3206951511
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https://pure.au.dk/ws/files/274021624/1_s2.0_S0264410X21011506_main.pdf
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English
| null |
Coverage and factors associated with receiving campaign polio vaccines in an urban population in Guinea-Bissau
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Vaccine
| 2,021
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cc-by
| 5,646
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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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Implementation of the Meningitis Vaccine Project in Africa: lessons for vaccine implementation programs
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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
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24. Means AR, Kemp CG, Gwayi-Chore MC, et al. Evaluating and optimizing the consolidated
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25. Kirk MA, Kelley C, Yankey N, Birken SA, Abadie B,
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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
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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
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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
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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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English
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Developing emulsion gels by incorporating Jerusalem artichoke inulin and investigating their lipid oxidative stability
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Food production, processing and nutrition
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cc-by
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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
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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
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purified inulin extract from JA); CI (commercial inulin from chicory root); CIE (crude inulin extract from JA) and control (without inulin). Data
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G. D. (2018). Inulin rich carbohydrates extraction from Jerusalem artichoke
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0480-y. Stoyanova, S., Geuns, J., Hideg, É., & Van Den Ende, W. (2011). The food additives inulin
and stevioside counteract oxidative stress. International Journal of Food Sciences and
Nutrition, 62(3), 207–214. https://doi.org/10.3109/09637486.2010.523416. Tavakoli, H. R., Naderi, M., Jafari, S. M., & Naeli, M. H. (2019). Postmarketing
surveillance of the oxidative stability for cooking oils, frying oils, and
vanaspati supplied in the retail market. Food Science and Nutrition, 7(4),
1455–1465. https://doi.org/10.1002/fsn3.982. Tchoné, M., Bärwald, G., & Meier, C. (2005). Polyphenoloxidases in Jerusalem
artichoke (Helianthus tuberosus L.). British Food Journal, 107(9), 693–701. https://doi.org/10.1108/00070700510615071. Tchoné, M., Bärwald, G., & Meier, C. (2005). Polyphenoloxidases in Jerusalem
artichoke (Helianthus tuberosus L.). British Food Journal, 107(9), 693–701. https://doi.org/10.1108/00070700510615071.
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Reflections on graduate professor training
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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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https://openalex.org/W2183944011
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0143940&type=printable
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English
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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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PloS one
| 2,015
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cc-by
| 7,727
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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-
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21092187 3. Eng CM, Guffon N, Wilcox WR, Germain DP, Lee P, Waldek S, et al. Safety and efficacy of recombinant
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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–
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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
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using three-dimensional stereotactic surface projections of fluorine-18-FDG PET. J Nucl Med. 1995: 36
[7]:1238–1248. PMID: 7790950 17. Fazekas F, Chawluk JB, Alavi A, Hurtig HI, Zimmerman RA. MR signal abnormalities at 1.5 T in Alzhei-
mer's dementia and normal aging. Am J Roentgenol. 1987; 149[2]:351–356. 18. Wahlund LO, Barkhof F, Fazekas F, Bronge L, Augustin M, Sjogren M, et al. A new rating scale for age-
related white matter changes applicable to MRI and CT. Stroke. 2001; 32[6]:1318–1322. PMID:
11387493 19. Schmidt R, Fazekas F, Kapeller P, Schmidt H, Hartung HP. MRI white matter hyperintensities: three-
year follow-up of the Austrian Stroke Prevention Study. Neurology. 1999; 53[1]:132–139. PMID:
10408549 20. Enzinger C, Fazekas F, Ropele S, Schmidt R. Progression of cerebral white matter lesions—clinical
and radiological considerations. J Neurol Sci. 2007; 257[1–2]:5–10. 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
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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
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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
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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
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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. Data Syst., 7, 83–99, 2018 References and Lakshmi, V.: Soil moisture at watershed scale: Remote
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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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BMC evolutionary biology
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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,
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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.
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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
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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
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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,
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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,
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International Institute for Sustainable Development: Winnipeg, MB, Canada, 2008; Volume 1. © 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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Effects of Non-Driving Related Tasks During Self-Driving Mode
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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
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focused on the relationship between lead time, velocity and
NDR-tasks. Indeed, the implications of these results are impor-
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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
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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
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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
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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
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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
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ный журнал Символ науки 2016; 26: 158-61). 3. Atlas of Diabetes, IDF Seventh Edition, 2015. [Electronic Source] http://www.idf.org
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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
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update of the Finnish diabetes risk score for prediction of incident type 2 diabetes. Diabetologia 2011; 54: 1004-12. 20
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Analysis of High Force Voice Coil Motors for Magnetic Levitation
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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.
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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
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The importance of a comparative thermophysiological study in female jeans
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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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https://openalex.org/W4361967281
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https://figshare.com/articles/journal_contribution/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/22487409/1/files/39939003.pdf
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English
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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
| null | 2,023
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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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https://europepmc.org/articles/pmc3102729?pdf=render
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English
| null |
ALTERED MERISTEM PROGRAM 1 Is involved in Development of Seed Dormancy in Arabidopsis
|
PloS one
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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
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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-
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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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BMC medical education
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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
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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
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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-
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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
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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
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acquisition, consulted during the entire process (regarding actual procedures
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Learning Objectives for Undergraduate Medical Education;
PME: Postgraduate medical education; UME: Undergraduate medical
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of mappers and data quality control. Mapping data
may be positively or negatively biased by certain fac-
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tion of intrinsic roles, CBME acceptance, institutional
culture and enthusiasm for teaching. Therefore, map-
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for curriculum depiction. It is rather an actual snap-
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changing field of UME, which must be regularly up-
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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-
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perspective curriculum evaluation (learned curricu-
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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
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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-
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Pendidikan. Yogyakarta: Deeppublish. Basir, C. (2009). Penyelesaian Sengketa Perbankan Syariah di Pengadilan Agama dan
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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). Metode Penelitian Kuantitatif, Kualitatif, dan R&D. Bandung: ALFABETA. Supardi D (2017) Statistik penelitian pendidikan Depok: PT Raja Grafindo Persada , D. (2017). Statistik penelitian pendidikan. Depok: PT Raja Grafindo Persada. Wawan, A., & Dewi , M. (2010). Teori dan Pengukuran Pengetahuan, Sikap dan Perilaku
Manusia. Yogyakarta: Nuha Medika. 88 Jurnal Al-Fatih Global Mulia
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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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Nature communications
| 2,024
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cc-by
| 467
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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
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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
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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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bioRxiv (Cold Spring Harbor Laboratory)
| 2,018
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cc-by
| 13,144
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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
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1
Downloaded from https://academic.oup.com/brain/adv doi:10.1093/brain/awaa045
BRAIN 2020: Page 1 of 15
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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
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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-
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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
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BMC complementary and alternative medicine
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cc-by
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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
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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
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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
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https://openalex.org/W2466011833
|
https://www.nature.com/articles/srep27091.pdf
|
English
| null |
miR-146a is essential for lipopolysaccharide (LPS)-induced cross-tolerance against kidney ischemia/reperfusion injury in mice
|
Scientific reports
| 2,016
|
cc-by
| 10,223
|
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-κB, leading to an increase in inflammatory cytokine expression such as IL-1β and TNF-α14–16. 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-κB-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-κB dependent22; Importantly, two key adapter molecules in the TLR4/NF-κB 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-κB 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
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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-κB 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-κB
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-κB (IκBs), nuclear translocation of
NF-κB p65/p50 and transcriptional activation of proinflammatory genes31. To determine the link of miR-146a
and NF-κB 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-κB 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-κB 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-κB 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-κB p65 nuclear translocation at all
time points. Either normal saline (NS) or LPS treated Sham scrambled control mice showed the same NF-κB
activity baseline (data not shown). Furthermore, the increased NF-κB activation was accompanied by a simul-
taneous decrease in cytosolic IkBα, an inhibitory protein that prevents translocation of NF-κB into the nucleus
(Fig. 7c,d, Supplementary Figure 2).h ( g
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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-κBp65/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-κB. www.nature.com/scientificreports/ All data were expressed as mean ± SEM. NF-κB 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-κB activity, disrup-
tion of chromatin remodeling15. Promoter analyses have shown that transcriptional up-regulation of miR-146a
induced by TLR4 is NF-κB 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
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Considering the regulation of TRAF6 and IRAK-1 in TLR4/NF-κB 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-κB system. We demonstrated that up-regulation of miR-146a in
LPS/IR stimulated kidneys was inversely correlated with activation of NF-κB p65/p50, degradation of IκBα. 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-κBp65/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-κB 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-κB activation and histology score. Knockdown of miR-146a caused a stronger nuclear translocation of
NF-κB 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-κB. translocation of p50/p50 homodimers and decreased levels of the active p65/p50 heterodimers38,39. However,
many studies have showed that NF-κB 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-κB decoy oligodeoxynucleotides in vivo and curtailed the
production of inflammatory cytokine genes, thereby attenuate kidney I/R injury41,42. The effects of NF-κB 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-κB 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-κB activation and histology score. Knockdown of miR-146a caused a stronger nuclear translocation of
NF-κB 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-κB. 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-κB, 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-κB 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-κB decoy oligodeoxynucleotides in vivo and curtailed the
production of inflammatory cytokine genes, thereby attenuate kidney I/R injury41,42. The effects of NF-κB 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-κB and hypoxia-inducible factor-2a (HIF-2a) signaling17. The current study indicates that
TLR4/ NF-κB 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-κB 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-κB 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-κB 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-κB 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-κB 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-κB 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-κB 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-IκB-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
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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
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14339–44 (2010). 27. Godwin, J. G. et al. 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
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https://openalex.org/W4377833255
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https://jurnal.ukdc.ac.id/index.php/BIP/article/download/396/274
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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
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cc-by-sa
| 4,681
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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. Pengaruh Motivasi dan Disiplin Kerja Terhadap Produktivitas
Kerja Karyawan Pada Hotel Merlynn Park. Skripsi. Universitas Negeri Jakarta.
Jakarta. DAFTAR PUSTAKA Baiti, K. N., Djumali, dan E. Kustiyah. 2020. Produktivitas Kerja Karyawan
Ditinjau Dari Motivasi, Disiplin Kerja, dan Lingkungan Kerja Pada PT
Iskandar Indah Printing Textile Surakarta. Edunomika. Vol. 4, No. 1. pp. 69-
87. Chaer, V. P. 2020. Pengaruh Motivasi dan Disiplin Kerja Terhadap Produktivitas
Kerja Karyawan Pada Hotel Merlynn Park. Skripsi. Universitas Negeri Jakarta. Jakarta. 75 Analisis Pengaruh Disiplin dan Motivasi Kerja Terhadap Produktivitas Kerja Karyawan Hotel Goodrich Suites Jakarta
Wendy Shelma Santika dan Thyophoida W. S. Panjaitan Djaya, S. 2021. Pengaruh Motivasi Kerja dan Kompetensi Terhadap Kinerja
Karyawan di Moderasi Kompensasi. Buletin Studi Ekonomi, Vol. 26, No. 1. Pp. 72-84. Ernawati, K. 2017. Pengaruh Disiplin Kerja Terhadap Produktivitas Kerja
Karyawan Non Medis di Rumah Sakit Islam Siti Aisyah Madiun. Skripsi. STIKES Bhakti Husada Mulia Madiun. Madiun. Ghozali, I. 2018. Aplikasi Analisis Multivariate dengan IBM SPSS 25. Badan
Penerbit Universitas Diponegoro, Semarang Gumelar, A. C. 2017. Pengaruh Disiplin Kerja, Motivasi Kerja, dan Penerapan
Standar Operasional Prosedur Terhadap Kinerja Karyawan Jawa Pos Radar
Kediri. Skripsi. Universitas Nusantara PGRI Kediri. Kediri. Hafid, A. P. 2018. Pengaruh Disiplin Kerja Terhadap Produktivitas Kerja
Karyawan Pada PT Rakyat Sulsel Intermedia Kota Makassar. Skripsi. Universitas Muhammadiyah Makassar. Makassar. Hafid, A. P., M. A. Pasigai., dan M. N. Rasyid. 2018. Pengaruh Disiplin Kerja
Terhadap Produktivitas Kerja Karyawan Pada PT Rakyat Sulsel Intermedia
Kota Makassar. Jurnal Profitability Fakultas Ekonomi dan Bisnis. Vol. 2, No. 2. pp. 70-79. Halim, J. dan F. Andreani. 2017. Analisis Pengaruh Motivasi dan Kompensasi
Terhadap Kinerja Karyawan di Broadway Barbershop PT Bersama Lima
Putera. Agora, Vol. 5, No. 1. Hidayat, R. 2021. Pengaruh Motivasi, Kompetensi, dan Disiplin Kerja Terhadap
Kinerja. Widya Cipta: Jurnal Sekretari dan Manajemen. Vol. 5, No. 1, pp. 16-
23. Irawan, D., G. Kusjono., dan Suprianto. 2021. Pengaruh Disiplin Kerja dan
Lingkungan Kerja Terhadap Kinerja Pegawai Negeri Sipil pada Kantor
Kecamatan Serpong. Jurnal Ilmiah Mahasiswa (JIMAWA). Vol. 1, No. 3, pp. 176-185. Javier, Faisal. 2022. Tingkat Hunian Kamar Hotel Bintang Februari 2022 Masih
dalam Tren Menurun Secara Bulanan. https://data.tempo.co/data/1389/tingkat-
hunian-kamar-hotel-bintang-februari-2022-masih-dalam-tren-menurun-secara-
bulanan. Diunduh 27 Agustus 2022, pukul 20.36. Kurniasari, R. 2018. Pemberian Motivasi serta Dampaknya Terhadap Kinerja
Karyawan Pada Perusahaan Telekomunikasi Jakarta. Widya Cipta: Jurnal
Sekretari dan Manajemen, Vol. 2, No. 1, pp. 32-39. Kuswibowo, C. 2020. Pengaruh Motivasi Kerja dan Disiplin Kerja Terhadap
Produktivitas Kerja Karyawan. Prosiding Simposium Nasional Multidisiplin. Universitas Muhammadiyah Tangerang, Vol. 2. 76 Jurnal Bisnis Perspektif (BIP’s), Volume 15, Nomor 1, Januari 2023 Mamanna, G. I., T. DAFTAR PUSTAKA Bernhard, dan T. S. Regina. 2022, Analisis Pengaruh Mood,
Motivasi dan Disiplin Kerja terhadap Produktivitas Kerja Pegawai pada Dinas
Kependudukan dan Pencatatan Sipil Kabupaten Minahasa, Jurnal EMBA, Vol. 10, No. 4, pp. 1728-1742. Ma´ruf dan U. Chair. 2020. Pengaruh Motivasi Kerja Terhadap Kinerja Karyawan
Pada PT Nirha Jaya Tehnik Makassar. Jurnal Brand. Vol. 2, No. 1, pp. 99-108. Pranitasari, D. dan K. Khotimah. 2021. Analisis Disiplin Kerja Karyawan. Jurnal
Akuntansi dan Manajemen. Vol. 18, No. 1, pp. 22-38. Pratiwi, E. A. 2018. Pengaruh Disiplin Kerja Terhadap Kinerja Karyawan di
Rumah Sakit Umum Darmayu Ponorogo. Skripsi. STIKES Bhakti husada
Mulia Madiun. Madiun. Sanjaya, R. 2018. Pengaruh Motivasi dan Disiplin Kerja Terhadap Kinerja
Pegawai Dalam Perspektif Ekonomi Islam (Studi di Dinas Sosial Tenaga
Kerja dan Transmigrasi Kabupaten Pesisir Barat). Skripsi. Universitas Islam
Negeri Raden Intan Lampung. Lampung. Santoso, A. B. dan N. K. Ramadhan. 2020. Pengaruh Disiplin Kerja dan Motivasi
Kerja Terhadap Produktivitas Kerja Karyawan Pada PT Rice Bowl Indonesia
di Plaza Asia Sudirman. Jurnal Disrupsi Bisnis, Vol. 3, No. 1, pp. 60-71. Sugiyono. 2018. Metode Penelitian Pendidikan: Kuantitatif, Kualitatif, dan R&D. Alfabeta. Bandung. Wau, J., S. Waoma, dan F. T. Fau. 2021. Pengaruh Disiplin Kerja Terhadap
Kinerja Pegawai di Kantor Camat Somambawa Kabupaten Nias Selatan. Jurnal Ilmiah Mahasiswa Nias Selatan, Vol. 4, No. 2, pp. 203-212. Yohanson, A. K., L. Hakim., dan Y. Wijaya. 2022. Kinerja Karyawan Hotel POP
Bandar Lampung Dilihat Dari Disiplin Kerja dan Pelatihan Kerja. Jurnal
Gentiaras Manajemen dan Akuntansi. Vol. 14, No. 2, pp. 111-122. 77
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